The Role of Globotriaosylceramide in Internalization and
Functions of Globotriaosylceramide-bound Protein Ligands
Aye Aye Khine
A thesis subrnitted in conformity with the requirements for the degree of Doctor of
Philosophy
Department of Laboratory Medicine and Pathobiology
University of Toronto
O Copyright by Aye Aye mine, 2000 National Library Biblïath&que nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Weilington Street 395, rue Wellington OttawaOlV KIAON4 O&awsOIJ KlAW Canada Canada
The author has granted a non- L'auteur a accordé une licence non exclusive Licence ailowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, dismiute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The Role of Globotriaosylceramide in Internalization and Functions of Globotriaosylceramide-bound Protein Ligands Doctor of Philosophy, 2000 Aye Aye Khine Department of Laboratory Medicine and Pathobiology University of Toronto
ABSTRACT
GIobotriaosylcerarnide(Gb,), the ce11 surface receptor glycosphingolipid for the
Escherichia coli-derived verotoxin(VT) mediates VT endocytosis and retrograde transport to the Golgi, endoplasmic reticulum and nuclear membrane. Since VT does not contain an ER retention sequence, Gb, itself may determine the retrograde transport of
Gb,-bound ligands. Intracellular -cking of VT is dependent on different fatty acid containing Gb, isoforms. Targeting of VTl to ER and nuclear membrane, and sensitivity to VT1 cytotoxicity are determined by Gb, isoforms with shorter fatty acid chahs, whereas longer fatty acid containing Gb, isoforms mediate VTI transport to the Golgi which is correlated with reduced VT 1 cytotoxicity.
The B-lymphocyte specific antigen, CD19 and the IFNARl component of type 1 interferon receptor are two integral membrane proteins, the extracellular domains of which have high amino acid sequence similarity to the Gb,-binding VT subunit B, suggesting the possible lateral association between CD19 or IFNARI and Gb, at the ce11 surface. Gb, is also known as a ce11 differentiation marker CD77 of a subset of germinal center B cells, which are readily entenng apoptosis. In the present study, antibody-crosslinked intemalized CD19 has also been found to undergo retrograde transport to ER and nuclear membrane in Gb,-positive cells only, providing further evidence of Gb,-dependent retrograde transport of a Gb,-bound protein ligand. CD19 mediated apoptosis and 1FN-a-mediated antiviral activity also has been found to be significant only in Gb,-positive cells, indicating that structural association with Gb, modulates the signal transduction and biological activities of CD19 and
IFNARI. Long chah fatty acid-containhg Gb, isoforms have been found to be more significant for 1FN-a-mediated antiviral activity.
VT is principally intemalized via clathrin-dependent receptor mediated endocytosis and partially via clathrin-independent caveolar-rnediated endocytosis. In the present study, reIative role and intracellular destination of the two pathways for the cytotoxicity have been compared. Clathrin-mediated pathway is the major mechanism of
VTl internalization while Golgi-independent caveolar-mediated transport to the peri- nuciear region can be an alternative pathway. Despite the similarity between VTl and
VT2 in the structure and receptor binding specificity to Gb,, VT2 internalization is more restricted to the Golgi-dependent pathway. 1 would like to express my gratitude to my supervisor Dr. C. A. Lingwood for his understanding, patience and guidance throughout this work. 1 also wish to thank my advisory cornmittee members Dr. S. Grinstine and Dr. T. Watts for their outstanding guidance and comments for my research and thesis, Dr. J. Shayman and Dr. G. Hannigan for accepting as the extemal examiners, and Dr. Y. T. Wang and Dr. D. F. Andrews for acting as chairpersons for the defense. 1 would also like to express my appreciation to Dr. C. Richardson, Amgen Research Institute, Princess Margaret Hospital, for giving me an opportunity to work as a post-doctoral fellow and his helptùl advise during the preparation of my thesis. The outstanding technical assistance for electronmicroscopy by Mr. S. Doyle, University of Toronto, EM facility, and for the photography by Mr. D. Aguilar, the graphic center, Hospital for Sick Children, are well appreciated. I am very grateful to al1 my colleagues in the laboratory, more specifically to Shirley for her secretarial assistance, Myl, Beth and Anita for their excellent technical support, Prateek and Patty for any help and assistance I constantly asked for, and Daniel, Heather, Linda and Mann for their great fiiendship. Finally, 1 would like to express my ultimate gratefûlness to my husband and my parents for their love, hope, strength, absolute trust and ceaseless support throughout this work.
iii For my dearest son Yezami
and the baby. TABLE OF CONTENTS
Abstract Acknowledgements Dedication Table of contents List of abbreviations List of figures List of tabies xiv Chapter 1: Introduction 1 1.1 Glyeospbingoiipids and Globotriaosylcer~mide 2 1.1.1 Glycosphingolipids 2 1.1.1.1 Structure 2 1.1.1.3 Classification 2 1.1.1.3 B iosynthesis 4 1.1.1.4 Degradation 13 1-1-15 Physical properties 14 1.1.1.6 Biological properties 14 1.1.2 Globotriaosyiceramide 19 1.1.2.1 Biological fimctions 19 1.2 Verotoxin-producing Escherichia coli and verotoxin 25 1.2.1 Verotoxin-producing Escherichia coli 25 1.2.1.1 Clinical manifestations of VTEC infection 26 1 2.1.2 Pathogenesis of VTEC infection 26 1.2.1 -3 Histo-pathology of hemorrhagic colitis and 27 hemolytic-uremic syndrome 1.2.1.4 Role of idammatory cytokines in pathogenesis of hemorrhagic colitis and hemolytic-uremic syndrome Verotostin Nomenclature and classification of verotoxin Structure and physico-chernical properties of verotoxin Functions of verotoxin Biological activities of verotoxin Antigenicities of verotoxin Functional receptors for verotoxin Pathogenesis of verotoxùi-associated diseases Intemalization of verotoxin Role for verotoxin in anti-cancer therapy B-lymphocyte differentiation antigen; CD19 B-ceIl aotigen receptor Structure Signal transduction Co-receptors of B-ce11 antigen receptor Biologicai functions CD19 Structure Signal transduction Biological functions Intemalization of CD 19 Relationship between Gb, and CD 19 Type I interferons and type 1 interferon receptor Classification of interferons Structure of type 1 [FN receptor Type 1 interferodreceptor-mediatedsignal transduction Type 1 interferon regulated proteins Type 1 interferon-mediated biologicai activities Intemalization of type 1 interferodreceptor Relationship between G4and type 1 interferon receptor 1.5 Vesicle-mediated protein transport 1.5.1 Biosynthetid secretory pathway 1.5.1.1 Structurd components 1.5.1.2 Regdatory components 1.5.1.3 Mechanism of vesicle movement 1.5.1.4 Protein sorting 1.5.1.5 Retrograde transport 1.5.2 Endocytic pathway 1S.2.l Clathrin-dependent endocytosis 1.5.2.1.1 Structurai component 1.5.2.1.2 Regdatory component 1 .5.2.1.3 Mechanism of vesicle movement 1 .j.ll.4 Protein sorting 1.5.2.2 Clathrin-independent endocytosis 1.5.2.2.1 Caveolar-mediated endocytosis 1.5.2.2.2 Clathnn-independent, caveolar-independent endocytosis 1.5.2.2.3 Macropinocytosis 1.5.2.2.4 Phagocytosis 1.5.3 lnternalizatioa of protein toxins Chapter 2: Objective and hypothesis Chapter 3: Functional role for Gb, in antibody cross-linked CD19 internalization and apoptosis Abstract Introduction Materials and methods Results Discussion Future directions Chapter 4: FunctionaI role for Gb, b interferon-a/ type I intederon receptor medhtd anti viral activity 4.1 Abstract 4.2 Introduction 4.3 Materials and methods 4.4 Results 4.5 Discussion 4.6 Future directions Chapter 5: Gb, dependent intracellular transport mechanisms of Verotoxin Abstract Introduction Materiais and methods Resul ts Discussion Future directions Chapter 6: The comparison of VTl and VT2 internaliution and intracellular targeting 6.1 Abstract 6.2 Introduction 6.3 Materials and methods 6.4 Results 6.5 Discussion 6.6 Future directions Chapter 7: Conclusion References
viii LIST OF ABBREVIATIONS
AP adaptor protein 2-5 AS 2'4' oligoadenylate synthetase BCR B-ce11 antigen receptor BFA brefeldin A CD clusters of differentiation COP coat associated protein CPE cyto pathic e ffect CT cholera toxin DAG diacyl glycerol DIM detergent insoluble microdomain DT diphtheria toxin e-IFNAR 1 extracellular domain of IFNARl EM electronmicroscopy EMCV encephalomyocarditis virus ER endoplasrnic reticulum ETA Pseudornonos exotoxin A FDC follicular dendritic ce11 FITC fluorescine isothiocyanate Gb, galabiosy lcerarnide Gb3 globotriaosylceramide Gb4 globotetraosylceramide GC germinal center GDP guanidine nucleotide diphosphate GTP guanidine nucleotide triphosphate GSL glycosphingolipids HC hemorrhagic colitis HUS hemolytic uremic syndrome IEM immuno-electronmicroscopy IFN interferon IFNARl interferon alpha receptor chain 1 IFNAR2 interferon alpha receptor chah 2 Ig imrnunoglobulin IL interleukin
IP3 inositol3 phosphate IRF- 1 interferon regulatory factor 1 ISG interferon-stimulated gene ISRE interferon stimulated response element ISGF3 interferon stimulated gene factor-3 LacCer lactosyl ceramide LPS lipopol ysaccharide LT E-coli heat labile toxin MEM minimum essential medium MHC major histocornpatibility complex m-Ig membrane immunoglobulin PBS phosphate baer saline PIP, phospho inositol2 phosphate PKC protein kinase C PKR interferon inducible doubIe stranded-RNA dependent protein kinase PLC phospholipase C PPMP 1-phenyl-2-hexadecanoylamino-3-morpholino- I -propanol RER rough endoplasmic reticdurn RME receptor mediated endocytosis RPMI- 1 640 Roswell park mernorial institute medium- 1640 PTK phospho tyrosine kinase RNA ribonucleic acid SLO streptolysin-O SLT Shiga-like toxin ST Shiga toxin Stat signal transducer and activator of transcription TBS tris buffer saline TGN trans-Golgi network TLC thin layer chromatography TNF tumor necrosis factor TT tetanus toxin VSV vesicular stomatitis virus VT verotoxin VTEC VT producing E. coli WGA wheat germ agglutinin LIST OF FIGURES
The structure of GSL Biosynthesis of GSL Mechanisms of lipid transport The structure of globotriaosylceramide The structure of verotoxin Verotoxin B subunit-Globotriaosylcerarnide interaction The structure of B-ce11 antigen receptor B-cell antigen receptor-mediated signal transduction Co-receptors of B-ce11 antigen receptor The structure of CD 19 Amino acid sequence sirnilarity between CD1 9 and VT binding subunit B Amino acids of VTl-B subunit shared with CD19 on VT 1-B crystal structure The structure of type 1 IFN receptor Type 1 interferon/ type 1 IFN receptor mediated signal transduction Amino acid sequence similarity between IFNARI and VT binding subunit B Arnino acids of VTI-B subunit shared with IFNARl on VT 1-B crystal structure Vesicle-mediated protein transport pathways Small GTPases-mediated membrane trafEcking cycle SNAREs-mediated membrane tdficking cycle Thin Layer chromatography of total GSL extracts fkom Daudi, VT-500 and PPMP-Daudi VTl cytotoxicity assay on Daudi, VT-500 and PPMP-Daudi Surface expression of CD 19 on Daudi, VT500 and PPMP-Daudi Surface redistribution of antibody crosslinked CD1 9 on Daudi, VT500 and PPMP-Daudi Intemaliztion of antibody crosslinked CD19 on Daudi, VT500 and PPMP-Daudi Quantitation of intemalized antibody crosslinked CD19 on Daudi, VT5OO and PPMP-Daudi Effect of VTl-B subunit CO-incubatiodinternalization on antibody crosslinked CD 19 internalization Intracellular targeting of intemalized CD19 on Daudi, VT500 and PPMP-Daudi Antibody crosslinked CD 19 induced apoptosis on Daudi, VT500 and PPMP-Daudi Quantitation of antibody crosslinked CD19 induced apoptosis 28. Effect of VT1 -B subunit CO-incubation/intemalizationon CD1 9- 117 mediated apoptosis 29. Stages of B-lymphocyte development Il9 30. The fate of B-lymphocytes in germinal center 126 3 1 .A. Total cellular Gb, on ver0 and VRP cells 143 3 1.B. Total cellular IFNARI on ver0 and VRP cells 144 32. Surface expression of Gb, and eIFNARl on vero cells and the VRP 145 cells 33 .A. Comparison of VT 1 cytotoxicity on Gb,-positive vero and Gb,-negative 146 VRP cells 33.B. Cornparison of IFN-a-mediated anti viral activity on 147 ver0 and VRP cells 34.A. Effect of VTl B CO-incubationon VTI cytotoxicity 149 34.B. Effect of VTlB CO-incubationon IFN-a for anti virai activity 150 35. Gb, dependency of IFN-a induced Stat f nuclear translocation 151 36. Western imrnunoblotting analysis of PKR expression in control 153 and IFNu treated vero and VRP cells 37. Total cellular Gb, expression and VT1 tlc overlay of vero and 155 MRC-5 cells 38.A. Cornparison of VT 1 cytotoxicity on ver0 and MRC-5 cells 156 38.B. Comparison of IFN-a antiproliferative assay on vero and MRC-5 cells 157 3 8.C. Cornparison of IFN-a antiviral assays against Echo l 1 virus and 158 VSV on ver0 and MRC-5 cells 38.D. Effect of inhibition of glycolipid synthesis with PPMP on antiviral 159 activity in MRCS cells 39.A. Total GSL expression and TLC overlay of Gb, on astrocytoma 161 ce11 lines 39.B. Effect of modification of Gb, cellular content on VTl cytotoxicity 162 on SF-539, XF-498 and butyrate or PPMP modified XF-498 cells 39.C Effect of modification of Gb, cellular content on EN-a anti 163 viral assay against Echo 11 virus on SF-539, XF-498 and butyrate or PPMP modified XF-498 cells 40. Effect of cytosolic acidification on FITC-conjugated VTl B 165 subunit intemalization and Stat 1 nuclear translocation 41. Effect of cytosolic acidification on VT1 cytotoxicity (vero cells) 166 42.A. Effect of cytosolic acidification on IFN-a-antiproliferative activity 167 ( vero cells) 42.B. Effect of cytosolic acidification on IFN-a-anti viral activity 168 (vero cells) 43.A. Effect of BFA on Golgi complex organization 191 43.B. Dose dependent effect of BFA on Golgi disruption 192 44.A. Effect of BFA on VTI induced total ce11 kiliing 194 44.B. Effect of BFA on VTI induced apoptosis 195 Effect of BFA on VTl induced protein synthesis inhibition Effect of %FAon FITC labeied VTlB intemalization Effect of BFA on VTl B (immunofluorescence) intemalization Effect of BFA on FITC-VTl B intemalization (double labeling with TGN marker) Effect of BFA on TGN and early endosorne markers distribution (double labeling) Effect of endocytosis inhibiton on FITC-conjugated VT 1B intemalization Effect of endocytosis inhibitors on VTI cytotoxicity Dose dependent effect of filipin on VTlcytotoxicity EfEect of BFA and filipin on VT1 cytotoxicity Effect of %FA, filipin and nocodazole on VTI cytotoxicity Effect of BFA on cytosolic translocation of VTI B (Immunofluorescence) in SLO pemeabilized cells Effect of BFA and filipin on cytosolic translocation of VTlB in SLO permeabilized cells VT 1 and VT2 cytotoxicity assay on vero cells Effect of BFA and filipin on VT1-induced cytotoxicity Effect of BFA and filipin on VT2-induced cytotoxicity Immunofluorescence labeling of internalized VTl and VT2 in ver0 cells Irnmuno-electronmicroscopyof intemalized VTl and VRin vero cells Effect of BFA and filipin in intemalization of VTl in vero cells Effect of BFA and filipin in intemalization of VTî in vero cells Total Gb, extraction of vero and VRP cells and VT1 and VT2 TLC overlay VTI and VT2 cytotoxicity assay on ver0 and VRP cells
xiii LIST OF TABLES Page 1. Classification of neutral GSL 5 3. Nomenclature and classification of verotoxins 32 3. Molecular weight and amino acid sequence similarity of verotoxins 33 4. Antiserum neutralization against different verotoxins 37
xiv Chapter 1 Introduction 1.1 Glycospbingolipid and Globotriaosylceramide
1.1.1 Glycosphingolipid
Glycosphingolipids are ubiquitous components of the plasma membrane, which are mainly present at the outer Ieaflet of the plasma membrane bilayer rather than in intracellular membranes.
1.1.1.1 Structure
GSL are the glycosides of N-acyl-sphingosine (ceramide). Sphingosine is a long chain amino alcohol (Figwe.1) and both the amino and the aicohol moieties of sphingosine can be substituted to produce various GSL (Gurr and Harwood, 1991). Severd other sphingosyl alcohols are also found in naturally occurring sphingolipids but sphingosine and dihydrosphingosine are the most common among animal tissue GSL (Kanfer and Hakomoril983). Addition of the fatty acyl group to the amino group of sphingosine results in a ceramide. The chah length of fatty acids may range from C 16 to C26 and rnay contain one or more double bonds andor hydroxyl substitute at C-2 (Cornish-Bowden et al., 1998). Further addition of one or more carbohydrate residues to the atcohol group of the ceramide produces GSL (Gurr and Harwood, 1991). Therefore, variations in sphingosine, fatty acids and carbohydrate give nse to a vast number of chemically distinct GSL. The carbohydrate moiety of a GSL forms a hydrophilic head group that protrudes fiom the lipid bilayer toward the outer environment. nie ceramide moiety is inserted into the outer leaflet of the lipid bilayer (Schwarzmann and Sandhoff, 1990). Fatty acid residue
B-D-Galactose Sphingosine
Figure 1. The structure of a glycosphingolipid 1.1.1.2 Classification
Based on the carbohydrate structure, GSL cm be classified into different categories. GSL are cla~sified~dependhg on the substituted groups of the carbohydrates or according to basic carbohydrate structures or based on the number of carbohydrate residues (Kanfer and Hakomon, 1983). According to substitution, GSL are classified as neutral glycolipids, sulfatides (sulphate ester-containing glycolipids), gangliosides (sialic acid-containing glycolipids) and phosphoinositido-gl ycolipids. According to the basic carbohydrate structures, GSL are classified into Globo- series, Lacto-series, Ganglio- senes, Muco-series and Gd-senes glycolipids (Table. 1). According to the number of carbohydrate residues, GSL are classified as mono-, di-, tri-. tetra-, penta- to poly-glycosyi ceramide.
1.1.1.3 Biosyntbesis
The first intermediate of a sphingosine base, 3-dehydrosphinganine, is formed by the condensation of palmitoyl-CoA with senne in the presence of pyridoxal phosphate. 3- dehydrosphinganine is irnmediately reduced to sphinganine by NADPH-dependent 3- dehydrosphinganine reductase. Sphinganine is acylated by dihydroceramide synthase, followed by introduction of the 4,s double bond by dihydrocerarnide desaturase, ieading to the formation of ceramide (Figure. 2) (Perry and Hannun, 1998). Finally, one or more carbohydrate groups are added to the C-1 hydroxyl group of ceramide by sequential transfening of a sugar group from nucleotide sugars such as UDP-galactose, UDP- g 1ucose to the growing oligosaccharide chain by respective gl ycosyltransferase enzymes (Rawn, 1989). The addition of a sugar residue by a glycosyltransferase is a highly specific and tightly regulated process. Ceramide, synthesized on the cytosolic face of ER is translocated into the lumen of ER and then carried to the Golgi apparatus (Lannert et al., 1998). The precursor of more complex GSL, glucosylceramide is synthesized on the cytoplasmic surface of Golgi Table 1. Classification of neutral glycosphingolipid Classification Oligosaccharides Names Symbols
1. Globserfes (major) Gb3a or GbOsc3a Gb3b or GbOse3b GMa or GbOsc4a Gb4b or GbOsc4b GbS or GhOxS
2. kto-stdu (major) GlcNAcPl+Xial~t4lc Lactatriaose Gal~1+30lcNAc~1+36d~14lc Lactatetraosc Gal~l4kNAc~l+30al~l~lc Lactontotetraosc Galfl1+4ûlcNAc~l+30alpl +4CllcNAcfll+3Gal~l4Glc Lactoheitaose
3. Gsnglioerks (major) GalNAcQl Malpl4Glc Ganglioiriaose OaiNAcQl+3GalNAcQ l~aI~14Glc Oanglloteiraose Gala1 4aljlI4GalNAcp 14GalNAcp 14aip1-+4Glc Gangliopcntaosc
- --
4. Mumseda (minor) Galpl -MalPl+4Glc Mucoiriaose Gal~l+4Galfll~alfll~lc Mucote traose
5, Cal-scrlcs (minor) Galal~al Galabiost Oala14aia14i1 Oalatriaose Figure 2. Biosynthesis of GSL H - I OOC - C - CH20H I @ NHi Palmitoyl CoA Serine
Serine palmitoyltransferase
CoASH *::- 4
2s-3- Ketosphinganine
Cont'd ...... Cont'd ......
Dihydroceramide synthasc b CoA
HO H I I CH3 (CH2),,- C - C - CH20H Dihydroceramide I I H NHC-R1 il O
D ihydroceramide desahvase b bJAO Ceramide (N-acy lsphingosine) Glucosylceramide (Glu P 1- 1 ceramide) in both early (cis- and medial) and late (trans- and TGN) sub-compartments of Golgi. Glucosylcerarnide is converted to lactosylceramide in the lumen of Golgi. Synthesis of complex GSL such as globotriaosylceramide, globotetraosylceramide, ganglioside etc takes place in the lumen of Golgi (Lannert et al., 1998). The mechanism of GSL transport fiom the site of synthesis to the site of residence cm be classified into two main classes; vesicular transport and alternative non-vesicular mechanism of lipid transport (Figure. 3) (Pagano, 1990). Like the vesicular-mediated transport of glycoproteins fiom ER to their destination, GSL also are transported by tightly regulated vesicular budding, docking and fùsion. The mechanism of vesicular mediated protein transport is described in section 1.5. By ùlis mechanism, membrane lipids fiom both cytosolic and lumenal leaflet of the membrane are transported fiom the donor compartment to the acceptor compartment (Helvoort and van Meer, 1995). Newly synthesized GSL are delivered to various destinations such as from ER to Golgi, from Golgi to ER recycling, through Golgi stacks, fkom Golgi to plasma membrane and fkom ER to plasma membrane (Figure. 3.A)(Pagano, 1990). Non-vesicular mechanism of lipid transport can be sub-divided into transbilayer (flip/flop) movement, transport of lipid monomers through the cytosol and lateral diffusion of lipid molecules between organelles (Figure. 3.B)(review, Pagano, 1990). To maintain the equivalent arnount of lipid in both leafiets of the bilayer membrane, lipids are translocated fiom one side to the other by a lipid iranslocase (Helvoort and van Meer, 1995). This transport mechanism may operate at ai1 the vesicular membrane compartments and plasma membrane. Lipid monomers cm also be transported between cytosolic leaflets of the membrane compartments by simple diffbion through the cytosol. Slower kinetics of lipid transport by this mechanism can be accelerated by cytosolic lipid transfer proteins such as phosphatidylinositoltransfer protein (PITI) (Pagano, 1990). This pathway can transport lipids not only between compartrnents of vesicular transport, but also between organelles, which are not connected by vesicles, such as mitochondria and peroxisomes (Pagano, 1990). In addition, lipid molecules on the cytoplasmic leaflet can be transported between organelles in close proximity by lateral diffusion through transient or permanent membrane bridges, evidenced by electronmicroscopy (Pagano, Figure 3. Mechanisms of lipid transport (3.A) Vesicular transport of iipids: Pathway 1, ER to Golgitransport; II, Golgi to ER mycling through an intermediate cornpartment; III, transport -through the Golgi stacks; N, Golgi to plasma membrane tramport; V, ER to plasma membrane transpoa; and VI, ER to mitochondria transport.' (3.B) Non-vesicdar transport of lipids: Pathway 1 and T[, tram-bilayer movement of lipids; III to WI, transport of Iipid monomen through the cytosol via Iipid transfer protein; and Vm,transport between organelles by lateml ciifhion. 1990). Although the intracellular &cking of GSL has ken generally assumed to be transported mainiy by a vesicular membrane movement, the involvement of glycolipid binding protein and/or glycolipid transfer protein also cannot be excluded (Sandhoff and Klein, 1994). 1.1.1.4 Degradation The GSL components fiom the plasma membrane enter the endosomaVlysosomal compartments by endocytic membrane flow and are degraded by various lysosomal glycohydrolase enzymes. Respective glycosidases cleave carbohydrate residues fiom GSL in a highly specific step-wise manner (Kanfer and Hakomori, 1983) and ceramidase degrades ceramide to sphingosine and fiee fatty acid (Sugita et al., 1972). However, the catabolism of membrane- bound GSL with short hydrophilic head group requires the assistance of small glycoprotein cofactors, sphingolipid activator proteins (SAPs), also known as saposins (Sandhoff and Klein, 1994). SAPs are usually water-soluble small glycoproteins and there are at least five types of SAPs, which are encoded by ody two genes (Sandhoff and Klein, 1994). One gene encodes GMz-activator. The other gene, sap- precursor, encodes SAP-A (saposin A), SAP-B (saposin B or SAP-1 or sulfatide- activator), SAP-C (saposin C or SAP-2 or glucosylceramidase activator protein) and SAP-D (saposin D or component C) (Sandhoff and Klein, 1994). SAP acts as a "liftase", lifting and extracting membrane GSL substrates with oligosaccharide head groups, which are too short to be reached by the water-soluble enzyme, and presenting it to an appropriate enzyme. The inhented deficiency of the hydrolases or SAPs causes the lysosomal storage disease of the respective GSL substrate (Sandhoff and Klein, 1994). The deficiency of glucocerebrosidase or mutations in SAP-C results in glucosyl ceramide storage disease, known as Gaucher's disease, characterized by appearance of large lipid-laden cells in spleen, liver, and bone marrow, and pigmentation of skin (Gurr and Harwood, 1991). The deficiency of ceramide tnhexosidase or mutations in SAP-B results in trihexosyl ceramide (Gb,) storage disease; Fabry's disease (Sandhoff and Kolter, 1996), characterized by skin rash, pains in the extrernities, pyrexia and progressive rend failure (Gurr and Hanvood, 1991 ). The deficiency of B-hexosaminidaseA or mutations in GM2 activator results in Tay Sachs disease (Sandho ff and Klein, 1994). 1.1.1.5 Physical properties The physico-chemical properties and the asymmetric localization of GSL play an important role for the structure and fünction of the cells. GSL may contribute to the structurd rigidity of the surface leaflet. As GSL have both hydrogen acceptors (amide carbonyl) and a variety of donors (hydroxyl groups of sphingosine, fatty acids and carbohydrates), GSL confer a rigid and uniquely ordered structural organization of the plasma membrane (Yamakawa and Nagai, 1978). GSL and cholesterol in the outer leafiet of the bilayer membrane are laterally organized into lipid microdomains, which form moving platforms or rafts (Simons and Ikonen, 1997). GSL associate with one another through weak interactions between the carbohydrate head groups, and cholesterol molecules fil1 in the gaps in between as spacers. GSL-cholesteroi rafts are insoluble in the detergent Triton X-100 at 4°C and thus referred to as detergent-insoluble. glycolipid-enriched complexes (DIGs). GPI-anchored proteins, transmembrane proteins and doubly acylated Src farnily tyrosine kinases associate with rafts and are incorporated into DIGs (Simons and Ikonen, 1997). Caveolae are flask-shaped membrane invaginations of 50-80 nrn diameter. GSL are the major lipid components of caveolae with which the integral membrane protein, caveolin is associated (Rothberg et al., 1992). 1.1.1.6 Biological properties Due to the localization of GSL, particularly on the outer leailet of plasma membrane, GSL act as a variety of ce11 surface antigens (Fenderson et al., 1990, Futerman, 1998), ce11 adhesion molecules (Hakomori and Igarashi, 1999, imrnuno- modulators (Bergelson, 1995) and receptors for some bacteria and bacterial protein toxins (Lingwood, 1993). In addition, GSL play a significant role in ce11 proliferation and di fferentiation (Hakomori and Igarashi, 1995). 1.1.1.6.1 Glycosphingoiipids as cell surface antigens During embryonic development, the zygote generates a large diversity of ce11 types. Phase-dependent changes of glycolipid composition and synthesis have ken observed during the differentiation of various ce11 types and have been described as developmental antigens (Kader and Hakomori, 1983). The exarnples are blood group ABH antigens, Forssman antigen, stage-specific embryonic antigens (SSEA-1,2,3) etc. In each case, the antigenic molecules have been identified as a glycolipid or the carbohydrate moiety of glycoproteins (Fenderson et al., 1990 and Solter and Knowles, 1978). Embryonic stem cells, deficient in GSL synthesis are able to differentiate into ectoderrnal, rnesodermal and endodermal denvatives but are unable to fom well differentiated tissues (Yarnashi ta et al., 1999). GSL are abundant in the nervous system and the composition of GSL varies with developmental stage, localization and cell type. Consequently, GSL shedded to the cerebrospinal fluid become the diagnostic markers of pathological alterations in brain, such as developmental abnonnalities, demyelination, neural cell destruction etc (Fredman and Lekman, 1997). 1.1.1.6.2 GlycosphingoIipids as tumor associated antigens Some specific types of GSL. which are chemically detectable in normal cells, are over-expressed on the surface of tumor cells and become immunogenic as tumor- associated antigens (Hakomon and Zhang, 1997). Some changes in glycolipid metabolism, associated with oncogenic transformation by oncogenic viruses or chernical carcinogens, or spontaneous tumors have been detected (Kanfer and Hakomori, 1983). nie glycolipid changes can be due to the blocking of the synthesis and accumulation of the precursors, or the induction of new glycolipid synthesis. Ganglio series GSL are preferentially found in tumors of neuroectodermal origin (Fredman, 1993). Globo series GSL are particularly associated with human embryonal carcinomas (Wenk et al., 1994), testicular tumors (Ohyama et al., 1990), ovarian hyperplasia (Arab et al.. 1997) and B lymphocyte neoplasia (Kalisiak et al., 1991). Since multidmg resistance receptor, MDR1 P-glycoprotein is a lipid translocase of broad specificity (van Helvoort et al., 1996), aiterations in GSL expression also are found to be associated with multidrug resistance phenotypes (Lavie et al., 1997, Arab et al., 1997). 1.1.1.6.3 Glycospbingolipids as cell adhesion molecules GSL on the ce11 surface can be specifically recognized by GSL on other cells as lectin-integrin ce11 adhesion mechanism. Ganglioside GM,-expressing cells specificaily adhere to plastic plates coated with Gg,, LacCer and Gb, (Kojima and Hakomori, 1991). Initial adhesion of GM,-expressing melanoma cells and LacCer-expressing endothelial cells is even predominant over integrin or lectin-mediated adhesion in a dynamic flow experimental system (Kojima et al., 1992). Such GSL-GSL interaction may initiate metastatic deposition, which may trigger a series of cascade reactions, promoting adhesion and migration of turnor cells (Kojima et al., 1992). 1.1.1.6.4 Glycosphingolipids as immunornodulators Sialic acid-containing GSL, gangliosides, exhibit imrnuno-modulatory effects by inhibiting T ce11 proliferative responses (Ladisch et al., 1992) andor by inhibition of human natural killer (NK) ce11 activity (Grayson and Ladisch, 1992). Most gangliosides, from the sirnplest monosialoganglioside to the most complex polysialoganglioside, strongly inhibit human T ce11 proliferative responses. Gangliosides containhg a terminal sialic acid are most potent, and some larger neutral GSL also retain some immunosuppressive activity (Jiadisch et al., 1992). In addition, the lipid moiety also cm influence the inhibition of immune ce11 function. Shorter fatty acid containhg gangliosides have been found to be more effective for the suppression of lymphocyte activation. H ydroxylation of the fatty acy 1 group decreases immunosuppressive activity (Ladisch et ai., 1995). Two gangliosides with the simplest carbohydrate structure, GM2 and GM, are very active inhibitors of human NK ce11 activity (Grayson and Ladisch, 1992). Consequently, it has ken postulated that shedding of gangliosides fiom tumors creates a highly immunosuppressive microenvironment, favoring an escape mechanism from imrnunosuppression of tumor growth by the host (Hakomori, 1996). 1.1.1.6.5 Glycosphingolipids as receptors for bacterial protein toxins The carbohydrate head groups of GSL, protruding to the extracellular environment may serve as functional receptors for a variety of bacterial protein toxins. A trisialoganglioside, GTl is a receptor for botulinum toxh (Kanfer and Hakomori, 1983). Gangliosides GDl and GT, function as tetanus toxin receptors. GMl is a hctional receptor for choiera toxin and E-coii heat labile toxin. Neutral GSLs, Gb, and Gb, are specific receptors of E. di-derived verotoxins (review, Monteccuco, 1994). 1.1.1.6.6 Role of glycosphingolipids in signal transduction GSL are known to have second-messenger fùnctions in a variety of signal transduction pathways. One of the exarnples of GSL-mediated signal transduction for ce11 proliferation is LacCer-mediated aortic smooth muscle ceil proliferation, leading to atherosclerosis (Chatterjee, 1997). Oxidized-LDLs specifically stimulate the biosynthesis of LacCer, which in turn activates NADPH oxidase to produce superoxides. Such superoxide molecules stimulate the GTP loading of p21 (ras) and subsequent kinase cascade (Raf-1, MEIU and p44MAPK). The phosphorylated form of p44MAPK translocates fiom the cytoplasm to the nucleus and induces c-fos expression, leading to the ce11 proliferation of aortic smooth muscle cells (Chattejee et al., 1997). The core structure of GSL; ceramide has been identified as an important second- messenger for various cellular functions such as ceil proliferation, ce11 differentiation' growth anest and apoptosis (Mathias et al., 1998). Ceramide can be generated either by sphinogomyelin hydrolysis or by de novo synthesis, in response to a wide variety of stimuli such as physiological stimuli (eg. cytokines and growth factors), stress stimuli (eg. ionizing radiation, W radiation), chemotherapeutic drugs (eg. Daunorubicin) etc. Cerarnide-mediated signaling has been found to be associated with MAPK pathway, SAPK pathway and the cascade of caspase proteases via activation of effector enzymes such as CAPK (ceramide-activated protein kinase), CAPP (ceramide-activated protein phosphatase) and PKCG (Mathias et al., 1998). 1.1.2 Globotriaosylceramide Glob~triaosylcerarnideis a member of globo- series neutral GSL (Table. 1). The ceramide portion of Gb, comprises a sphingosine base and a fatty acid of various chah lengttis. The characteristic sequence of carbohydrate head group is Ga1 (al-4) Gai (P1-4) Glc (j.3 1- 1 )-Cer (Figure. 4). 1.1.2.1 Biological functions Ce11 surface Gb, fwictions as a receptor for verotoxins, as a ce11 surface antigen and as a signal transduction molecule. 1.1.2.1.1 Globotriaosylceramide as a verotoxin receptor Verotoxins are Escherichia coli denved protein toxins, which are known to be associated with hemorrhagic colitis (HC) (Riley et al., 1987) and hemolytic uremic syndrome (HUS) (Karmali, 1989). The primary site of damage in both cases is the endothelium of small blood vessels (of the large intestine in HC and of the glomerular capillaries in HUS) (Richardson et al., 1988). There are four types of VT, namely VTl, VT2, VT2c and VT2e. VTI, VT2 and VT2c are associated with hurnan diseases and specifically bind to ceil surface Gb, (Lingwood et al., 1987 and Waddell et al., 1988). VT2e on the other hand, specifically recognizes both globotetraosylceramide (Gb,) and Gb,, to a lesser extent (DeGrandis et al., 1989 and Samuel et al., 1990). Depending on the ce11 lines, VTl binds to Gb, with a binding affinity 10 to 50 fold higher than that of VT2 (Tesh et al., 1993 and Jacewicz et al.. 1999) and VT2c (Head et al., 199 1). VT specifically binds to Gb, and the terminal Ga1 (al-4) Gai residue of the carbohydrate head group is critical for receptor recognition. Removal of the terminal galactose or substitution with N-acetyl gdactosarnine abolishes the toxin binding activity (Lingwood et al., 1987 and Waddell et al., 1988). The ceramide portion as well is essential Ga1 (al+ Gal (f31-4) Glu R = Fatty acid residue Sphingosine Figure 4. The structwe of globotriaosyiceramide for VT/Gb, specific recognition and VT is unable to bind to digalactosyl diacylglycerol (DGDG), which is a Ga1 (al-6) Gd containhg glycero-lipid (substitution of glycerol for sphingosine) (Lingwd et al., 1987 and Waddell et al., 1988). Although VT may bind to other Gd (al-4) Gai containing GSL such as galabiosyl ceramide (Waddell et al., 1988), Gb, oniy is the functional receptor and reconstitution of VT-resistant cells with Gb, alone is suficient to sensitize the cells to VT induced cytotoxicity (Waddell et ai., 1990). VT binding to the carbohydrate moiety is also influenced by the lipid moiety of Gb, and the swounding lipid environment. Fatty acid heterogeneity (Pellizzari et al., 199 1), chain length and unsaturation (Kiarash et al., 1994) markedly affect VT binding to Gbj in a model phospholipid bilayer surface in microtiter wells. Short chain-containing Gb, species are not efficient in VT binding. C6:O-containing Gb, does not bind to VT (Pellizzari et al., 1991), and Cl2 and C14-containing Gb, have oniy minimal binding (Kiarash et al., 1994). Cl6 to C24-containing Gb, homologues recognize VT effectively and Gb; with unsaturated fatty acids have higher binding capacity (Kiarash et al., 1994). VT binding affinity to Gb, is greater for a mixture of semisynthetic Gb, homologues than for an individual homologue, and the proportion of Gb, homologues in the mixture also affects the binding afinity (Pellizzari et al-, 1991). Different types of VT have preferential binding to Gb, homologues. C20:O and C22:l-containing Gb, have the greatest binding capacity for VT1, and C18:O and Cl 8:l-containing Gb, for VT2c (Kiarash et al., 1994). VTI and VT2c binding to Gb, is increased as a function of decreasing phosphatidylcholine acyl chah length of auxiliary phospholipid~cholesterol bilayer in a microtiter well model system (Arab and Lingwood, 1996). The lipid cornponent and the lipid environment of Gb, likely affect the surface exposure and relative conformation of the carbohydrate moiety. After ce11 surface receptor binding, VT enters the ce11 by receptor mediated endocytosis (Khine and Lingwood, 1994) and induces cytotoxicity. Ce11 cycle dependent surface expression of Gb, also determines the susceptibility of host cells to VT induced cytotoxicity. Although the total cellular Ievel of Gb, is the same, surface exposure of Gb, and sensitivity to VT cytotoxicity is higher in log phase ce11 culture and at the G,/S boundary of the ce11 cycle (Pudymatis and Lingwood, 1992). Different fatty acid chain length Gb, isoforms influence the intracellular targeting of VT and sensitivity of the host cells to the toxin (Arab and Lingwood, 1998). Cells containing higher levels of the shorter fatty acid Gb, isoforms are more sensitive to VTI and the toxin is targeted to the ER, nuclear membrane and nucleus. In contrast, those with longer fatty acid Gb, isoforms are less sensitive to VTI and toxin targeting is ody to the Golgi (Arab and Lingwood, 1998 and Lingwood et al., 1998). Studies in a rabbit model have identified that localization of VT binding to Gb, determines the VT-induced pathological changes. Clinical symptoms and microangiopathic lesions in central nervous system, gastrointestinal tract and lungs are found to be associated with Gb, expression in the corresponding organs. Lack of the lesions in kidney, heart, spleen and liver also is associated with the absence of Gb, (Zoja et al., 1991). Clinical features, sirnilar to those observed in HUS, DIC and ITP (section 1 21.1) have been observed in a baboon model, infllsed with a sublethal dose of E-colr' (Taylor et al., 1997). Human rend tissue is a rich source of Gb3.Gb3 is present in both cortex and medulla but is higher in cortex (Boyd and Lingwood, 1989). Despite the increased incidence of HUS in infants, expression of Gb, is reduced in infant kidney, compared to adult kidney tissue. Since elevated level of cytokines such as TNF-a, IL-1, IL6 and IL-8 have been detected in HUS (van de Kar et al., 1995 and Karpman et al., 1995), local production of such cytokines in kidney may alter the expression of Gb, (described in section 1.2. t .4). Verotoxin binding to human renai tissue identifies the presence of Gb, in the distal convoluted tubules, adjacent to glomeruli, and in the collecting ducts in both adult and infant specimens. However, glomerular Iabeling is detected only in infant sections, which could be a risk factor favoring the development of HUS in early c hildhood (Lingwood, 1994). 1.1.2.1.2 Globotriaosylceramide as a cell differentiation antigen During B lymphocyte development, pre-B cells are differentiated fiom progenitor cells in the bone marrow and released into the blood circulation, where maturation of B- cells takes place. Upon T-ce11 dependent activation, mature B-cells are activated and selectively differentiated into antibody-producing plasma cells, in an antigen-specific manner (Stites and Terr, 1991). However, activated B-cells, which are not selected for plasma ce11 differentiation, undergo apoptosis in germinal centers of the lyrnphoid tissue (Liu et al., 1989). During this process, the specific stages of B-ce11 maturation are reflected by changes in the expression of severat B-ce11 differentiation antigens. Gb, has been identified as CD77, which is expressed only on the germinal center B cells (Mangeney et al., 1991). 1.1.2.1.3 Globotriaosylceramide as a P blood group antigen The human P blood group system consists of three antigens; P, Pl and P'. There are 5 phenotypes of P blood group; Pl, Pz, P, pk,, pk2, which are defined by different combination of these three antigens. The molecular structure of P, Pl and Pk antigens have been identified as Gb,; GaiNAc (B 1-3) Gai (a1 -4) Gal (f3 1-4) Glc-ceramide), globoside; Ga1 (a1-4) Ga1 (w-4) GlcNAc (p 1-31 Ga1 (f3 1-4) Glc-ceramide, and Gb,; Ga1 (al -4) Ga1 (p 1-4) Glc-ceramide, respectively (Naiki and Marcus, 1975). 1.1.2.1.4 Globotriaosylceramide as a tumor-associated antigen Increased expression of Gb, is usually associated with some neoplasias and thus Gb, functions as a tumor-associated antigen. Burkitt's lymphoma is derived fiom germinal center B cells and Burkitt's lymphoma cells specifically express the Burkitt's lymphoma-associated antigen, which is chemically defined as Gb, (Wiels et al., 1984). Increased expression of globo- series GSL such as Gb,, Gb, and Gb, are found to be associated with primary testicular germ ce11 turnors, especially seminomas and embryonal carcinomas (Olie et al., 1996). Elevation of Gb, content is associated with human embryonal carcinomas (Wenk et ai., 1994) and testicular tumors (Ohyama et al., 1990), both benign and malignant serous, mucous and endometroid tumors of ovary and very siçnificantly correlated with cimg-resistant ovarian tumors and muiti-drug-resistant (MDR)ovarian ce11 lines (Arab et al., 1997). 1.1.2.1.5 Role of Globotriaosylceramide in signal transduction Gb, can function as a signal transduction molecule for the induction of apoptosis. Anti-Gb, antibody, immobilized on tissue culture dishes can induce apoptosis of Burkitt's lymphorna ce11 lines, which is preceded by CAMP-dependent protein kinase activation, increased intracellular CAMP levels and increased intraceilular Ca+2concentration (Taga et al., 1997). Gbj also plays a modulatory role for the biological functions of potentiai Gb,- associated ce11 surface receptors. The extracelluiar domain of the B-ce11 differentiation antigen CD19 (Maloney and Lingwood, 1994) and the IFNARI chah of type 1 interferon receptor (Lingwood and Yiu, 1992) have high amino acid sequence sirnilarity to VT1 receptor binding subunit B, suggesting the possible lateral association between these molecules and Gb, on the ce11 surface. Since each molecule is a member of a multi- molecular signal transduction complex, Gb, rnay act as an accessory molecule for the fùlly functi0na.l complete receptor complex. The role of ce11 surface expression of Gb, has been found to be associated with CD 19-mediated B-cell homotypic adhesion (Maloney and Lingwood, 1994) and IFN-dtype 1 IFN receptor-mediated growth inhibition (Ghislain et al., 1994). Verotoxin and verotoxin-producing Escherichia coli Verotoxins are a group of protein toxins, which are denved fiom Escherichia coli and known to be associated with hernorrhagic colitis (Rdey et al., 1987). Due to the distinct cytopathic effect of the toxin on ver0 cells, it was first described as verotoxin (Konowalchuk et al., 1977). As the toxin has more than 99% arnino acid sequence simi larity, similar subunit composition, isoelectric point and biological activities as Shigeella dysentriae-derived shiga toxin, it is also known as shiga-like toxin or E-coli shiga toxin (O'Brien et al-, 1983). 1.2.1 Verotoxin-producing Escherichia coli Enteropathogenic E-coli saains are classified into four groups, on the basis of their mode of pathogenesis. Enterotoxigenic E-coli (ETEC) produce heat labile toxin (LT) or heat stable toxin (ST) or both. Enteroinvasive E-coli (EIEC) invade and propagate wi thin the intestinal epithelium. Enteropathogenic E. coli (EPEC) adhere to the intestinal epithelial cells and produce a characteristic histopathological feature known as the attaching and effacing Iesion (A/E lesion). Al1 three types of E.coli are associated with watery diarrhea caused by either increased intestinal secretion or impaired absorption of water or both. Enterohemorrhagic E-coli (EHEC) on the other hand, induce bloody diarrhea (review by O'Brien and Holmes, 1987). Certain strains of E-coli serotypes such as OZ6:H 1 1, 011 1:H8, O1 1 1:H NT (Marques et al., 1986), 0157:H7, 0157:H- etc. (Scotland et al., 1985) are verotoxin- producing E-coli and produce one or more types of verotoxin. Arnong growing number of VTEC strains, 0157:H7 strains are the most commonly isolated strains associated with hemorrhagic colitis (Remis et al., 1985) and hemolytic uremic syndrome (Karmali, 1989). 1.2.1.1 Clinical manifestations of VTEC infection Symptoms induced by VTEC infection may Vary fiom asymptomatic infection to fever. abdominal cramps, mild watery diarrhea, bloody diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Hemorrhagic colitis due to VTEC presents with abdominal cmps, bloody diarrhea, tenesmus, vomiting and Little or no fever (Riley et al.? 1987). About 10% of children with VTEC associated hemorrhagic colitis proceed to diarrhea associated HUS (D+ HUS) (Karmali, 1989). Among more than 50 serotypes oCVTEC associated with HC and HUS, Exoli 0157:H7 is the most cornmon serotype and has been particularly associated with the outbreaks (Karmali, 1989). HUS is prirnarily a pediatric disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute rend failure (Robson et al., 1993). D+ HUS develops during or afier the episode of diarrhea and usually presents with pallor, oligouria, peripheral edema and macroscopic hematuria. Irritability, abdominal tendemess, hypertension and hepatosplenomegaiy may also be present (Robson et al., 1993). Other multi-systemic complications such as disseminated intravascular coagulopathy @K), idiopathic thrombocytopenic purpura (ITP), central nervous system complications like a decreased level of consciousness and seizures, pancreatitis. cardiomyopathy and myocarditis may follow HUS (Robson et al., 1993). 1.2.1.2 Pathogenesis of VTEC infection In the gastrointestinal tract, EPEC and some strains of VTEC adhere to the mucosal epithelium of the intestine by the attaching and effacing lesion, which is further associated with signal transduction events and underlying cytoskeletal recruitrnent (Rosenshine et al., 1992 and Isrnaili et al., 1995). AIE lesions followed by loss of epithelial microvilli result in functional impairment of the epitheliai cells, leading to malabsorption and osmotic diarrhea @o~enberg and Kaper, 1992). The signal transduction events, particuiarly the rise in intracelluiar calcium concentration (Isrnaili et al., 1995) also contribute to the watery diarrhea associated with chloride secretion. Secretions of cytokines such as IL-8 fiom the intestinal epitheiium may also promote the colitis (Huber et al., 199 1). However, the signal transduction events following EPEC and VTEC are different. Both organisms induce elevation of inositol trisphosphate and intracellular calcium level (Dytoc et al., 1994 and Ismaili et al., 1995) but in contrast to EPEC, VTEC do not induce tyrosine phosphorylation of epithelial ce11 proteins (Ismaili et al., 1995 and Ismaili et al., 1998). Afier the ingestion of contaminated food or water, E-coli adheres to the colonic mucosq multiplies and causes ce11 death, resulting in diarrhea. In case of VTEC infection, VT can translocate to the intestinal mucosal vasculature and causes microvascular damage, resulting in hemorrhagic colitis (Ruggenenti et ai., 1997). Afier the entry of VT to the blood circulation, VT cm be disseminated to virtually any target organ and bind to the VT receptor present on the endothelial cells of the target organs. Microvascular damage of the target organs results in damage and hctional impairment of the organs involved (Ruggenenti et al., 1997). Endothelial damage in the kidney is the main source of HUS pathogenesis, which precedes platelet-endothelial ce11 interaction, platelet thrombi formation in the renal microvasculature and subsequent acute renal failure (Kaplan et al., 1990). Within the renal glomerulus, mesangial cells are in direct contact with adjacent glomerular endothelial cells and also with the blood circulation through the pores in the glomerular endothelium (van Setten et al., 1997a). Gb, is present on both platelets (Cooling et al., 1998) and mesangial cells (Robinson et al., 1995 and van Setten et al., I997b). Platelet consumption and erythrocyte disruption in the injured microvasculature also results in thrornbocytopenia and hemolytic anemia (Ruggenenti et al., 1997). 1.2.1.3 Histo-pathology of hemorrhagic colitis and hemolytic-uremic syndrome The principal histologic features of microangiopathy associated with HC and HUS are swollen endothelial cells, widening of the subendothelial space and microvascular thrombosis (Ruggenenti et al., 1997). The glomerular endothelial cells of kidneys are the main targets of damage in HUS and fibrinogen deposition of renal arterioles also is common (Robson et al., 1993). As a result, cortical necrosis, tubular necrosis and focal necrosis of glomedi are fiequently obsewed (Robson et ai., 1993). Microthrombi and microvascular occlusions are found in other organs such as the lungs, heart. liver. brain, pancreas etc. which are presurnably associated with extra-renal complications in HUS (üpadhyaya, et al., 1980). 1.2.1.4 Role of inflammatory cytokines in patbogenesis of hemorrbagic colitis and hemolytic-uremic syndrome Although the vascular endothelial cells are the main targets of VT in HC and HUS, in vitro studies have shown that endothelial cells such as hurnan vascular endothelial cells (HVEC), human urnbilical vein endotheliai cells (HUVEC) (Louise et al., 1991), human saphenous vein endothelial cells (HSVEC) (Tesh et al., 1991), hurnan glomerular microvascular endothelia1 cells (GMVEC) (van Setten et al., 1997) etc express low level of Gb, and the cells are not sensitive to VT. However, Gb, expression and VT cytotoxicity can be restored by culturing the EC in the presence of LPS or cytokines such as TNF-a and IL-1 (van de Kar et ai., 1992, Obrig et ai., 1993 and van Setten et al., 1997) which induce endothelial gaiactosyl transferase activity (van de Kar et al., 1995). The possible sources of cytokine production in response to VT are monocytes and macrophages. Although non-adherent human monocytes (van Setten et ai., 1996) and peritoneal macrophages (Tesh et al., 1994) express only low Ievel of Gb, and are refiactory to VT cytotoxicity, the cells produce pro-infiammatory cytokines such as ïNF- a,IL-1 P, IL-6 (van Setten et al., 1996 and Tesh et al., 1994) and IL-8 (van Setten et al., 1996) afier VT treatment. During the course of VTEC infection, increased levels of plasma IL-8 and urinary TNF-a,IL-1 and IL-8 have been detected in advanced stage of infection such as HUS and TTP, indicating the local secretion of cytokines in the kidneys (van de Kar et al., 1995 and Karpman et al., 1995). Therefore, systemic VT and pro- inflammatory cytokines presurnably act together to induce endothelial damage. Human colonic epithelial ce11 lines such as Caco-2 and TS4 also produce IL-8 in response to VT, in N-glycosidase activity dependent manner, which could be the possible mechanism of the gut epithelium to attract neutrophils in order to activate neutrophil dependent vascular injury (Yamasaki et al., 1999). In contrast to the EC of larger veins (Louise et al., 1991 and Tesh et al., 1991), rnicrovascular endothelial cells such as human microvascular endothelial cells (HMVEC) (Ohrni et al., 1998) and human intestinal microvascular endothelial cells (HIMEC) (Jacewicz et al., 1999) constitutivety express high level of Gb, and are highly sensitive to VT. Cytokines do not enhance the sensitivity to the toxin in these cells (Jacewicz et al., 1999). Microvascular endothelium could be the first endothelium to encounter the toxin during VTEC infection. Verotoxin The first described member of verotoxin farnily was found in culture filtrates of a number of E-coli straiw (Konowaichuk et al., 1977). Unlike ETEC derived heat-stable enterotoxin, the new toxin was heat-labile but still different fiom ETEC heat-labile enterotoxin in regard to cytotoxic effect on vero cells. Heat-labile toxin affected cells were enlarged, thick-walled, refiaçtile with several filamentous tendrils. The morphological effects eventudty faded away after 3 days. The cellular function was not irnpaired and thus it was a cytotonic toxin. In contrast, VT was cytotoxic and VT-treated vero cells were rounded and floated fiee in the medium. The irretrievable cytopathic effect was observed within 24 hr and which was advanced with time. Due to its distinct cytopathic effects on ver0 cells, the toxin was tenned verotoxin (Konowalchuk et al., 1977). 1.2.2.1 Nomenclature and classification of verotoxin In 1983, O'Brien and LaVeck reported a toxin derived fiom EPEC strain H30 that showed identical isoelectric points and comparable biologicai activities to Shigella dysentriae-derived Shiga toxin (ST). The cytotoxicity of this toxin on Hela cells could be neutralized by antibody prepared against ST and thus the toxin was narned E-coli Shiga- like toxin (SLT) (O'Brien et al., 1983). Later, it was concluded that VT discovered by Konowalchuk, 1977, and SLT by O'Brien, 1983, were the same and the names VT and SLT have been used interchangeably (O'Brien et ai., 1983). In 1985, another toxin with similar cytotoxic pattern, produced by E-coli serotype 0 157:I-i-, strain E3SS 11 was identified. Unlike the toxin denved fiom strain H30, this toxin could not be neutralized by antibody to ST (Scotland et al., 1985). As a consequence, VT neutralizable with anti-ST was terrned VT1 and the other as VT2. Some strains of E.coli serotype 0157:H7 such as E30480 and 933 produce both toxiw (Scotland et al., 1985). In. 1988, a toxin with verocytotoxicity, which also could not be neutralized by anti-ST was purified fiom E.coli serotype K-12, strain NM522 and was named SLTII (Downes et al., 1988). However, cytotoxicity induced by SLTlI could be completely neutralized by anti-VT2 but VT2 was only partially neutralized by anti-SLTII (Head et al., 1988). Another type of toxin with vero-cytotoxicity was identified fiom E-coli serotype 0 138, 0 139 or 014 1, cornmonly isolated fiom pigs with edema disease and the cytotoxicity was neutralizable by anti-SLTII. Thus, this toxin was termed SLTIIv (SLTII- variant) or VTE (edema disease producing VT) (Marques et al., 1987). In 1994, the nomeiiclature of vero-cytotoxins was intemationaily standardized as described in Table. 2 (O'Brien et al., 1994). 1.2.2.2 Structure and physico-chemicai properties of verotoxin Al1 members of the VT family consist of a single subunit A covalently associated with a pentameric subunit B (Figure.5). The approximate molecular weights of subunits A and B are 32 kDa and 7 kDa, respectively with slight variation among different toxins (O'Brien and Holmes 1987) (Table. 3). The nucleotide sequence homology between subunits A and B of VTI and VT2 is 57% and 60% respectively and amino acid sequence homoiogy is 55% and 57% respectively (Jackson et ai., 1987). The nucieotide sequence homology between subunits A and B of VT2 and VT2c is 98.6% and 95.5% respectively (Ito et al., 1991). Since ST and VTl are alrnost identical with only one amino acid difference in the subunit A, ST is the prototype of the toxin family (Cdderwood, 1994). The isoelecûic points for VTl, VT2, VT2c and VT2e are 6.72 (Petric et al., 1987), 5.2 (Downes et al., 1988), 6.5 (Head et al., 1988) and 9 (MacLeod et al., 1991), respectively. The subunit A of VT is a globular subunit (Kovlov et al., 1993). The subunit B is a doughnut-shaped pentarner with a central pore, and each monomer comprises two three- stranded anti-parallel B sheets and an a helix (Stein et al., 1992) (Figure. 5). The pore of VT-B subunit is non-polar (Stein et al., 1992), and a stretch of 9 non-polar arnino acid residues at the C-terminus of subunit A penetrate into the pore and form a non-covalent association between the two subunits through a tryptophan (Haddad and Jackson, 1993). SLT Former Reference 1 Classification system system (Canada &UK) strain Type 1 VTI SLTI SLTI (neutralized SLTII VT2 SLTIIc neutralized SLTIIc SLTII sLnr by anti-ST SLTIIe VTENT2v SLTIIv Table 2. Nomenclature and classification of verotoxins Amino acid Toxins Subunit A Subunit B sequence simila ri ty L to ST Table. 3 Molecular weight of verotoxin farnily and amino acid sequence similarity to ST Figurr.5 Tbstnrtw of VTl hddcxin. (Fmscr et al., 1 W) The possible çarbohydrate binding site of a pentarnenc B subunit is the clefi formed by the P sheet interaction between two adjacent monomers. Amino acid residues, lining the binding clef& with polar and acidic side chains could form hydrogen bonding with polar groups of the carbohydrate and those with arornatic side chains could stack against sugar rings (Stein et al., 1992). 1.2.2.3 Functions of verotoxin The subunit A is the enzymatic moiety? which is responsible for the cytotoxic activities of VT (Igarashi et ai., 1987) and the subunit B recognizes the ce11 surface receptor (Donohue-Rolfe et al., 1984). After the internalization of the toxin, 32 kDa subunit A is proteolytically nicked to a 27.5 kDa active Al fragment. The Al fhgment of subunit A cleaves the N-glycosidic bond of the adenine residue at position 4324 in 28s ribosomai RNA and prevents elongation factor4 binding to 605 ribosomal subunit, thereby inhibiting protein synthesis (Igarashi et al., 1987). Tyr,,, Tyr,,,, Glu,,,, kg,,, and Trp,, have been shown to be important for the enzymatic activity of the subunit A (review, Jackson and O'Brien, 1994). The cytotoxicity induced by VT can also be partly due to the induction of apoptosis (Mangeney et al., 1993, Inward et al., 1995, Arab et al., 1998 and Kiyokawa et al., 1998). The role of subunit B is to mediate the binding of the holotoxin to specific receptors on the sensitive cells and the entry of the toxin into the cells (Sandvig et al., 1989). Subunit B alone has the same binding specificity and binding &ity as the corresponding holotoxin (Head et al., 1991 & Tyrell et al., 1992), and cmenter the ce11 and target to the same intracellular destinations as does the holotoxin (Khine & Lingwood, 1994). In addition to receptor binding fûnction, higher concentration of subunit B alone can also induce apoptosis in Burkitt's lymphoma ce11 lines (Mangeney et al., 1993). The subunit B is also responsible for the biological variability of different VTs by defining the cytotoxic titer (Head et al., 1991), cytotoxic specificity (Head et al., 1991 and Weinstein et al., 1989) and antigenicity (Head et al., 1991) of the respective hototoxins. 1.2.2.4 Biological activities of verotoxin ,411 members of the verotoxin family demonstrate similar biological activities such as cytotoxicity to vero cells, lethality to laboratory animais such as mice, rats and rabbits. and accumulation of fluid in rabbit intestine with some variations in their potency. The CD,, of VTI, VT2 and VT2e for ver0 cells are 6 pg (Noda et al., 1987), 1 pg (Yutçudo et al., 1987) and 0.5 pg (MacLeod & Gyles, I&1, 1990), respectively. The vero- cytotoxicity of VT2c has been reported as 100-1000 times less toxic than VTI cytotoxicity (Head et al., 1991). The LD,, of VTI, VT2 and VT2e on mice are 400 ng (Tesh et al., 1993), 1 ng (Tesh et ai., 1993) and 200 ng (Samuel et a1.,1990), respectively. Acute rend necrosis, following the ingestion of VT in mice, is more extensive in VT2-treated mice than in VT1-treated mice (Tesh et al., 1993). In rabbits, VT induces accumulation of fluid in the intestines, diarrhea, flaccid paralysis of fore and/or hind limbs and lethaiity. VTl-induced diarrhea is watery and VT2c induces bloody diarrhea (Head et al., 1988). LD,, value is 0.2 &Kg (Petric et al., 1987) and 30 pg/Kg (Head et al., 1988), respectively for VTI and VT2c. 1.2.2.5 Antigenicities of verotoxin Despite the similarity in subunit structural composition and receptor binding specificity, the members of VT family are serologically distinct. VTl induced cytotoxicity can be completely neutralized by anti-ST (O'Brien et al., 1983), partially neutralized by anti-VT2c but not by anti-VT2 (Head et al., 1988). Neither VT2 nor VT2c cytotoxicity is neutralized by anti-VT1 or anti-ST (Head et al., 1988). VT2c cytotoxicity is completely neutralized by anti-VT2 but VT2 cytotoxicity is only pstrtially neutralized by anti-VT2c (Head et al., 1988). VT2e cytotoxicity can be neutralized by anti-VT2 but Toxin Table 4. Antiserum neutralization against verotoxins not by anti-ST (Marques et al., 1987). The antisenim neutralization against different verotoxins is summarized in Table. 4. The target tissue uptake of both VTI and VT2 can be protected in rabbits immunized with either VTI toxoid or VT2 toxoid, or with either the VT 1A or VT2A subunit (Bielaszewska et al., 1997). 1L2.6 Functional receptors for verotoxin VT1, VT2 and VT2c specifically recognize globotriaosylceramide (Gb,) mainly and gatabiosylceramide (Gbl) as well (Lingwood et al., 1987 and Waddell et ai., 1988). VT2e binds specificaily to globotetraosylceramide (Gb,) and to a lesser extent to Gb, as well (DeGrandis et al., 1989 and Samuel et al., 1990). Site-directed mutagenesis and biochemical studies have shown that a stretch of aspartic acid residues at position 16-18 (Jackson et al., 1989), Phe,, (Clark et al., 1996) and Lys,, (Jackson et al., 1990 and Khine & Lingwood, 1994) of VTl-B subunit are important for receptor binding. Asn,,, Gln, and Lys, of VT2e-B subunit also are found to be significant for receptor binding specificity of VT2e to Gb, (Tyrell et al., 1992). The predicted binding sites for Gb,, within the pentamenc subunit B have been determined by molecular rnodeling studies using the crystal structure of VTl-B subunit binding to deoxy Gb, analogues (Figure. 6) (Nyholm et al., 1996). There are two potential Gb, binding sites; one within the clefi between two adjacent monomers (site 1) and the other within a shallow depression on the lower surface, opposite from the subunit A (site II). The two binding sites lie on either side of Phe,,. 1.2.2.7 Pathogenesis of verotoxin-associated diseases VT1, VT2 and VT2c are found to be associated with human diseases whereas VT2e causes edema disease in pig (Linggood & Thompson, 1987). Single mutation of Asp,, to Asn, in VTl-B resulted in binding to Gb,, in addition to Gb, (the binding phenotype of VT2e). The double mutation of Gln, to Glu and Lys, to Gln in VT2e-B Figure. 6 Veroiacm B shmi-OlobariP~lcec~niidemierpaiai. (Nyhdm u al, 1996) results in selective loss of Gb, binding while Gb, binding is still maintained (the binding phenotype of VTl) (Tyrrell et al., 1992). The mechanism of VT pathogenesis has ken widely studied on ST and VTl prototypes. As for other protein toxins, the VT-induced mechanism of host ce11 pathogenesis is by a four-step mechanism; receptor binding, endocytosis, rnembi-ane translocation and enzymatic target modification (Montecucco et al., 1994). 1L2.8 In temalkation of verotoxin Mechanisms of protein toxin intemalization, in general are described in section 1.5.3. After binding to the glycolipid receptor Gb,, evenly distributed on the ce11 surface, VT/receptor complexes undergo cap formation in Daudi Burkitt's lymphoma cells (Khine and Lingwood, 1994). VT, concentrated into clathrin coated pits enter the cells by receptor mediated endocytosis (Sandvig et al., 1989 and Khine and Lingwood, 1994). Depending on the ce11 line, VT can also enter the cells by clathrin-independent, caveolar- mediated endocytosis (Schapiro et al., 1998). The toxin is transported to the endosomal cornpartment, but escapes Eiom degradation in late endosomes and lysosomes, by direct transport from early and recycling endosomes to TGN via AP 1-containhg clathrin coated vesicles (Mallard et al., 1998). From TGN, the toxin undergoes retrograde transport to earl ier compartrnents of Golgi, ER and nuclear membrane for su bunit translocation (Sandvig et al., 1994 and Khine and Lingwood, 1994). The intracellular targeting of VT cmbe determined by different fatty acid chain length-containing Gb, isoforms (Arab and Lingwood, 1998). The mechanism of toxin translocation has not ken identified yet but a proposed mechanism is described in section 1.5.3. 1.2.2.9 Roles for verotoxin in anti-cancer therapy In 1976, Farkas-Himsley and Cheung detected an anti-neoplastic activity in extracts of one strain of E-coli (HSCIO) and described it as bactenocin (Farkas-Himsley and Cheung, 1976). Later, the active cornponent, responsible for this activity was identified as VT1 (Farkas-Himsley et al., 1995). This finding has introduced a new area of VT research as a new therapeutic for cancer. Gb, is absent or minimal in normal ovaries. The studies on ovarian cancer ceIl lines and ovarian tumors revealed that expression of total Gb, was elevated in both benign, malignant and metastatic ovarian tumors and more markedly, in multi-dmg resistant tumors (Farkas-Himsley et al., 1995 and Arab et al., 1997). In addition, FITC-conjugated VTl overlay of ovarian tumor fiozen sections showed that the turnor tissue and the vascular endothelium within the tumor mass were strongly labeIed with the toxin (Farkas-Himsley et ai., 1995 and Arab et ai., 1997). Similarly, FITC-VTl or FITC-VTI B labeling of tumor cells and the lumen of the blood vessels in the tumor mass of human brain tumor fiozen sections also has been detected (Arab et ai., 1999). Therefore, VT1 treatment may offer duel targeting of both the tumor and the blood supply. Consequently, VT could be used as an antineoplastic agent when standard therapies for MDR ovarian tumors have failed. Gangliosides and some neutral GSL exhibit immunosuppressive activity (Ladisch et al., 1993) and shedding of GSL fiom the tumors may create a highly immunosuppressive microenvironment (Hakomori, 1996). Shedding of Gb3 may also create such environment which cannot be accessed by the immune system but selective overexpression of Gb, in tumors could be a specific target for VT. Human astrocytoma ce11 Iines also are Gb,-positive and VT-sensitive. Among which SF-539 ce11 line is the most VT-sensitive ce11 line in vitro (Arab and Lingwood, 1998). Intra-tumor injection of VTI induced rapid apoptosis of human astrocytoma (SF- 539) xenografi subcutaneous tumor and blood vessels in nude mice and cornplete regression of tumor was achieved within 10 to 30 days (Arab et al, 1999). In the human hematopoietic system, Gb, expression is restricted to a subset of B- cells (germinal center B-cells) and derived cancers such as human non-Hodgkin's Iymphomas (NHL), especially follicular lymphomas. VTI treatment of murine bone marrow ex vivo effectively cures severe combined irnmunodeficient mice of human B- ce11 lymphoma xenografi while sparing normal hematopoietic precursor cells CaCasse et al., 1996). Therefore, VT1 rnay be potentially used for the purging of human bone marrow before autologous bone marrow transplant in the case of Gb,-positive B-ce11 lymphomas (LaCasse et al., 1996, LaCasse et al., 1999). Major histocompatibility complex (MHC) class 1 molecules present endogenous peptide derived from cytosolic and nuclear proteins to CD8', cytotoxic T lymphocytes (CTL). In vivo, endogenous antigens expressed by tumor tissues are transferred fiom the tumor celis to bone martow-derived antigen presenting cells (APC) and efficientiy processed via an exogenous MHC class 1-restricted pathway for presentation to CTL (Haung et al., 1994). Shiga toxin B-fragment fused to a CD8 epitope of the Mage 1 (a mode1 tumor antigen) antigen can be internalized, processed and presented to CTL by APC such as peripheral blood mononuclear cell-derived dendritic cells and B- lymphoblastoid cells. The ability of ST-B subunit fusion protein to introduce antigens into the exogenous MHC class 1-restricted paîhway can be a potential method ?O use it as an non-living, non-toxic vector in cancer vaccine development (Lee et al., 1998). B-lymphocyte differeatiatioa antigen; CD19 CD 19 is a key member of the multimeric signal transduction cornplex, which functions as a CO-receptorof the B-ce11 antigen receptor @CR). 1.3.1 B-ce11 antigen receptor 1.3.1.1 Structure The B-ce11 antigen receptor is a membrane-anchored immunoglobulin (dg), sheathed by a non-covalently associated Iga/Igp hetrodimer. The mig portion is the antigen-binding component and the Iga/Igp portion is the signal transduction subunit (Figure. 7) (Stites and Terr, 1991). immunoglobulins are tetramolecular glycoprotein complexes, composed of 2 disulfide-bonded 50 to 70 kDa heavy (H) chah subunits, each of which is linked by interchain disulfide bonds to a 23 kDa light (L) chain (Figure.7). The polypeptide subunits of each H or L pair are identical, Each polypeptide contains an amino-terminal, variable (V) region; and a carboxy-terminal, constant (C) region. V domains of H and L chains together fom the antigen-binding site of dg. IgM is the most comrnon mIg present on the B-ce11 surface. Aithough IgM and IgD are the predominant mIgs, other classes of Ig (A. D and E) may also be present on the ce11 surface and may function as antigen receptors (Stites and Terr, 1991). Dunng B-ce11 development, B-cells are differentiated fiom progenitor cells in the bone marrow, and re-arrangement of H and L chah genes takes place during pre-B ce11 stage. The complete comptent Igs are expressed as early as on the virgin or immature B- cells, which have not been exposed to antigens. At this stage, B-cells migrate fkom the bone marrow into the blood and peripheral lymphoid tissues (Stites and Terr, 1991). Figure 7. The structure of Bell antigen receptor 44 AAer interaction of antigens with dgs, mature B cells are activated and ultirnately differentiated into plasma cells, which produce large amount of secretory Igs. The surface IgD is lost upon B-ce11 activation and surface IgM, upon plasma ce11 differentiation. Mer the sameexposure to antigen, some population of B-cells remain for years as memory B- cells (Stites and Terr, 1991). Both signal transduction components of BCR, Iga and IgP have a glycosylated Ig-like extra-cellular domain, a single-spanning transmembrane domain and a cytoplasmic domain (Kurosaki, 1997). The cytoplasmic domains of Iga and Igp contain a sequence motif, known as an ITAM (immunoreceptor tyrosine-based activation motif), which is characterized by six conserved amino acids in approximately 26 amino acid residues @/EXXXXXXXD/EXXYXXL/IXXXXXXXYXXL/I)(Kurosaki, 1997). 1.O. 1.2 Signal transduction As for many other ce11 surface receptors, signal generation and rapid tyrosine phophorylation of cellular proteuis can be initiated upon crosslinking of BCR (Peaker, 1994). However, the cytoplasmic tail of BCR has only 3 amino acids and does not have intrinsic tyrosine kinase activity. Therefore, mIg generates signals via the cytoplasmic tails of Iga and Igp (Kuroski, 1997), or depending on the type of stimuli, via signal transduction machinery of the nearby CO-receptors(O'Rourke et al., 1997). Upon BCR Iigation, several cytoplasmic protein tyrosine kinases are activated. There are two main classes of PTK, involved in BCR signal transduction; Src-PTK such as Lyn, Fyn and Blk, and non-Src-PTK such as Syk, Btk and Csk (Kurosaki, 1997). Src- PTK are initially pre-associated with Iga tail of resting BCR, without phosphorylation. Afier BCR ligation, the stimulatory Tyr,,,of Lyn, is auto- andor trans-phosphorylated. The C-terminal inhibitory Tyr,?, also is phosphorylated by balanced regulation between Csk-mediated phosphorylation and CWS-mediated dephosphorylation (Kurosaki, 1997). Parallel phosphorylation of Iga4gf3 ITAM recruits and activates additional Lyn and activated Lyn further phosphorylates Iga/IgB into a doubly phosphorylated rnolecule. Then, IgdgB ITAM recruits and phosphorylates PD-kinase, Btk and Syk (Figure. 8). Syk can be activated by the same mechanism as Src-PX activation, in Src-PTK- independent manner. Activated Syk Merrecniits and activates Vav. PLC-y and several coupling molecules such as Shc. Vav catalyzes GDP/GTP exchange on Rac-1, which Mer activates cJun amino-terminal kinase (JNK), also known as SAPKinase (Kurosaki, 1997). Vav is involved in BCR-mediated cytoskeletal architecture changes such as BCR capping and recruitment of accessory molecules to BCR (Fischer et al., 1998). Activated PLC-y hydrolyzes PIPl to DAG and IP,. DAG subsequently activates PKC, and IP, releases Ca+' fiom intracellular stores. followed by a sustained Ca-' influx (Kurosaki, 1997). Shc and other nucleotide exchange factors activate GDP-Ras to GTP- Ras (Kurosaki, 1 997). Activation of Syk is dso involved in the Ras paîhway via phosphorylating and inactivating Ras-GAP (GTPase activating protein), leading to decreased hydrolysis of GTP-bound active Ras to GDP-bound inactive Ras (Kurosaki, 1997). Sustained activation of Ras subsequently activates MEK [mitogen-activated, ERK(extracel1ular regulated kinase)-activating kinase], followed by MAPK (mitogen activated protein kinase) activation (review, Kurosaki, 1997). 1.3.1.3 Co-receptors of B-cell antigen receptor There are at least three membrane CO-receptorsof B-lymphocytes, which respond to a variety of ligands and may enhance or inhibit BCR signals (Figure. 9). The CD 1%CD2 1 -CD8 1 complex is the most important signal transduction co- receptor complex, which enhances BCR signals. CD 19 is the signal transduction subunit of the complex and CD21 is the complement C3d receptor. The Gd, attached to a potential antigen may crosslink CD 1%CD2 1-cornplex and mIg of BCR and thereby enhances the signal transduction events (Doody et al., 1996) (Figure. 9). CD22 is a B-cell specific membrane protein which can bind to Siaa2-6GalPl- 4GlcNAc of glycoconjugates, present on a variety of ce11 types. Since the cytoplasrnic domain of CD22 has both ITAM and ITIM (immunoreceptor tyrosine-based inhibitory motif) (YSLL), CD22 can both enhance and inhibit BCR signaling, depending on the Figure 8. BceU antigen receptor-mediated signai transduction 47 Figure 9. Co-receptors of Be11 antigen receptor type of stimuli (O'Rourke et al., 1997). Ligation of CD22 enhances BCR signals via activation of Lyn, Syk, PI3-kinase and PLC-y via ITAM. Upon CO-ligation,CD22 can recruit and activate SH2 domain containing phosphotyrosine phosphatase (SHP-1 ) via ITIM, in addition to enhancernent of BCR signaling via ITAM (Doody et al., 1996 and O'Rourke et al., 1997) (Figure. 9). FqRII, an Fc receptor, suppresses signaling through BCR by CO-ligationof BCR and FcyRII with antigen-IgG complex (Figure. 9). The cytoplasrnic tail of FcyRII has an ITIM, which can activate SHP-1 for the negative regulation of BCR signaiing (Doody et al ., 1996 and O'Rourke et al., 1997). 1.3.1.4 Biological functïons The antigen binding subunit of BCR is responsible for the specific antigen binding, endocytosis and delivery of the internalized antigen for processing to the MHC class II-rich endosomal compartments. The mIg constitutively transports antigens fiom the plasma membrane to the peptide loading cornpartment, in a manner independent of Igdgp (Knight et al., 1997) and crosslinking of BCR (Song et al., 1995). Depending on the type of stimuli, specific stage of B-cells and CO-stimulationwith other CO-receptors,the biological responses following BCR signal transduction may range from B ce11 activation to B-ce11 apoptosis or rescue of B-cells fiom entering apoptosis (Rothstein, 1996). Although the signal transduction mechanisms for BCR-mediated B- ce11 activation such as B-ce11 proliferation and B-ceIl activation have been well studied, the mechanism for other two responses have not been well defined yet. In contrast to BCR crosslinking-mediated mature B ce11 activation, the crosslinking of immature B cells may induce ce11 death by apoptosis. This phenornenon may be involved in the elimination of self-reactive immature B-ceI1s when they interact with self-antigens (Norvell et al., 1995). However, mature B-cells also may undergo apoptosis in response to hyper-crosslinking of mIg by using plate-bound antibodies, antibody-biotin-avidin system or by using excess amount of ligating antibodies, which reflects the surface/membrane-bound antigen (Tsubata et al., 1994, Chaouchi et al., 1995 and Rothstein, 1996). Although resting B-cells do not express Fas, the expression can be up-regulated after addition of LPS, CD40 ligand or IL-4, rendenng activated B-cells susceptible to Fas-mediated apoptotic ce11 death (Bras et al., 1997). Fas ligation triggers apoptotic signais by activating cysteine proteases; ICE (IL-lp-converthg enzyme) cascade and specific proteolysis of PARP (poly ADP-ribose polymerase) or by down-regdation of antiapoptotic intracellular molecules such as Bcl-2/Bcl-x. Crosslinking of BCR prevents Fas-induced apoptosis by inactivating ICE cascade and/or maintainhg normal Bcl-2/Bcl- x Ievels and thereby rescues B-cells fiom apoptosis (Bras et al., 1997). CD19 is a B-ce11 specific antigen, which is expressed on the B-ceIl surface fiorn as early as pre-B cell stage, until terminal differentiation of activated B-cells into antibody-producing plasma cells. CD19 is a key member of the ce11 surface signal transduction complex, which serves as a CO-receptorof BCR (Fugue. 9) (Stites and Terr, 199 1). 1.3.2.1 Structure CD19 is a 95 kDa, integral membrane glycoprotein expressed only by B- lymphocytes and follicular dendritic cells. CD19 cDNA, encodes a 540 amino acid protein which traverses the membrane only once (Figure. 10). The N-terminal extracellular domain has 275 amino acids and contains two Ig-like domains separated by a smaller disulfide-linked domain. The transmembrane region contains a typical hydrophobic sequence of 22 amino acids lacking charged residues, which is immediately followed by two charged amino acids. The cytoplasmic domain is highly charged and contains 242 amino acids with 9 potential tyrosine residues for phosphorylation during signal transduction (Bradbury et al., 1993). The CD19 gene encodes at least 15 exons. Four exons encode the extracellular domain and one exon encodes the transmembrane domain. Six out of nine exons, encoding the cytoplasmic domain are identical in sequence across mouse, guinea pig and human species, indicating the significance of cytoplasmic tail as a signal transduction dornain (Zhou et al., 199 1). As a fùctional compIex, CD19 non-covalently associates with CD21, CD81, which is also known as TAPA- 1 (target of antiproliferative antibody- 1), Leu- 13 in some ce11 types and a number of unidentified proteins (O'Rourke et al., 1997) (Figure 9). CD2 1 is a 140 kDa molecule and a complement (C3d) receptor. CD81 is a 20 kDa serpentine molecule, which contains four transmembrane spanning domains, and Leu- 13 is a 1 6 kDa ce11 surface protein (review, Cambier et al., 1994). CD19 directly interacts with CD81 and CD2 1, and with Leu- 13 through CD8 1 (Bradbury et al., t 993) (Figure. 9). 1.3.2.2 Signal transduction The endogenous ligand and in vivo fbnction of CD19 have not been elucidated. However, crosslinking of ce11 surface CD19 with monoclonal antibodies induces internalization of the molecule and generates signaling events, indicating CD19 as an important receptor of B-lymphocytes (Zhou et al., 1991). CD19 is phosphorylated upon BCR ligation and amplifies the signals of BCR. CD 19-rnediated signal transduction mediates activation of Src-PTK, non Src-PTK, PI3- kinase and Vav, as the same mechanism as BCR-mediated signal transduction, described in section 1.4.1.2 (07Rourkeet al., 1997). In addition to Syk-mediated Vav activation, CD19 can directly recmit and activate Vav. The signaling ability of CD19 does not depend on the existence of a hctional BCR and can induce similar signal transduction in a totally BCR-independent mariner (Uckun et al., 1993). <. 1.3.2.3 Biological functions The engagement of ce11 swface CD19 with monoclonal antibody activates a tyrosine kinase signal transduction pathway, which induces homotypic adhesion through LFA- 1 (Ieukocyte bction-associated molecule- 1)/ ICAM- 1 (intercellular adhesion molecule-1) dependent or independent mechanisms (Bradbury et al., 1993). The transmembrane region of CD19 anaor association with CD81 through the transmembrane domain is essential for CD 19-mediated homotypic adhesion (Bradbury et al., 1993). As does BCR-mediated signaling, CD19 also can mediate B-ce11 proliferation and differentiation or inhibition of B-ce11 proliferation, depending on the nature of stimuli and specific stage of B-cells. Antibody crosslinking of CD19 alone induces stimulatory signals of B-ce11 growth (Bradbury et al., 1993) but antibody binding to CD19 cm also induce inhibitory signal to oppose mitogen-mediated B-ce11 activation (Bradbury et al., 1993). Upon CO-ligationof CD19 and BCR, CD19 induces stimulatory signals and lowers the threshold of antigen required for BCR-mediated B-ce11 activation. This phenomenon is critical for the in vivo B-ce11 response to a very low concentration of antigen (Carter and Fearon? 1992). Hyper-crosslinking of CD1 9 may also inhibit proliferation of B-cells by inducing ce11 cycle arrest at G1 and G2/M phases (Ghetie et al., 1994), or by induction of apoptosis (Chaouchi et al., 1995). Co-crosslinking of CD19 and BCR potentiates the BCR- mediated apoptosis, Mer indicating the CO-receptor role of CD19 for both BCR- mediated B-ce11 activation and ce11 death (Chaouchi et ai., 1995). The binding of specific antibodies to ce11 surface antigens usually results in a down regulation of the antigens and this process is known as antibody-induced antigenic modulation (AIAM). CD19 also undergoes AIAM upon antibody ligation (Pulczynski et al., 1993). Crosslinking of ce11 swface CD19 and subsequent incubation at 37°C induces patching and capping of CD19 prior to the htemalization, indicating the receptor mediated redistribution of the ligand for the receptor mediated endocytosis. Antibody crosslinked CD19 enters the ce11 via coated pits or by means of macropinocytosis, and which is later transporteci to perinuclear uncoated vesicles, resembling endosornes and receptosomes, and ultimately to lysosomes (Pulczynski et al., 1993). 1.3.2.5 Relationship between Gb, and CD19 Gb,, which is also known as B-cell antigen CD77, is expressed only at the germinal center stage of B-cells (Mangeney et al., 1991). After excluding the two Ig like domains and the intervening disulfide-linked domain, the remaining amino acid sequence of the extracellular domain of CD19 molecule has striking sequence homology to the Gb, receptor binding subunit of VT. Overall sequence similarity of CD 19 to VT 1 -B subunit is 41%. but 46% with the consensus sequence of VT-B subunits and 49% if conservative substitutions are included (Figure. 1 l), suggesting the possible lateral association of CD19 and CD77/Gb3 on B-ce11 surface (Maioney and Lingwood, 1994). Co-capping of antibody crosslinked CD 19 with Gb, indicates the possible physical association between CD19 and Gb,. In addition, CD19 positive I3 cells specificdly bind to a Gb,-based oligosaccharide rnatrix, suggesting that CD19 and CD77/G& could be endogenous ligands for each other. Since both CD19 and Gb, are expressed on germinal center stage B-cells and follicular dendritic cells, CD19/Gb3 mediated B-ce11 adhesion could be the initial mechanism to anchor B-cefls in lymphoid follicles. Anti-CD19 induced B-cefl homotypic adhesion is found to be significant only in Gb,-positive cells and Gb, reconstituted Gb, deficient mutant cells, suggesting that CD19-Gb, interaction could be the mechanism for homotypic adhesion of B-cells in the germinal center (Maloney and Lingwood, 1994). 1 VTIB: TPDCVTG-• --- - KVBYTKYND *DDTFTVKVOGDKBLFTNRWN- - - - Figure Ilk Amino acid sequence sirnilarity beîween CD19 and W binding subunit B Figure. 11B AminoacickofVTLB sharedwithCD19mW1B ctystaldrwture. green, shared anan0 acids. Shad6d area, Ob3 site. Gingw004 1996) 57 Type I interferons and type 1 intederon receptor Interferons are a family of cytokines, which are derived from a \vide variety of ce11 types and capable of interfering with the infection of cells by vinws (Pfeffer, 1997). 1.41 Classification of interferons Interferons are a family of distinct proteins, many of which are struchually related in amino acid sequence and three dimensional structure but are still differerent in the type of stimuli which induce them, the type of cells which produce them, and their biological potencies (Baron et al., 1987). There are five major antigenically distinct species of interferons; IFN-alpha (a), iFN-beta (P), IFN-omega (o),IFN-tau (r) and IFN-gama (y)(PfeRer, 1997). IFN-a, -P, - o and -r are collectively called type I interferons. Type 1 IFNs are acid-stable, similar in primary and three dimensionaf structure, and biological activities, share a common gene locus on human chromosome 9, and bind to the same receptor. IFN-y* also termed type II interferon. is acid-labile, recognizes a distinct receptor, and the gene is located on chromosome 12 (Pfeffer, 1997). IFN-a is a 16 to 27 kDa, 165-166 amino acid-containing glycosylated protein, produced by leukocytes, in response to components of foreign cells, or transformed or infected cells (Baron et al., 1987). There are at lest 13 subtypes of IFN-a,with 75-80% arnino acid sequence identity (Pfeffer et al., 1998). IFN-P is a 20 kDa, 166 amino acid-containing glycosylated protein, produced by fibroblast and epithelial cells, in response to foreign nucleic acids including those, derived from viruses. There is only one subtype of IFN-P. IFN-a and -P share considerable homology at the nucleotide (45%) and amino acid (30%) levels (Baron et ai., 1987). IFN-w is a 22.5 to 24.5 kDa, 172 amino acid-containing glycosylated protein, produced as a major component of human leukocyte interferon by virus-infected leukocytes (Adolf, 1995). There is only one species of human IFN-a but genes of other IFN-o subtypes have been identified in cattle, horse and sheep. IFN-o shares about 60% amino acid sequence identity with 1FN-a species but only 29% with IFN-P (Adolf et al., 1991). IFN-r is a 17.5 to 19 kDa, 172 amino acid-containhg glycosylated protehs, expressed by trophoectoderm of the preimplantation trophoblast of ruminant ungulate species, such as cattle and sheep, for a few days during early pregnancy (Roberts, 1996). IFN-r shares 70% and 50% amino acid sequence identity to IFN-o and IFN-a, respectively (Li and Roberts, 1994). IFN-y is a 166 arnino acid-containing glycosylated protein. The apparent molecular weight varies with degree of glycosylation fiom 15.5 to 25 kDa. Due to the more potent immunornodulatory property of IFN-y, compared to type 1 IFN, it is also known as immune IFN (Pfeffer et al., 1998). There are two subtypes of IFN-y, principally derived fiom activated T-lymphocytes and natural killer (NK) cells, in response to foreign antigens or mitogens. IFN-y does not share significant homology with IFN-a or IFN-P at either the nucleotide or the amino acid sequence (Baron et al., 1987). 1A2 Structure of type 1 interferon receptor The type 1 interferon receptor is a multi-subunit transmembrane glycoprotein receptor with a single transmembrane spanning region and belongs to class 1 cytokine receptor family. The functional type I receptor complex comprises at least 2 transmembrane chains; IFNAR1 and IFNAR2, and Janus family kinases (Jaks), associated with their cytoplasmic domains (Haque and Williams, 1998). Both IFNARl and IFNAR2 genes are located on hurnan chromosome 2 1 (Williams and Hyque, 1997) (Figure. 12). The type 1 IFN receptor is present in almost every ceIl type, at a rather low abundance (1 00 to 5000 copies per cell) (Cohen et al., 1995). There are two components of type 1 IFN-receptor interaction; a high affinity low copy number component (K, = 4.9 x 10-"~,400 siteskell in Daudi cells) and a low affinity high copy number component (K, = 4.2 x IO-~M,1000 sites/cell in Daudi cells). High affinity receptor binding is essential for IFN- mediated biological activities (Cohen et al., 1987). IFNARI is a 557 amino acid-containing glycoprotein with a 100 amino acid cytoplasmic domain and a 21 amino acid transmembrane domain (Haque and Williams, 1998). Due to the variation in glycosylation, the apparent molecular weight varies fiom 105 to 135 kDa in different celf types (Constantinescu et al., 1995). IFNARI plays a role in both ligand binding and more significantl y in intracellular signaling. However, reconstitution of human IFNARl to murine cells is not sufficient for the high affinity receptor binding to al1 species of type 1 IFN, indicating the requirement of an additional factor/s for the hlly fhctional type 1 IFN receptor complex (Pestka, 1997). IFNAEU has 3 isofonns, al1 of which are encoded by the same IFNAR3 gene. IFNAR2a chain is a soluble form of the receptor consisting of the extracellular domain only of IFNAR2b and IFNARk, both of which are transmembrane forms of the receptor. IFEI'AR2c has a large cytoplasmic tail compared with the short domain of IFNAR2b (Haque and Williams, 1998). A high affmity type 1 IFN receptor is composed of IFNARI and IFhTAR2c. IFNARSb can bind to a wide variety of type 1 IFNs but the role of IFNAWb in signal transduction is still controversial (Hoiland et al., 1997). IFNAR2c is a universal ligand binding domain of the type 1 IFN receptor and also mediates signai transduction, when it is CO-expressedwith IFNARl in murine cells. IFNAR2c is a 5 15 arnino acid transmembrane glycoprotein with apparent molecular weight of 102 kDa (Cohen et al., 1995). Upon ligand binding, IFNARl and IFNAR2c associate and this event triggers the signal transduction (Cohen et al., 1995). However, the human fetal lung fibroblast cell line, MRC-5 expresses both IFNARl and IFNAR.2, together with essential tyrosine kinases' Jakl and Tyk2, but still exhibits limited ligand binding capacity and biological activities (Ghislain et al., 1995). Reconstitution experiments of murine celis with transfectants have suggested that an additional component(s), encoded on human chromosome 21 is necessary to confer high affinity receptor binding and full biological activities (Ghislain et ai., 1995). An additional factor encoded by a gene located on the human chromosome 21, which is still distinct fiom IFNARI, IFNAR2 and Mx gene (invoived in IFN-induced antiviral responses), is necessary for signaling pathways prior Figure, 12 The structure of type 1 IFN receptor wmplex 6 1 to the activation of IFN-responsive genes. This novel factor is designated as a type 1 interferon signaling factor 21 (ISF21) (Holland et ai., 1997). The signaling function of ISF2I is independent of ligand-receptor interaction, processing and receptor down- regulation of IFNARI and IFNAR2 (Holland et al., 1997). 1A.3 Type 1 interferon receptor-mediated signal transduction Protein tyrosine kinases; Tyk2 and Jakl are constitutively associated with IFNARI and IFNAR2, respectively. Upon ligand binding, IFNAR2 associates with IFNARI and the subunit association mediates transphosphorylation and activation of Tyk2 and Jakl. Then, Tyk2 phosphorylates and activates Tyr,, on the IFNARl cytoplasmic domain, which in tuni serves as a docking site for Stat2 (a 113 kDa, cytosolic, signal uansducer and gctivator of gene transcription). Tyk2 phosphorylates Stat2, and receptor-bound, phosphorylated Stat2 fùrther recruits and phosphorylates Stat 1. Stat 1 comprises 2 isofonns, 91 kDa Statla and 84 kDa Statl P (Schindler et al., 1992). Phophorylated Statl and Stat2 form a hetro-dimer and together with another cytosolic factor p48, complete the formation of interferon stimulated gene factor-3 (ISGF-3). The ISGF3 complex translocates fiom the cytoplasm to the nucleus, interacts with upstream regulatory DNA elements of the interferon stimulated genes (ISGs), also known as interferon stimulated response element (ISRE), and transcriptionally activates the expression of ISGs (review, Haque and Williams, 1998). Interferon-a induced phosphorylation and activation of cytosolic phospholipase A? also facilitates ISGF-3 formation and subsequent activation of ISRE-containing genes (Figure. 13) (Ftati et al., 1996). As an alternative pathway, phosphorylated Statl rnay form a homodimer which activates the IFN-a-inducible genes without ISRE element, such as interferon regulatory factor-l (RF-1) via binding to their promoter regulatory DNA element, known as interferon-y activation site (GAS) (Figure. 13) (Haque and Williams, 1994). Stat proteins are a family of cytosolic transcription factors, which are activated by Figure. 13 Type 1 interferon-Type 1 IFN receptor mediated signal transduction diverse polypeptide ligands. Seven members of marnmalian Stat proteins, known as Statl, Stat2, Stat3, Stat4, StatSa, StatSb and Stat6, have ken identified. Type I IFNs are known to activate both Statl and Stat.2 but 1FN-y activates Statl only. Epidermal growth factor (EGF) and IL-6 activates Stat3, IL-3 activates Stat5, and IL4 activates Stat6. Tyrosine phosphorylated Stat proteins dirnerize and translocate into the nucleus to activate specific genes (Schindler et al., 1992). Differential activation of Stat proteins by different ligands is considered as one of the mechanisms by which specificity and diversity of cytokine-induced signal transduction is achieved (Fasler-Kan et al., 1998). During recent years. the role of type 1 IFN in activation of Stat proteins other than Statl and Stat2 has ken reported. SMcm be activated by IFN-a in a wide varïety of ce11 types. Despite the presence of Stat 1 and Stat2, Stat3 deficient cells also are found to be defective in IFN-a mediated biological activities such as antiproliferation and anti viral activity (Yang et al., 1998). The SH2 domain-containing portion of Stat3 duectly associates with tyrosine phosphorylated IFNARI, without prior phosphorylation by Janus family tyrosine kinases (Yang et al., 1996). Stat3 homodimer or Statl-Stat3 hetrodimer cm bind to GAS-related consensus sequence of ISGs (Zhong et al., 1994). The activation of Stat4. Stat5 and Stat6 are cell-type specific, which could be responsible for cell-type specific responses to IFN-a (Zhong et al., 1994 and Fasler-Kan et al., 1998). Stat4 expression is restricted to testis, thymus and spleen only (Zhong et al., 1994). IFN-a induced Stat5 and Stat6 activation has been observed in Daudi Burkitt's lymphoma cells only, but not in acute monocytic leukemia ce11 line (THP-1) and promonocytic ce11 line (U937). Most of the activated StatS and Stat6 form homo-dimers and a minor part form hetro-dimers (Fasler-Kan et al., 1998). As phosphorylated Stat2 serves as an adapter molecuie to recruit Statl to the type 1 IFN receptor, the recruitment of StatS and Stat6 to the IFNAR may be achieved by adaptor molecules, particularly expressed in Daudi cells (Fasler-Km et ai., 1998). Another alternative pathway of type 1 IFN signaling is CrkL (an SH2/SH3- containing protein that provides a link to downstream pathways which mediate growth inhibition) pathway. StatS is constitutively associated with type 1 IFN receptor-associated TyH. Upon stimulation with 1FN-a and IFN-P, phosphorylated form of Tyk2 serves as a docking site for CrkL. CrkL-Stat.5 complex translocates to the nucleus and binds to the GAS element to regulate gene transcription of ISG without ISRE (Fish et al., 1999). The specificity of cytokine-mediated Stat activation is not only conducted by selective phosphorylation of different Stat proteins, but also precisely regulated by a family of PIAS @rotein inhibitor of activated Stat) proteins (Liu et al., 1998). For example, PIAS-1 but not other PIASs, associates specifically with Statl only fier ligand stimulation, and inhibits DNA binding activity of Statl and Statl-mediated gene activation in response to IFN-a (Liu et al., 1998). During IFN-induced, Stat-mediated signal transduction, not only the phosphorylation and activation of Stat proteins, but also the sustained activation of Stat proteins is critical for the subsequent gene activation and biological activities. Comparable initial tyrosine phosphorylation of multiple Stat proteins has been detected in both IFN-a sensitive and resistant cells. However, prolonged phosphorylation and DNA- binding of Statl and other Stats is maintained for 24 to 32 hours in sensitive cells, compared to only 4 to 8 hours in resistant cells (Grimley et al., 1998). 1.4.4 Type 1 intederon regulated proteins There are more than 30 IFN-inducible genes, some of which encode enzymes, critical for IFN-mediated biological activities. 2-5 oligoadenylate synthetase (2-5 AS) is one of the enzymes, principally induced by both type 1 and type II IFN. Synthesis of 2-5 AS is induced by IFN and activated by cellular or viral RNA. Activated 2-5 AS catalyzes the conversion of ATP into growing 2- 5 A, (n = 2 to 15). 2-5 A activates 2-5 A-dependent RNAase-L, which degrades cellular or viral RNA. The activity of 2-5 AS activity is negatively regulated by 2-5 phosphodiesterase, which degrades 2-5 AS into AMP and ATP. There are different isoforms of 2-5 AS such as 40, 46, 69 and 100-kDa proteins. The 40 kDa isofonn is found to be important to confer IFN-mediated biological activities (Samuel, 1991). Another significant IFN-regulated protein is a 68-kDa, interferon-inducible double-stranded RNA dependent protein kinase @KR) (Meurs et al., 1990). The synthesis of PKR is induced by IFN and PKR is activated by cellular or viraI-ds-RNA dependent auto-phosphorylation. The substrate of PKR is protein synthesis initiation factor02 (eIF2) and phosphorylation of eIF2 inhibits protein synthesis at the initiation step of translation (Williams, 1996). Mx proteins also are IFN-reguiated prote&, encoded by a gene located on the distal part of the long arm of chromosome 21. There are 2 isofoms of human Mx proteins; M.xA and MxB, and oniy MxA is induced upon IFN stimulation. MxA is a 75.5 kDa protein with intrinsic GTPase activity, which is ubiquitous and abundant in the cytoplasm, and induced selectively by type 1 IFN but not by type II IFN. The exact mechanism of action of Mx protein is not well understood yet but likely by modifying cellular functiow required dong the virai replication pathway (Horisberger, 1995). 1.4.5 Type 1 interferon-mediated biological activities Type 1 interferons induce their biological activities via synthesis and activation of IFN-regulated proteins. There are three major biological functions induced by type 1 IFN; antiproliferative activity, anti viral activity and immwio-modula.tory activity. Interferons are not essential for the maintenance of normal ce11 growth but IFN- induced tumor ce11 antiproliferation and differentiation activities are very significant for the growth control of IFN-sensitive tumors such as hairy ce11 leukemia, non-Hodgkin's lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia etc (Sen and Lengyel, 1992). Type 1 IFN-induced antiproliferative activity is mainly mediated by synthesis and activation of 2-5 AS and PKR enzymes (Sen and Lengyel, 1992). IFN-induced anti-tumor activity cm also be achieved by direct lysis of tumor cells, activation of immunocompetent cells and inhibition of oncogene expression (Baron et al., 1987). Type I IFN exhibits antiviral activity against a wide variety of viruses by various mechanisms. IFN-mediated 2-5 AS activation is selective for picornaviruses such as mengo and enchephalomyocarditis only (Samuel, 1991). Mouse Mx 1 protein effectively interferes with the replication of infiuenza virus only and human MxA protein, with vesicular stomatitis and influenza viruses only. However, PKR enzyme can be involved in the protection against a wide varïety of vinises such as adenovinis, human i mmunode ficiency virus, influenza virus, poliovinis, reovinis, vaccinia virus etc (Samuel, 1991). In contrast, vinises also can attempt to overcome the antiviral mechanisms of IFNs by a large variety of counter-mechanisms. For example, EMC and herpes simplex vinws can inhibit the 2-5 AS, RNAase-L system by inactivating RNAase-L. AdenoMral VA-1 RNA (virus associated RNA 1) binds to PKR enzyme and impairs its activation by dsRNA. Poliovirus accelerates the proteoIytic cleavage of PKR. Influenza virus activates the pre-existing latent cellular inhibitor of PKR. Some viruses, such as adenovirus, encode proteins which inhibit the transcriptional activation of the IFN-inducible genes by blocking the activation of ISGF-3 (review, Sen and Lengyel, 1992). Type 1 IFN modulates immune responses by various mechanisms. Type 1 IFN increases the expression of class 1 MHC molecules on B-cells. Since cytolytic T- lymphocytes (CTL) recognize foreign antigens presented by class I MHC, type I IFN enhances the effector phase of the cell-mediated immune response by promoting CTL- mediated killing (Stites and Terr, 1991). Another well characterized mechanisrn of IFN- mediated immuno-modulation is activation of the cytotoxic activity of natural killer (NK) cells to kill virally-infected cells (Pfeffer et al., 1998). IFN-a interferes with the synthesis of various cytokines and thereby acts as an anti-infiammatory agent. IL-1 is a proinfiammatory cytokine, which is an important mediator of fever, hypotension and acute-phase reaction. A specific inhibitor of IL-1 ; IL- 1Ra, blocks the binding of IL-1 to IL-I receptor. IL-I Ra has ken shown to reduce the severity of sepsis, arthritis, colitis and other infiaarmatory processes in several animal models (Tilg, 1997). IFN-a mediates anti-infiammatory effect against IL-1 by reduction of IL- 1 synthesis, induction of IL-1Ra synthesis and suppression of endotoxin-induced IL-1 synthesis. IFN-a can also exhibit anti-inflarnmatory activity by modulating other cytokines such as IL-8, IL-1 O and TNF (review by Tilg, 1997). Internalization of type 1 IFN/receptor IFN-a interacts with high-aEnity receptor binding sites on the ce11 surface at 4°C and upon temperature shift to 37OC, IFN accumulates in clathrincoated pits and later in receptosomes (Zoon et al., 1983). However, the number of such high-affmity binding sites are less than 1Oûûkell in most cell types and consequently, direct visualization of the receptor-bound IFN and fate of the ce11 bound-IFN is very difficult. Therefore, biochemical approaches using radiolabeled ligand molecules are usually applied (Branca et ai.. 1982). After IFN-dreceptor intemalkation, there is only Limited recycling of the receptors and surface available receptors are down regulated by rapid degradation. Recovery of IFN-a binding activity requires de novo synthesis of receptors (Sranca et al., 1983). The intracellularly secreted 1FN-a can activate ISGF-3 formation and can induce anti viral activity at a lesser extent. However, ce11 surface high-affinity receptor binding and subsequent ligand/receptor intemakation are essential for the IFN-a mediated antiproliferative and anti Mral activities (Killion et ai., 1994, Anderson et ai., 1982 and Yonehara et al., 1983). 1.4.7 Relationsbip between Gb, and type 1 interferon receptor The study on Daudi Burkitt's lymphoma cells and Gb, deficient Daudi mutant cells have shown that Gb, deficient VT-resistant cells are also cross-resistant to IFN-a induced antiproliferative activity (Cohen et al., 1987). Comparable to the amino acid sequence of the B-cell differentiation antigen; CD 19, the extracellular dornain of IFNARl transmembrane subunit dso has 43% arnino acid sequence similarity to VTI binding subunit B (figure. 14), suggesting the possible lateral association between IFNARl and Gb, on the B-ce11 surface (Lingwood and Yiu, 1992). IFN-a binding studies to receptors on Gb,-positive Daudi Burkitt's lymphoma cells and Gb,-deficient Daudi mutant cells have shown that although both high-affuiity and low-affinity receptor binding components are still maintained in both ce11 lines, receptor binding capacity of IFN-a is 100 fold reduced in Gb,-deficient cells. The reduced binding capacity reflects an inappropriate presentation of the receptor on the ce11 surface in Gb, deficient cells. IFNa induced ISGF-3 binding to the 2-5 AS gene and IFN-a-mediated growth inhibition also are defective in Gb,-deficient cells, indicating a role for Gb, in IFNa-mediated signal transduction (Ghislain et al., 1994). The binding of a fusion protein eIFNAR1-IgG (extracellular domain of IFNARl and Fc portion of IgG) to Gb2 and Gb, of the cellur glycosphingolipid extract, separated on thin-layer chromatography also suggests the possible physical association between eIFNARl and Gb2/Gb, on the ce11 surface (Ghislain et al., 1994). Since type 1 IFNR is a multimolecular functional complex (Uze et al., 1995), Gb, may function as a component of the rnultisubunit type 1 IFN receptor to facilitate the receptor-mediated signai transduction. 1 VTIB : TPDCVTGKVEYTKYNDDDTFTVKV- GDKELFTNRWN - - + Figure. 14A Amiw acid sequeaxe simhrity between IFNARI and VI' binding subunit B Figure- 14B A.mimacidsafVT1B dmnit sharedwithiFNAR1 onVTlB crystal structure. Green, shared anrino acids. Shaded ami, Gb3 site. Uw-4 1996) Vesiele-mediated protein transport Eukaryotic cells consist of numerous membrane-bound structures with distinct specialized hctions. To maintain the normal structure and hction of the cells, protein and lipid molecules need to be transported in either an outward direction during biosynthesis or an inward direction during endocytosis. The molecules are pac kaged into membrane-bound vesicles and transported from one cornpartment to another by a tightly regulated mechanisrn known as vesicle-mediated membrane transport. Fundamentally, vesicles bud fiom the donor membrane, and dock to and fuse with the acceptor membrane. There are two major pathways of protein transport namely the biosynthetic/secretory pathway and the endocytic pathway (Figure. 15) (Borgne and Hoflack, 1998). 1S. 1 Biosynthetid secretory pathway During protein synthesis, secretory and membrane proteins are CO-translationally inserted into the endoplasmic reticulum. The proteins are packaged into vesicular intermediates and transported f?om the ER to the cis- end of the Golgi. In the lumen of the ER and the Golgi, newly synthesized proteins are processed, folded and modified to become fully functional (Nickel and Wieland, 1998). At the trans- end of the Golgi, proteins are sorted and delivered to their final destinations accordingly as secretory proteins, plasma membrane proteins, lysosomal enzymes etc (Pelham and Munro, 2993). Regarding the functional aspect of the pathway and the direction of vesicle movement, from inside to outside of the cell, this pathway is termed the biosynthetic/secretory or anterograde pathway. f'tAswA MEMBRANE BIOSYNTHEI2C PATHWAY Figure. 15 Vesicle-mediated protein transport paîhways Pathway 1, transport of proteins to plasma membrane; 2, secretory granules; 3, AP-1- clathrin coated vesicle-mediated transport of M6P-containing ligaad/MPR to early andior late endosomes; 4, AP-24athrin coated vesicle-mediated endocytosis hmthe plasma membrane; and 5, retrograde transport of ER resident proteins fiom Golgi compartments to ER 1.5.1.1 Structural compooents Al1 the membrane transport vesicles, which are budding fiom different compartrnents, are coated with specific coat-protein complexes. There are two types of vesicles, narnely clathrin-coated vesicles and non-clathrin coated vesicles, also known as COP-coated vesicles. The clathrin-coated vesicles are Merclassified into clathrin-AP1 and clathrin-AP2 vesicles, and the COP-coated vesicles into COPI and COPII vesicles. The coat consists of different combination of cytosolic subunits, which are specifically assembled on the cytoplasmic side of the budding vesicles. Clathrin-AP2, clathrin--Ml, COPI and COPII coats are specifically associated with plasma membrane, TGN, Golgi and ER membranes, respectively (Figure. 15) (Rohan and Wieland, 1996 and Schekman and Orci, 1996)- Two closely related adaptor complexes; AP1 and AP2, are heterotetrarners consisting of two -100-kDa proteins called adaptins (aand f32 for AP2 and y Ad p 1 for API), and one copy each of -50-kDa (p) and -20-kDa (6) proteins. Adaptors are recruited ont0 the appropnate membrane presumably by specific adaptor receptors, prior to the binding to cytoplasmic tails of the targeted proteins on the same membrane. There are three types of sorting signals in the cytoplasmic tails and the adaptors specifically recognize a YXX0 (0 is a bulky hydrophobie amino acid) motif, a di-leucine motif or a FXNPXY motif (review, Robinson, 1994). Clathrin consists of three copies each of heavy and light chahs, fonning a three-legged structure known as a tnskelion. Cytosolic clathrin binds to either plasma membrane or TGN by interacting with the membrane bound adaptor molecules AP2 or API, respectively (review, Robinson, 1994). The mechanism of plasma membrane coated pit formation by clathrin and AP2 will be descnbed in section 1S.2.1.1. The fiuictions of clathrin-AP2 and clathnn-AP 1 vesicles are to transport proteins fiom the plasma membrane to early endosomes, and fiom the TGN to early anaor late endosomes, respectively (Borgne and Hoflack, 1998). COPI vesicles comprise seven polypeptide subunits for the structural component: u (160 kDa), f3 (1 10 Da), P' (102 kDa), y (98 ma), 6 (61 ma), E (31 Da) and 5 (20 Da). The subunits can be pre-assembled as a soluble cornplex, called the coatomer, and are recruited from the cytoplasm to the Golgi membrane by activation of the small GTP- binding protein, ARï (ADP-ribosylation factor). COPI vesicles transport proteins from ER to Golgi, bidirectionally within Golgi compartments and fiom Golgi to ER (Review, Schekman and Orci, 1996). COPII coat consists of five subunits; Sarlp (21 kDa), Sec23p (85 kDa), Sec24p (105 kDa), Secl3p (33 kDa) and Sec3lp (150 kDa). The subunits are recruited to the ER membrane upon activation of the SarIp-specific small GTP-binding protein SAR. COPII vesicles emerge from the ER and fiise with the cis end of the Golgi (Review, Saiama and Schekman, 1995). 1.5.1.2 Regulatory components In addition to the structurai components for vesicle formation, there are two major classes of proteins for the specificity of vesicle docking and fusion. The first class comprises two soluble factors; NSF (N-ethylmaieimide-sensitivefactor) and soluble NSF attachment protein (SNAP), and two integral membrane proteins, SNAREs (SNAP receptors) which are necessary for membrane docking and fusion (Review, Hay and Scheller, 1997). V-SNAREs (vesicle-associated SNAREs) are present on the budding vesicles fiom the donor membrane and t-SNAREs (target-associated SNAREs), on the acceptor membrane. NSF is an ATPase, and NSF/SNAP complex interacts with the v- SNARE/t-SNARE complex on the acceptor membrane (Rothrnan and Warren, 1994). The second class consists of smail GTPases, known as Rab proteins, which catalyze SNARE-mediated vesicle fusion (Novick and Zerial, 1997). There are more than 40 members of Rab farnily of proteins in yeast and mamrnalian cells, and the distribution of Rab proteins to different membrane compartments is unique. For example, Rab1 and Rab2 are associated with ER and cis-Golgi, Rab6 on Golgi and TGN, Rab 4 and Rab5 on early endosornes etc (review, Stow, 1995). Moreover, another family of small GTPases; ARF (ADP-ribosylation factors), also are required for the assembly and disassembly of cytosolic coat proteins on the membrane (Donddson and Klausner, 1994). Like di GTPases, both Rab and ARF exist Doaor Membrane Y'v ~soFweny(gnoup Figure. 16 GTPase-mediated membrane tdficking cycle as an inactive GDP-bound form in the cytoplasm and as a membrane-associated, active GTP-bound form. Association with GDP-dissociation inhibitor (GDI) maintains the inactive GDP-bound form. GDI-displacement factor (GDF) and guanidine nucleotide exchange factor (GEF) catalyze the GDP/GTP- exchange activity and GTP-bund active form binds to the donor membrane. Upon arrivd of the vesicles on the target membrane, GTP is hydrolyzed to GDP after the interaction of GTP-bound Rab/ARF with GTPase activating protein (GAP) and the GDP-bound form is released into the cytoplasm (Martinez and Goud, 1998) (Figure. 16). 1.5.1.3 Mechanism of vesicle movement Vesicle budding is triggered by activation of ARE Following the attachment of GTP-ARF ont0 the donor membrane, cytosolic coat proteins are recruited to the membrane for the coat assembly, which allows the formation of a budding vesicle. V- SNAREs als~are recruited to the budding vesicles via preceding attachrnent of GTP-Rab. The vesicles canying the cargo proteins move to the target membrane by simple diffusion or along the cytoskeletal fibres (Review, Rothman and Wieland, 1996). The vesicles dock to the acceptor membrane by specific interaction between V-SNAREs and t-SNAREs. Hydrolysis of GTP-Rab to GDP-Rab cataiyzes the SNARE complex formation and vesicle fusion. Following the binding of NSF/SW to the SNARE cornplex, hydrolysis of ATP by NSF disassembles the SNARE complex (Review, Hay and Scheller, 1997) (Figure. 17). The Golgi membrane nucteotide exchange activity by GEF is specifically inhibited by a fungal metabolite, brefeldin A @FA) (Donaidson and Klausner, 1994), which causes dissociation of P-COP fiom Golgi membrane, followed by disintegration of Golgi structure and redistribution of Golgi enzymes to ER (Scheel et al., 1997). As a result, cis-, medial and trans- Golgi fuse with ER and TGN fuses with early endosornes (Pelharn, 1991 and Wood et al., 1991). Figure. 17 SNAREsaiediated membrane trafiicking cycle 1.5.1.4 Protein sorting After the CO-translationaltranslocation of newly synthesized proteins across ER membrane, the chaperone system facilitates the proper folding of newly synthesized proteins until the folding is complete. Mis-folded and incompletely folded proteins are withheld in the ER by chaperone-mediated retention. Some ER membrane proteins are transported to the cis- end of Golgi via COPI1 vesicles (Nickel and Wieland, 1998). While the proteins are moving through different compartments of Golgi via COPI vesicles, Golgi resident proteins are retained in Golgi (Nilsson and Warren, 1994). Proteins in the secretory pathway are precisely sorted at the TGN, to target to the plasma membrane, secretory granules or lysosomes (Borgne and Hoflack, 1998). The transport of plasma membrane proteins and some secretory proteins to the plasma membrane is a default pathway and is mediated by Ml-clathrin coated vesicles. Some secretory proteins are packaged in the secretory granules and are released in response to extemal signals. Otherwise, signal-mediated sorting mechanisms speci@ the movement or lack of movement of the proteins (Rothman and Wieland, t 996). The sorting signais are discrete peptide domains with 4 to 25 residues or the conformationally determined epitope. A transport signal concentrates the protein in a budding vesicle as a cargo, which is accurately directed to the specified compartment (Rohan and Wieland, 1996). Transport signals on the cytoplasmic side bind directly to a coat protein, and those on the lurnenal side bind to the lurnenal domain of an intennediary protein, the cytoplasmic tail of which in tum binds to the coat. Lysosomal enzymes bearing mannose-6-phosphate signal at the lumenal end bind to mannose-6- phosphate receptor (MPR), the cytoplasmic di-leucine signal of which, is further recognized by AP1 adaptor of AP1-clathrin vesicles and the enzymes are transported from TGN to late endosornes (Hunziker and Geuze, 1996). Another type of sorting signal, known as a retention signal specifies lack of movernent and restricts proteins fiom entering a budding vesicle. Retention signals are usually compartment specific, such as ER versus cis-, medid- and tram-Golgi cornpartments. The trans-membrane (TM) domains of Golgi proteins serve as retention signals but the exact mechanism of retention is not well understood yet. MediaI-Golgi enzymes associate with each other, forming complexes, which are too large to be transported via vesicles. This phenonmenon is known as "kin recognition". Another factor of Golgi retention is the length of Golgi protein TM domain. The bi-layer membrane of plasma membrane is thicker than Golgi membrane. The shorter TM domain of Golgi proteins (17 residws) segregates the proteins from transport to the plasma membrane (the average TM domain of a plasma membrane protein has 21 arnino acid residues) and retains them in Golgi (Nilsson and Warren, 1994). 1S. 1.5 Retrograde transport Some ER resident proteins require Mer addition and modification of oligosaccharide residues in the Golgi apparatus (Pryer et al., 1992). Such proteins carry retneval signals and are subject to retm to the ER in the opposite direction of the anterograde pathway. Consequently, this pathway is termed "retrograde transport". There are two types of retrieval signals; a C-terminal tetra peptide KDEL and a C- terminal KIUCX (X is any arnino acid) (Review, Nilsson and Warren, 1994 and Pelharn, 1995). The KDEL sequence is specifically recognized by KDEL-receptor on the Golgi membrane. KDEL receptors are distributed across the Golgi stacks and most concentrated at the cis- end of the Golgi. The KKXX sequence on the other hand, is recognized by the coatomer of COPI vesicles. Endocytic pathway Endocytosis is the mechanism by which cells take up extracellular material by a variety of different pathways. The roles of endocytosis in physiological processes include nutrient uptake, maintenance of ce11 polarity, antigen presentation, regulation of ce11 surface receptor expression, etc. Microorganisms such as viruses and toxins also utilize endocytic pathway to gain access into the cells. The different mechanisms of endocytosis cmbe classified into two major classes (Lamaze and Schmid, 1995). 1. Clathrin-dependent endocytosis II. Clathrin-independent endocytosis 1. Caveolar-mediated endocytosis 2. Non-coated vesicle-mediated endocytosis 3. Macropinocytosis 3. Phagocytosis 1.5.2.1 Clathrin-dependent endocytosis Macromolecules, bound to ce11 sudaçe receptors are concentrated laterally before intemalization and as a result, this mechanism is termed receptor-mediated endocytosis (RME). RME was the first described and is the most extensively studied mechanism of endocytosis. It has ken generalized that this pathway is the only efficient intemalization mechanism for ce11 surface receptors and the tenns RME and clathrin-mediated endocytosis have been used interchangeably (Lamaze and Schmid, 1995). The concentrated ligandheceptor complexes in the coated pits are invaginated into coated vesicles and transported to early endosomes, late endosomes and ultimately to lysosomes, where intemalized materials are catabolized (Mukhejee et al., 1997). Structurai compoaenb Clathrin coated pits are readily detectable by conventional electronmicroscopy as membrane invaginations with an electron-dense bristle coat (Lamaze and Schmid, 1995). Coated vesicles range in size fiom -100-150 nm in diameter. The main constituents of the clathrin coat of the ceIl membrane are clathrin and AP2 adaptor, described in section 1 .S. 1.1. Clathrin-AP1 vesicles also shuttle between TGN and early or late endosornes for the delivery of lysosomal proteins and recycling of MPR. COPI components also have been found to be recniited to the eariy endosomal membrane (Aniento et al., 1996). 1.5.2.1.2 Regulatory components Dynamin is a 100-kDa cytoplasmic protein with GTPase activity. Protein kinase C (PKC) phosphorylates and activates its intrinsic GTPase activity in vitro. As other members of GTPases, redistribution of the GTP-bound active form of dynarnin to the neck of the deepiy invaginated coated-pit is required for coated vesicle pinching fiom the plasma membrane (Damke, 1996). Other regdatory components of vesicle budding, docking and hion such as SNARE components, ARF and Rab GTPases also play important roIes in the mechanism of endocytosis (Mukhe jee et al., 1997). 1S.2. 1.3 Mechanism of vesicle movement Depending on the ce11 type, clathrin-coated pits occupy -2% to 60-70% of the ce11 surface. Some receptors are constitutively associated with coated-pits (LDL receptor) (Anderson et al., 1982), but other receptors such as EGF receptor, become concentrated in coated-pits upon ligand binding (Dunn and Hubberd, 1993). Recruitment of ceIl surface recepton is mediated by binding of cytoplasmic internalization signal motifs (tyrosine- based, leucine-based, lysine-based etc.) with AP2 adaptors. Lipid and lipid-linked proteins also enter the membrane invaginations, nonslectively or selectively. Binding of ST to giycospingolipid receptor, Gb, causes aggregation of Gb, in clathrin coated-pi&, prior to the toxin-bound receptor endocytosis. The mechanism of lipid receptor concentration codd be due to the association of the receptor with other tram-membrane proteins, which contain an intemalization motif at the cytoplasmic tail or altematively, an aggregate of toxin-band lipid receptor may have a preference for coated-pit localization (Sandvig et ai., 1989). After adaptor-mediated clathrin recruitment to the plasma membrane, deeply invaginated coated-pits are formed and GTP-bound dynamin facil i tates the formation of coated vesicles. While the internaiized molecules are rapidly directed to early endosomes, the vesicles are uncoated by a 70-kDa uncoating ATPase (Braeli et al., 1984). The vesicle budding. docking and fision fiom one endosomal compartment to another is mediated by SNAREs and smdl GTPases. 1.5.2.1.4 Protein Sorting Internalized ligand/receptor complexes are sorted in early endosomes, also known as sorting endosomes, for receptor recycling to the plasma membrane or delivery to late endosomes and lysosomes. Recycling of internalized receptor via the endocytic recycling compartment is a default pathway for membrane components. Sorting mechanisms are necessary for the retention of proteins in early endosomes and subsequent delivery to late endosomes and lysosomes (Mukhe rjee et al., 1997). There are two general types of sorting mechanisms: physical sorting based on properties such as pH and the geometry of the compartrnent, and signal-mediated sorting based on protein-protein recognition. A pH-dependent conformational change in the receptors releases ligands fiom the receptors, after which receptors usually recycle back to the plasma membrane and ligands are degraded. Some ligand-receptor dissociation may take place at a pH lower than early endosomal pH, protecting the recycling molecules from degradation in early endosomes. For example, M-6-P bearing lysosomd enzymes dissociate fiom MPR at pH 5.8 (Mukhe rjee et al., 1997). Recycling compartments bud off as narrow-diameter tubuies fiom the early endosomes. The molecules with diarneter larger than recycling compartments are excluded from the recycling (Review, Mukherjee et al., 1997). As in a signal-mediated sorting mechanism, the cytoplasrnaic region containing the sorting signals such as the di-leucine motif of MPR may play a role in targeting of the receptor to late endosomes and lysosomes (Johnson and Kodeld, 1992). In addition, aggregation and oligomerization of proteins also segregate the receptors in early endosomes, protecting them fiom recycling. The delivery of late endosornai contents to lysosomes occurs by the tùsion of late endosomes to pre-existing lysosomes, and endocytosed macromolecuies are dtimately digested in Iate endosomes and lysosomes. The recycling cornpartment not only retums membrane components to the plasma membrane, but also recycles the membrane cornponents within the earl y endosomal system (Ghosh and Maxfield, 1995), in order to maintain the distinct protein compositions of various endosomal compartments (Mukhejee et al., 1997). 1.5.2.2 Clathrin-independent endocytosis The clathrin-independent, alternative pathway is usually demonstrated by strategies whkh selectively inhibit clathrin-dependent endocytosis such as incubation in hypertonic media (which prevents clathrin coated-pits formation), potassium depletion, cytosolic acidification (which prevents coated vesicles pinching off) (Heuser et al., 1989), use of anti-clathrin antibodies (Doxsey et al., 1987) or expression of the temperature- sensitive mutant of dynamin (Damke et al., 1995). However, inhibition of clathrin- mediated endocytosis itself may induce clathrïn-independent endocytosis, as a response to maintain cellular surface-to-volume ratio (Mukherjee et al., 1997). 1.5.2.2.1 Caveolar-rnediated endocytosis Caveolae are uniform omega- or flask-shaped membrane invaginations of 50-80 nm in diameter. Quick-freeze, deepetch images of caveolae reveal a granular, spiraling coat structure not detectable by conventional electron microscopy (Rothberg et al., 1992). The cytoplasmic surface of caveolae is often decorated with a coat composed of delicate filaments arranged in striations. The occupancy of caveolae on the plasma membrane varies with ce11 type. A large fiaction of the plasma membrane surface is covered by caveolae in some specialized cells such as endothelium (Mukherjee et al., 1997). Various receptors such as P-adrenergic receptor, cholera toxin receptor (GMI), tetanus toxin receptor (GD1 and GT1) and many GPI-anchored proteins are pre-associated with caveolae and the receptors can be further concentrated upon ligand binding (Mayor et al., 1994). The stnicnual componerits of caveolae comprises the integrai membrane protein caveolin also known as VIP21, and high concentrations of certain lipids such as cholesterol and glycosphingolipids. Sterol binding dmgs such as filipin cause precipitation of cholesterol and disnipt the organization and fûnction of caveolae (Rothberg et al., 1992). The SNAREs, NSF, SN*, GTPases (Schnitzer et al., 1995) and dynamin (Henley et al., 1998) also play a regulatory role in caveoiar-mediated transport, which also involves classical budduig, docking and fusion of vesicles (Schnitzer et al., 1995). Caveolar-mediated endocytosis may target to early endosornes as in the classical endosomaVlysosomal pathway (Tran et al., 1987) or target directly to Golgi and ER via microtubule dependent or independent mechanism (Conard et al ., 1995). Very recently, vascular EGF (VEGF) induced nuclear translocation of caveolin-1 in vascular endothelid cells also has been reported (Feng et al., 1999). Potocytosis is an alternative mechanism of caveolar-mediated endocytosis, by which lower molecular weight molecules and ions are delivered directly to the cytoplasm, bypassing the vesicle-mediated transport. The caveolae close off after concentrating various molecules in caveolae, creating a very high concentration of the molecules in the sealed-off caveolae and driving the diffision of the molecules directly to the cytoplasm by the concentration gradient (Anderson, 1992). Clathrin-independent, caveolar-independent endocytosis Non-clathrin coated membrane invaginations, lacking caveolar marker caveolin, may pinch off to form smooth vesicles of -100nrn in size, and may carry the fluid phase marker enzyme, horseradish peroxidase (HRP) (Cupers et al., 1994). Another type of clathrin-independent endocytosis is via surface tubules for entry into macrophages (STEM), of -250 nrn in diarneter and 1 pm or more in length. Very- low-density lipoprotein (VLDL) particles are delivered to late endosomes and lysosomes via this mechanism (Myers et aI., 1993). The plant toxin, ricin enters the ce11 by the clathrin-independent, caveolar- independent mechanism (Lord and Roberts, 1998). 1.5.2.2.3 Macropinocytosis Macropinosomes are non-clathrin-coated vesicles of 0.5-200 pm in diarneter, which are found constitutively in macrophages and many tumor cells or in other cells &er stimulation with growth factor or phorbol esters. The formation of macropinosomes is associated with membrane nimes, which are fonned by bands of out-wardly directed actin potymerization resulting in folds or circular, cupshaped extensions of the cytoplasm. The ruffles close off to form large vesicles and take up large volumes of fluid non-selectively. Macropinosomes may acidiw and shrink or merge with lysosomal compartrnents or may remain isolated and recycle most of their contents to the ce11 surface (review, Mukherjee et al., 1997). 1.5.2.2.4 Pbagocytosis Phagocytosis is a receptor-mediated, actin-dependent and clathrin-independent intemalization of large particles and microorganisms into neutrophils, monocytes and macrophages. The ingested particles are degraded in phago-lysosomes, afier the fusion of phagosomes to lysosomes (Rabinovitch, 1995). 1.5.3 Interoalization of protein toxins Many pathogenic bacteria and plants produce subunit protein toxins, which induce their pathogenic effect- Generally, the toxuis comprise an enzymatic subunit A and receptor binding subunit B. The subunit B of toxins such as DT (diphthena toxh), ETA (Pseudomonas exotoxin A) and TT (tetanus toxin) has two domains; a C-terminal receptor binding domain and an N-terminal domain involved in membrane translocation. In contrast, the binding subunits of other toxins such as CT (cholera toxin), LT (Exoli heat labile toxin)? ST and VT are a doughnut-shaped pentameric oligorner. The toxins usually gain access into the cells via a four-step mechanism; receptor binding, endocytosis, intracellular trafEcking and membrane translocation (Review, Monteccuco et al., 1994). Specific receptors for the protein toxins could be either glycoprotein or glycolipid or both. DT and ETA are the examples of protein receptor binding toxins. DT and ETA bind to heparin-binding EGF-like growth factor precursor and a,-rnacroglobulin receptor, respectively (Monteccuco et al., 1994). The examples of glycolipid receptor binding protein toxins are GMl binding CT and LT, Gb, binding ST and VT, and GD, and GTl binding TT (Review, Monteccuco et al., 1994). Ricin, a plant toxin binds to the terminal galactose residue of both glycoprotein and glycolipid (Sandvig and van Deurs, 1996). Afier receptor binding, the toxins enter the ce11 via clathrin-dependent mechanism or clathin-independent mechanism. Protein receptor binding DT and ETA enter the cells by clathrin-dependent endocytosis, whereas lipid receptor binding CT, LT and TT enter the cells by clathrin-independent endocytosis such as caveolar-mediated endocytosis (Monteccuco et al., 1994 and Mayor et al., 1994). Despite the glycolipid nature of the receptor, ST and VT enter the cells by clathrin-dependent endocytosis (Sandvig et al., 1989 and Khine and Lingwood, 1994). Ricin uses both clathrin-dependent and clathrin- independent, but non-caveolar mediated mechanism (review, Lord and Roberts, 1998). Intracellular toxins are transported to their respective sites of membrane translocation via the endosornaVlysosomal pathway or via retrograde transport to the compartrnents of the biosynthetic/secretory pathway such as Golgi, ER and also to nuclear membrane and nucleus, depending on the ce11 lines. DT and TT are the examples of the toxins which utilize acldic compartments of endosomal system for the membrane translocation. Consequently, cytotoxicity of DT and TT can be prevented by agents that inhibit the vacuolar-ATPase proton pump such as bafilomycin-A (Monteccuco et al., 1994). In contrast, CT, LT, ETA. ST, VT and ricin undergo retrograde transport (review, Montecucco, 1998). Therefore, the cytopathic effect or cytotoxicity induced by these toxins cm be prevented by agents that disrupt the Golgi structure such as brefeldin A @FA) (Donta et al., 1993, Sandvig et al., 1991) and ilimaquinone (Madhusoodana et al., 1995). The C-termuid residues of CT subunit A comprise the ER retrograde transport signal tetrapeptide, KDEL. LT and ETA also carry C-terminal KDEL-like sequences, RDEL and REDLK, respectively (review, Lord and Roberts, 1998). ST and VT however, do not have such sequence, suggesting that association with Gb, itself is critical for the retrograde transport. In the endosomal cornpartment, the subunits of toxins such as DT and TT dissociate. A conformational change of the B subunit, triggered by acidic pH, enables the B subunit to form a transmembrane channel to allow the passage of enzymatic subunit A to the cytoplasm (Monteccuco et ai., 1994). The mechanism of membrane translocation for the toxins, which undergo retrograde transport, has not ken well defined yet. However, it has been speculated that at least subunit A is partially unfolded in the ER lumen by ER-resident chaperones such as calnexin, and undergoes retrotranslocation across the ER membrane by the ER-associated degradation (ERAD) pathway, which is a quality control mechanism to remove mis-folded proteins in the ER. Such proteins are ubiquitinated on their lysine residues and degraded in proteosornes. However, a total lack of or the presence of very few lysine residues on the subunit A could be a mechanism for escaping ubiquitin-mediated protein degradation (Hazes and Read, 1997). Cbapter 2 Objective and Hypothesis 2.1 Objective The main objective of the present study is to study the role for Gb3 in internalization and biological activities of Gb,-bond protein ligands. 2.2 Rationale Globotnaosylcerarnide is a ce11 surface glycolipid, the terminal Ga1 (a 1-4) Ga1 residue of the carbohydrate moiety of which is specifically recognized by the binding subunit B of verotoxin. The extracellular domains of the B-ce11 specific antigen CD19 and the IFNARl chah of type 1 interferon receptor have high amino acid sequence similarity to the VT-B subunit, suggesting possible lateral association between these molecules and Gb, on the ce11 surface. The endogenous ligand for CD19 is still unknown but antibody crosslinking of CD1 9 causes the surface redistribution of CD19 to patches and caps. Gb, was found to CO-capwith CD 19, suggesting a physical association between two molecdes on the ceIl surface (Maloney and Lingwc~d., 1994). In addition, CD 19-positive Daudi Burkitt's lyrnphoma cells could specifically bind to Gb,-containing oligosaccharide matrices, suggesting that Gb, and CD19 may interact intercelldarly as well (Maloney and Lingwood, 1994). Functionally, antibody crosslinking-induced CD 19-mediated hornotypic adhesion of B-cells was found to be more significant in Gb,-positive Daudi cells, compared to Gb,-deficient Daudi mutant, VT500 cells (Maloney and Lingwood. 1994), suggesting that the structural association of Gb, with CD19 may rnodify the biological funçtions of CD19. Among the different stages of B-ce11 developrnent, co- expression of CD19 and Gb, occurs only during the germinai center stage. Gb, is a marker of GC B-cells, readily entering spontaneous apoptosis. CD19 can also induce apoptosis upon antibody crosslinking. Therefore, the role of Gb, in CDl9-mediated apoptosis has been examined in the present study. The fusion protein containing the extrocellular domain of IFNARI, linked to the Fc fragment of IgGl bound to terminai Ga1 (a 14) Gal- containing Gb, and Gb, on thin layer chromatography, indicating the possible physical association between e-IFNARI and Gb, on the ce11 surface (Ghislain et al., 1994). IFNa mediated growth inhibition activity dso was found to be significant only in Gb,-positive Daudi cells (Cohen et al., 1987 and Ghislain et al., 1994). Since two major biological actions of type 1 IFN; growth inhibition and anti viral activities, are not always associated (Ghislain et al., 1995 and Pfeffer et al., 1996), the possible role of Gb, in IFN-a mediated anti viral activity has been investigated in the present study. Afier binding to the receptor Gb,, VTI mainly enters the ce11 via clathrin- dependent receptor mediated endocytosis (Sandvig et al., 1989 and Khine and Lingwood, 1994) and undergoes retrograde transport to ER and nuclear membrane in a BFA- sensitive, Golgi dependent rnanner (Sandvig et al., 1991 and Donta et al., 1995). Recently, the internalization of VTl via clathrin-independent, caveolar-mediated endocytosis dso has been reported (Schapiro et al., 1998). The intracellular transport mechanism of the caveolar-mediated pathway can be either via a Golgi-dependent (Tm et al., 1987) or a Golgi-independent mechanïsm (Smart et al., 1994). The significance of these two pathways in VT1 intemalization and VT1-induced cytotoxicity has been examined in the present study. Both VTl and VT2 specifically bind to Gb, but the protective effect against VTI and VT2-induced cytotoxicity by the Golgi-disrupting agent, BFA was found to be different, suggesting possible differential intracellular transport mechanisms for the two closely related VTs. In the present study, the role of Golgi-dependent retrograde transport and Golgi-independent, caveolar-mediated transport in VTI and VT2 internalization and intracellular targeting have ken evaluated. 2.3 Hypothesis Association of Gb, with protein ligands on the ce11 surface facilitates the internalization and retrograde transport of Gb,-bound protein ligands, and Gb, functions as an accessory molecule for the signal transduction and biological activities of Gb,- bound protein ligands. 2.4 Specific aims ( 1> To study the role of Gb, in antibody crosslinked CD19 intemalization and CD 19-mediated apoptosis. (2) To examine the role of Gb, in IFN-cdtype I IFNR -mediated antiviral activity. (3) To evaluate the role of different intracellular transport pathways in VT 1 internalization and VT1 -induced cytotoxicity. (4) To compare the role of different intracellular transport pathways in internalization of VTl and VT2. Chapter 3 Functional role for Gb3 in antibody cross-liaked CD19 interoalization and apoptosis Abstract A region of the N-terminal extracellular domain of the B-ce11 restricted ce11 differentiation antigen, CD19, has high arnino acid sequence similarity to the receptor binding subunit B of VTI, which specifically binds to the ce11 surface glycolipid Gb,, Cocapping of antibody crosslinked CD 19 and Gb, provides evidence for the association of the two molecules on the Daudi Burkitt's lymphoma ce11 surface, and CD 19- mediated homotypic adhesion was shown to be Gb, dependent (Maloney and Lingwood, 1994)- The role of Gb, in antibody cross-linked CD19 internalization and CD19-induced apoptosis is now investigated. Initiai ce11 surface distribution, antibody-induced redistribution, intemalization and subsequent intracellular routing of CD19 were studied by imunofluorescence and postembedding irnrnunoelectronrnicroscopy in Gb,-positive and Gb,-negative cells. Daudi Burkitt's lymphoma cells were used as Gb,-positive cells, and as Gb,-negative celfs, Daudi mutant VT500 cells and Daudi cells treated with PPMP; an inhibitor of Gb, synthesis, were used. The antibody ligated CD 19 intemalization was found to be delayed in Gb,-aegative cells. Intemaiized CD19 was targeted to the nuclear envelope in Gb,-positive cells, in a manner similar to that reported for VT, but not in Gb,- negative cells. Induction of apoptosis following cross-linking of ce11 surface CD19 was found to be more significant in Gb,-positive cells than in Gb,-negative cells. VTlB subunit could inhibit intemalization of CD 19 and CD 19-mediated apoptosis in Gb,- positive cells. The nuclear targeting of intemalized CD 19 and induction of apoptosis following CD19 cross-linking only in Gb,-positive cells indicates a role for Gb,- dependent CD19 retrograde transport fiom the B ce11 surface via the ER to the nuclear envelope in CD 19-mediated signal transduction for apoptosis. Introduction CD 19 is a 95 kDa imrnunoglobulin superfamily integral membrane glycoprotein present on the ce11 surface of hurnan B lymphocytes fiom the early stage of B-ce11 development to the terminal differentiation to plasma ceils (Stites and Terr, 1991). On mature B-lymphocytes, CD1 9 forms a multimolecular signal transduction complex together with CD21, CD81 and Leu43 and hctions as a signal transduction subunit of the complex (Tedder and Isaacs, 1994). The structure of CD 19 consists of an extracellular region with two Ig-like domains, a single transmembrane domain and a large C-terminal intracellular tail (Zhou et al., 1991). The extended cytoplasmic domain of CD19 contains conserved tyrosine residues and serves as a signal transducer of the complex by tyrosine phosphorylation (Carter and Fearon, 1991 ). B-ce11 antigen receptor (BCR) is a membrane imrnunoglobulin (dg) which interndizes the antigen for subsequent presentation to T-cells and transduces antigen induced transmembrane signals leading to B-ce11 activation, anergy or deletion (Cambier et al., 1994 ). Since dghas only a 3 amino acid long cytoplasmic tail, associated Iga and IgP chahs function as signal transduction domains of BCR. The CD19 multi-molecular complex serves as a CO-receptorfor the signal transduction of BCR (Cambier et al., 1994). Depending on the type of ce11 surface CD 19 ligation, CD 19-mediated biological actions may Vary fiom growth inhibition, growth activation to induction of apoptosis (Rothstein, 1996). Although the endogenous ligand for CD19 has not ken identified yet, monoclonal antibody binding to CD19 cm induce CD19 intemalization and signal transduction events such as tyrosine kinase activation, PLC activation, increased intracellular [Ca-'] etc. Antibody ligation alone induces B-ceIl homotypic adhesion (Bradbury et al., 1993) and monoclonal antibody-induced signal transduction mechanism cm be fiuther enhanced by crosslinking of ceIl surface CD19 using both primary and secondary antibodies (Bradbury et al., 1993). Co-crosslinking of CD 19 and BCR synergistically reguiates B-ce11 activation and proliferation (Carter and Fearon, 1992). In contrast, hyper-crosslinking of CD19 or CD19 and BCR together may induce B-cell apoptosis (Chaouchi et ai., 1995). The N-terminal extracellular region of CD19 shows a striking amino acid sequence sirnilarity (4146% of VT18) with the verotoxin receptor binding subunit B. Disulfide linkage of intervening VT-dissimilar sequences brings the VT-like sequences into close proximity. In the membrane bilayer, the ceramide portion of Gb, is inserted into the outer leafiet of the bilayer while the catbohydrate moieties are extruding toward the outer environment (Gahmberg and Hakomori, 1973). VT-B binds to the terminal Ga1 a 1->4 Gal residue (Lingwood et al., 1987) of Gb3. Gb, is specificaily expressed only on Burkitt's lymphoma cells (Nudelman et al., 1983) and a subset of germinal center tonsillar B-lymphocytes (a normal counterpart of BL cells) (Gregory et al., 1987). Gb, has ken defined as the B-ce11 differentiation antigen CD77; an antigen of the germinal center (GC) B-cells prone to enter apoptosis (Mangeney et al., 1991). The arnino acid sequence of VT-B shared by CD19 lies within the glycolipid receptor binding cleft between subunit B monomers (Maloney and Lingwood, 1994, Lingwood, 1996), suggesting a potential association of CD19 and Gb, on the B-ce11 surface. Cocapping of antibody crosslinked CD 19 and Gb, has indicated the potential association between CD19 and Gb, (Maloney and Lingwood, 1994). In addition, anti-CD19 induced homotypic adhesion in Gb,- positive cells (Maloney and Lingwood, 1994) dso suggests the possible fùnctional relationship between CD19 and Gb,. In the endocytic pathway, receptor-ligand complexes taken up fiom the plasma membrane by clathrin coated vesicles are delivered to the acidic early endosomes where receptors and ligands are uncoupled (Smythe and Warren, 1991). Receptors may be recycled directly to the plasma membrane or via the tram-Golgi network (Pryer et al., 1992), and ligands are directed to late endosornes and ultimately degraded in lysosomes (Smythe and Warren, 1991). The catalytic subunit A of some subunit bactenal toxins translocates across the membranes of endosomal/lysosomal compartments into the cytosol (Montecucco et al., 1994). In contrast, some protein toxins including VT or VTB subunit can be transported to TGN (Mallard et ai., 1998) and undergo retrograde transport to RER and the nuclear membrane (Sandvig et al., 1994, Khine and Lingwood, 1994). Although the ER retention sequence (KDEL tetra peptide) bearing toxins such as CT, LT, ETA undergo retrograde transport to ER via KDELKDEL-receptor specific interaction (Montecucco, 1998), only Gb, bound VT and the VTlB subunit have been known to undergo retrograde transport to the ER and nuclear membrane without the presence of an ER retention sequence, suggesting the unique rote of Gb, in this process. Although a functional relationship between CD 19 and Gb, during a specific stage of B-cell developrnent is not yet established, the GC is the only stage at which CD19 and Gb, are coexpressed on B-cells. (Tedder and Isaacs, 1994 and Lindhout and deGroot, 1995). The GC is the site where mature B-cells which do not recognize the specific antigen undergo apoptosis (Liu et al., 1989). Since Gb, serves as a signal transducer for B-ceIl apoptosis (Mangeney et al., 1993), and CD19 ais0 mediates apoptosis after antibody crosslinking (Chaouchi et al., 1995 and Myers et al., 1995), CD19 and Gb, may be fùnctionally related during GC B-ce11 apoptosis. 3.3 Materials and Methods 3.3.1 Materials Chemicals: Glutaraldehyde, paraformaldehyde, osmium tetroxide, uranyl acetate and Epon 8 12 were from Polysciences inc. (Warrington, PA ). Sodium metaperiodate, gelatin from cold water fish skin, Triton X-100, ethidium bromide and RPMI were fiom Sigma (St. Louis, MO). Formaldehyde was fiom Fisher scientific (Pittsburgh, PA) and PPMP ( 1-p hen y 1-2-hexadecanoylamino-3-morpholino- 1-propanol) was from Matreya Inc. (Pleasant Gap, PA). Acridine orange was fiom Molecular Probes. Verotoxin: VTI and VTlB subunit were purified fiom the recombinant E.coZi strains pJLB 28 and pJLB121 respectively ( Petric et ai., 1987 and Ramotar et al., 1990). The purity of the toxins was checked by Tricine SDS-PAGE and the cytotoxicity of VT1 was tested on vero cells. Immunoreagents: Anti CD19 (clone Bq) (mouse monoclonal) was fiom Coulter Electronics (Hiaieah, FL). Cy-3 conjugated donkey anti mouse (DAM) anti body and anti FITC antibody were fkom Sigma. Unconjugated and 15 nm gold conjugated goat anti mouse (GAM) were fiom Zymed Laboratories Inc. (South San Francisco, CA). Cells and cell culture: Daudi Burkitt's lymphoma cells and Daudi mutant VT500 cells (Cohen et al., 1987) were maintained in RPME-1640 medium supplemented with 10% fetal calf senun, 10 mM HEPES, 1 mM sodium pyruvate, 1 mM sodium glutamate and 50 pg/ml Gentamycin, at 370C in 5% CO> Some Daudi cells were grown in the presence of 2.5 pM PPMP, for 6-10 days to deplete Gb3 synthesis (Inokuchi et al., 1987). PPMP was dissolved in 100% ethanol as a stock solution (25mM) and then as a working solution in PBS (250pM). 1: 100 dilution of 250 pM PPMP in PBS was added to the ce11 culture medium (2.5 FM, final concentration) (Rosenwald et ai., 1992). 3.3.2 Methods 3.3.2.1 Total glycosphingolipid extraction and thin layer chromatography 2x10' cells in suspension were pelleted by centrifugation at 800 rpm for 10 min and washed one more tirne with PBS (0.1M phosphate buffer saline, pH 7.4). The ce11 pellet was resuspended in 0.5 ml of PBS and the suspension was added to 9 ml (20 volume) of chlorofod methanol ( 2/l, v/v) . Total cellular lipid was extracted by shaking vigorousiy overnight. The extract was partitioned against 1.3 ml of PBS (chloroform/methanoVPBS,2: 1:0.6, v/v/v) and centrifuged at 2000 rpm for 10 min to separate into upper and lower phases. The lower phase was collected and dried under nitrogen. The dried sample was resuspended in 1 ml of chloroform/methanol (98/2, v/v) and passed ont0 the silica A mini colurnn. The colurnn loaded with the lower phase was washed with 3 volume of chloroform and glycosphingolipid was eluted with 10 volume of acetone/methanol (9/1, v/v). nie eluted fiaction was dried under nitrogen and resuspended in 0.1 to 0.5 ml chlorofonn/methanol (Zl, v/v) and separated on tlc by chlorofonn/methanoVwater (65/25/4 by volume). The separated GSL were visualized by orcinol spray (Lingwood et al., 1987). 3.3.2.2 VTI cytotoxicity assay 500 pl of 1 Sx104/ml ce11 suspension was added in 24 well ce11 culture plate and serial dilutions of VTl were added. The ce11 culture plates were Merincubated at 370C for another 48 hr. At the end of the incubation penod, the viable cells in each sample were counted by trypan blue exclusion method and the percentage of live cells was calcdated. 3.3.2.3 Immunofluorescence microscopy The absence of Gb, on PPMP-treated Daudi and VTSOO cells was verified by staining the cells with FITC labeled VTl-B subunit as described (Khine and Lingwood, 1994).To study the initial surface distribution and antibody induced internalization of CD 19, Daudi. PPMP-treated Daudi and VT500 cells (5x105 cells in 1.5 ml eppendorf tubes) were incubated on ice with 10 pg/ml (1 00 pl in 1% BSA in PBS) anti CD 19 primq antibody for 30 minutes and unbound antibody was washed with 1 ml of cold PBS. Then, cells were labeled with Cy-3 conjugated DAM secondary antibody (100 pl in 1: 100 in 1% BSA in PBS) for 30 minutes at 40C for initial surface iabeling or for 30 to 150 minutes at 370C for intemalization (Maloney and Lingwood, 1994). As a control, celts were incubated with mouse IgG instead of CD19 primary antibody, followed by the secondary antibody. After washing and fixing the cells in 4% paraformaldehyde in PBS for 30 minutes at room temperature, the pellet was resuspended in the mounting medium (DAKO) and mounted on the slides. The preparations were exarnined and photographed under incident UV illumination using Polyvar fluorescence microscope on EL 135, 400 ASA film at magnification xlOO or by confocal microscopy at magnification x120. 3.3.2.4 Preparation of cells for electron microscopy To study the subcellular targeting of intemalized CD19, 2x1 o6 cells were used for each EM preparation. The cells were treated with primary antibody at 40C as described above. Afier washing with cold PBS the cells were incubated at 370C for 3 hr with GAM secondary antibody and fixed. The volume of the incubation medium was 1 ml at al1 steps. To induce apoptosis, cells were incubated in the presence or absence of 10 pghl anti CD19 and 30 &ml unconjugated GAM antibodies (as described above) at 370C for 24 hr. Fixation: One ml of 2.5% glutaraldehyde: 2% pdormaldehyde in TBS was directly added to the washed ce11 pellet for fixation at room temperature for 30 minutes. Afier washing with 1.5 ml of PBS, the pellets were postfixed in 1% osmium tetroxide in phosphate buffer for 30 minutes and washed with sterile water, Then, the pellets were further postfixed with 2% uranyl acetate in sterile water for 15 minutes (Pulczynski et al., 1993). Dehydration: The celis were dehydrated in 50% to 100% serial ethanol, 10 minutes each for two times. Embedding: Dehydrated cells were serially infiltrated with 50% to 75% Epon in 100% ethanol for 30 minutes each and finally with 100% Epon for 1 hr with 2 changes. The pellet was transferred into the gelatin capsule filled with 100% Epon and polymerized at 650C. 3.3.2.5 Immuno-gold Iabelling of the sections Afier sectioning of the polymerized blocks using an ultramicrotome, the sections for the study of internalization were immunogold labeled. The sections were treated with saturated sodium meta-periodate for 30 minutes to unmask antigenicity by de-osmication (Stirling and Graff, 1995) and washed in distilled water. Then, the sections were blocked by floating on a &op of O. 1% BSA, 0.2% fish skin gelatin in 50 mM TBS for 30 minutes and immunolabeled with 150 dilution of anti CD19 primary antibody followed by 150 dilution of GAM-15 nm gold in the blocking buffer for 1 hr each at room temperature with thorough washing in dH,O at each step. As controls, some sections were treated with unrelated primary antibody (anti-FITC antibody) followed by GAM-gold secondary antibody conjugate or with secondary antibody alone. Finally, the sections were stained with 5% uranyl acetate and then with Reynold's lead citrate for 6 minutes each and anaiyzed with Hitachi EM 600 at 75kV. The photographs were taken with Eastman fine grain release positive film and pnnted on Polycontrast III RC at 59400 fold final magnification for the internalization study and 18000 fold magnification for the apoptosis study. 3.3.2.6 Cytochemical staining of apoptotic celis To examine the morphological changes in the nuclear chromatin, cells undergoing apoptosis were identified by acridine orangdethidium bromide staining of nuciei (Vasconcelos et al ., 1994). Mer incubation in the presence or absence of anti CD 19 and GAM for 21 hr at 370C, cells were pelleted. Afier washing with PBS, cells were resuspended in the nuclear stain (100 pghl acridine orange and 100 &ml ethidium bromide) and examined under UV illumination as descnbed above. Cells with condensed chromatin and apoptotic Mies were scored as apoptotic cells. A minimum of 200 cells were counted for each experiment. 3.4.1 Gb, affects the redistribution and internalization of antibody crosslinked CD19 Gb, is by far the major neutmi glycolipid in Daudi cells (Cohen et al., 1987 ). In order to study the possible influence of Gb, in CD19 internalization, the initial surface distribution and antibody crosslinked redistribution of CD19 was investigated in Gb3- posotive and Gb3-negative cells- As Gb,-positive celis, Daudi Burkitt's lyrnphoma cells were used and as Gb,-negative cells, VTSOO and PPMP-treated Daudi cells were used. VTSOO cells are Daudi mutant, Gb, deficient, VT resistant cells. The synthesis of Gb, in Daudi wild type cells was also inhibited by a cerarnide analogue, PPMP which inhibits -ducosyl cerarnide synthesis and thereby depletes glycolipids (Inokuchi et al., 1987). The total expression of Gb, was exarnined by total GSL extraction of the cells, followed by detection with orcinol spray (Figure.18). Deficient expression of Gb, was observed in VT500 (aImost 100% reduction) and PPMP-treated Daudi cells (-90% reduction), compared to wild type Daudi. As a hctional assay, VT1 cytotoxicity on Gb,-positive and Gb,-negative cells was performed. While VT1 induced ce11 death in Daudi cells, Gb, deficient cells were resistant to VT1-induced cytotoxicity (Figure. 19). Then, the surface expression of CD19 on Gb,-positive and Gb,-negative cel1s was investigated. Daudi, VTSOO and PPMP-treated Daudi cells were labeled with anti CD 19 primary antibody followed by Cy-3-DAM secondary antibody for indirect immunofluoresence, at 40C for initiai surface CD19 distribution. In Daudi cells, CD19 was found on the ce11 surface with some intervening-nonfluorescent areas (Figure. 20,a) as has been previously reported (Pulczynski et al., 1993) (Maloney and Lingwood, 1994). Comparable levels of CD 19 surface expression were observed in both Gb,-positive and Gb,-negative ce11 lines (Figure. 20,a,b and c). Antibody crosslinking-induced CD19 redistribution was Mer investigated by incubating the antibody-crosslinked-cells at 370C for 2.5 hr and examined by fluorescence microscopy. The crosslinked CD19 was Std Daudi VT500 PPMP-Daudi Figure. 18 Thin layer chromatography of total GSL s.xîracîs from Daudi. C'T500 and PPMP-Daudi. The extract from 1slO(7) of cells was loaded in each lane. 1 Daudi VTSOO PPM P-Daudi Figure. 19 VTl cytotoxicity assay on Daudi, VT-500 adPPMP- Daudi. Figure. 20 Surface expression of CD1 9 on Daudi, VT5ûû and PPMP-Daudi. CD19 indirect immunofluorescence a t M,examined by confocal laser scanning microscopy. Daudi(a),VT'T5M)(b) and PPMP-Daudi(c). As a control, mouse IgG was used instead of anti-CD19(data not shown). found as patches and caps or as a single fluorescent area at the center of the ce11 (Figure. 21), which was confirmed as intemalized CD1 9 by confocal microscopy (Figure.22). Intemalization of CD19 was more significant in Gb,-positive Daudi cells than in Gb,- negative cells. In VT500 and PPMP treated Daudi cells, although internalized CD19 was present in some population of cells, patches and caps were still present (Figure. 2 1 ,b and c), indicating a delay in CD19 intemalization, and an involvement of Gb3 in the surface redistribution and intemalization of antibody crosslinked CD19. The delay in CD 19 intemalization was Merconflrmed by tirne course observation of crossluiked CD19 internalization at 370C for 30 to 150 minutes by both conventional fluorescence and confocal microscopy (Figure.23). CD19 internalization was found as early as 30 minutes in Gb,-positive cells and the population of cells with intemaiized CD19 increased significantly at every time point. In Gb,-negative cells, the delayed internalization was observed at later time points. To identi@ the requirement of Gb, for CD19 intemalization, the effect of FITC labeled VTl B CO-incubationon CD 19 intemalization was examined at 370C incubation for 2.5 hr. The intemalization of CD19 was markedly reduced in the presence of FITC VTlB CO-incubation andor internalization (Figure. 24). The utilization of surface available Gb3 by Gb3-VT1 B intemalization delayed the antibody crosslinked-CD 19 redistribution and intemalization, supponing the idea that G4 was necessary for CD19 internalization. 3.4.2 Gb, directs the retrograde transport of cell surface CD19 to the nuclear membrane it has been reported that CD19 was intemalized der antibody crosslinking by receptor mediated endocytosis in other Burkitt's lymphoma ce11 lines (Pulczynski et al., 1993). We studied the intracellular localization of CD19 in Daudi, VT500 and PPMP treated Daudi cells following ce11 surface CD19 ligation, by postembedding IEM. The Figure.2 1 Surface redistribution of antibody crosslinked CD 19 on Daudi, VT500 and PPMP-Daudi. CD 19 indirect i mmunofluorescence at 370C for 2.5 hr anti body crosslinking, examined by conventional fluorescence microscopy. Daudi(a), VTSûû(b) and PPMP-Daudi(c). Arrows indicate the cells with intemdized CD19. Daudi VTSOO PPMP-Daudi Figure. 22 Ln temalization of antibody crosslinked CD1 9 on Daudi, VT5ûû and PPMP-Daudi. CD1 9 indirect immunofluorescence at 370C for O to 150 min antibody crosslinking, esamineci by confocal laser scanning microscopy.Daudi(a,d), VT500(b,e) and PPMP-Daudi(c,f). O 30 60 90 120 150 Time (minutes) Figure. 23 Quantitation of intemaiized antibody crosslinked CD1 9 on Daudi, VT500 and PPMP-Daudi. (n = 4) Figure 24 Effec t of VT1B conincuba tion/ internaliza tion on antibody crossiînked CD1 9 intemalization @audi celis). Surface ezcpression(a) and intemalized(b) CD 1 Sin the absence Ot FITC-VTlB conincubation and coincubation of FITC-VTlB(c) and antibody aosslinked CD19 (d) double labeling, at 370C for 2.5 hr. Figure. 25 Intracellular targeting of internalized CD19 on Dau4 VT50 and PPMP-Daudi cells. The cells were antibody crosslinked at 370C for 3hr, processed and examined by immuno electron microscopy. Control Daudi(A), antibody crosslinked Daudi(B), VTSOO(C) and PPMP-Daudi(D). Nu;nucleus, C; cytoplasm, NM;nudear membrane, RER; rough endoplasrnic re ticulum cells were incubated with crosslinked anti CD19 for 3 hr at 370C and processed for EM. The target of CD19 intemaiized at 370C was found to be different in Gb,-positive and Gb,-negative cells. In Daudi cells, intracelluar CD19 was found mainly in ER and the nuclear membrane (Figure.25,B) but such labeling was not detected in control Daudi cells not treated with anti CD19 (Figure.25,A). In Gb3-negative cells, although CD19 was found in endosorne- and lysosome-like membranous structures, there was no labeling in the nuclear membrane (Figure. 25,C and D). The ER and nuclear membrane targeting of CD 19. particularly in Gb,-positive cells, indicates that similar to Gb, mediated retrograde transport of VT. internalized CD19 also could undergo retrograde transport to ER and nuclear membrane in Gb, dependent manner, without the requirement of ER retention signal KDEL. This indicates a unique role of Gb, in the retrograde transport of Gb,-bound ligands. 3.4.3 Functional relatiooship between Gb, and CD19 for induction of apoptosis Gb,-positive and Gb3-negative Daudi cells were incubated with crosslinked anti- CD19 at 370C for 24 hr and cellular morphology was compared by EM. Afier 24 hr incubation of the cells with anti-CD19 and GAM antibodies at 37OC, morphological characteristics of apoptosis were found in Daudi cells. In control Daudi cells, normal morphology of the cells, such as microvilli on the plasma membrane, diffuse chromatin distribution in the nucIeus, well defined cytopiasmic ultrastructure etc was found (Figure. 26.A). In contrast, morphology of apoptotic cells such as loss of microvilli, condensation and rnargination of chromatin in the nucleus, nuclear fragmentation and apoptotic body formation was observed in anti-CD19 treated Daudi cells. This was later followed by secondary necrosis and loss of cytoplasm (Figure. 26,B). On the other hand, much les extensive apoptosis was present in Gb,-negative cells (Figure. 26, C & D), indicating the significance of Gb, expression for CD 19-mediated B-ce11 apoptosis. CD19 mediated apoptosis was quantitated by cytochemical naining of the cells with the nuclear stain, acridine orange and ethidium bromide solution, after 24 hr treatrnent of the ceils with crosslinked anthdies. Membrane permeable acridine orange stained green for the nuclei of live cells and ethidium bromide stained red for dead ce11 nuclei when the membrane integrity was no longer intact. Either in liveldying or dead cells, the cells with chromatin condensation and apoptotic body formation were scored as apoptotic cells. By this criterion 30% of Daudi cells were apoptotic while only 10% and 12% of VTSOO and PPMP-treated Daudi cells respectively undement apoptosis following surface iigation of CD19 (Figure. 27). To confi the role of Gb, in this process, the effect of surface Gb, utilization by VTlB binding and/or internalization on CD19-mediated apoptosis was investigated. VTl B alone can induce apoptosis in some ce11 lines and Daudi cells are particularly sensitive to VTlB induced apoptosis but at relatively higher dose (1 pg/rnl), while VTl could induce apoptosis at 1 ng/ml (Mangeney ey al., 1993). Treatment of Daudi celis with 50 ng/ml of VTlB induced minimal apoptosis (Figure. 28) but CO-incubation of the cells with VTlB effectively prevented antibody crosslinked CD19-mediated apoptosis (Figure. 28). Figure. 26 Antibody crosslinked CD19 induced apop tosis in Daudi VT500 and PPMP-Daudi. The cells were antibody aosslulked at 370C for 24 hr, processed and examined by electron microscopy. Control Daudi(A), antibody crosslinked Daudi(B), VTWC) and PPMP-Daudi(D). 1 crosslinked CD19 1 Daudi VTSOO PPMP-Daudi Figure. 27 Quantitation of antibody crosslinked CD 19 induced apoptosis. (n= 4) conîrol VT-B alone crosslinked CD19 Figure. 28 Effect of VT 1 B subunit CO-incubation/intemaiizationon CD 19-mediated apoptosis. (n = 4) 3.5 Discussion CD19 is a B-ce11 restricted ce11 surface antigen, expressed fkom the pro-B ce11 to mature B-cell stage (Stites and Ten; 1991) (Figure. 29). Although the endogenous ligand for CD1 9 is still unknown, the crosslinking of surface CD 19 with monoclonal antibody to CD1 9 and secondary antibody induces CD19 intemdization (Pulczynski et al., 1993). In addition, engagement with monoclonal anti-CD 19 alone or crosslinking of CD19 also triggers a series of signal transduction cascades leading to increased tyrosine phosphorylation, inositol phospholipid metabolkm and [cafZ]i mobilization throughout B-ce11 ontogeny (Carter, 1991, Uckun et al., 1993, Tedder and Isaacs, 1994). The ability of CD1 9 to undergo internalization and to transmit signals indicates that CD19 could be an important receptor for B-ce11 development. The signaling ability exists independently, before the expression of other associated ce11 surface antigens and later synergizes with the signa1 transduction of other antigens after their expression ( Uckun et al., 1993). This indicates that CD 19 is a signal transducer molecule, which is stnicturally and functionally associated with other B-ce11 surface antigens at different specific stages of B-ce11 development. Glycolipid biosynthesis also varies as a fûnction of B ce11 differentiation (Wiels et al., 199 1, Taga et al., 1995) but in contrast to CD19, Gb, is expressed at only one specific stage of B-ce11 development, that is on a subset of germinal center B-cells (Figure 29) (Mangeney et al., 1991). The amino acid sequence similarity between CD19 and the Gb,-binding VTI-B subunit and cocapping of CD19 with Gb, strongly suggest a structural relationship between these two ce11 swface antigens (Maloney and Lingwood, 1994). Since CD 19 fùnctions as a member of a multi-molecular complex, Gb, may serve as one of the components of the CD19-containing multimolecular signal transduction complex (Fearon et al., 1995), at least dwing the GC B-ce11 stage. Following the demonstration of the cocapping of Gb, and CD19 after antibody crosslinking of CD19 (Maloney and Lingwood, 1994), the role of Gb, in antibody crosslinked CD19 redistribution and intemalization was Merinvestigated in this study. As Gb,-negative Figure. 29 Stages of B-lymphocyte development cells, the Gb, deficient Daudi mutant ce11 line VT500 and chemically induced Gb, deficient Daudi cells were used.Gb, synthesis of wild type Daudi cells was inhibited by the cornpetitive inhibition of GSL synthesis with a ceramide analogue, PPMP (Inokuchi et al., 1987). PPMP inhibits the enzyme UDP-G1c:cerarnide glucosyl transferase, which catalyzes the transfer of glucosyl residue to the ceramide, to produce glucosylcerarnide, which is the precursor of most GSL, including Gb, (Abe et al., 1992). Immunofluorescence labeling of CD19 on wild type Daudi and VTSOO showed an equal level of ce11 surface CD19 expression (Figure. 20,a and b). The slightly more intense and more uniform CD19 labeling on PPMP-Daudi ceIl surface (Figure, 20, c) indicated that CD19 surface expression rnight be influenced by Gb, and some unknown minor glycolipid species other than Gb,, the syntheses of which dso were inhibited by PPMP. In addition to Gb,, anotther Gal al4Ga1 containing glycolipid Gb, has been found to be potentially associated with the extracellular domain of IFNARI, which shares the same VT1-B like sequence as CD19. Gb2 synthesis cm also be blocked by PPMP (Ghislain et al.. 1994)- After initial binding of a ligand to its receptor, the ligand-receptor complex is redistributed to patches by energy independent spontaneous crosslinking (Rosen, 1979), followed by energy dependent intracellular microfilament-assisted capping of the complex at one pole of the cell. After capping, the complex undergoes either internalization or shedding from the surface (Rosen, 1979). The clustenng of ligand- receptor complex is mediated by a family of cytoplasmic proteins known as adaptors (AP), which bind to the cytoplasmic internalization signal motif of the receptor and recniit cytoplasmic clathrin proteins to the plasma membrane (Brown and Greene, 1991, Robinson, 1994). At physiological temperature, the eukaryotic ce11 membrane cm be regarded as a continuously flowing two dimensional lipid sea, in which integral membrane proteins are free to diffuse independently (Rosen, 1979). It has been hypothesized that lipid molecules are inserted into the plasma membrane fiom the cytoplasm at one site and are resorbed back to the cytoplasm at a different site, allowing a continuous flow. When the ceIl surface antigens or receptors are cross-linked fiom outside of the cells, the conformational change of the antigens or receptors at the cytoplasmic end is believed to be recognized by cytoplasmic adaptors and these molecules are selectively aggregated into patches. When the ligandkeceptor complex is large enough, the rate of lateral diffusion of the complex may not overcome the lipid flow, and the complex drifts dong the Iipid flow to one pole of the cell, where the lipid molecules could be resorbed back to the cytoplasrn (Bretscher, 1976). The delayed kinetics in surface redistribution and internalization of crosslinked CD19 in Gb,-negative cells compared to the internalization of CD19 in Gb,-positive wild type Daudi cells (Figure.21 to 24) indicates the influence of Gb, in the surface redistribution and endocytosis of CD 19. Physical association of the integral membrane proteins with lipid molecules may facilitate the kinetics of antibody-crosslinked ce11 surface CD19 redistribution. In eukaryotic cells, both the biosynthetic secretory pathway and the endocytic pathway utilize vesicular traffic. In the endocytic pathway, the transported molecules fiom the ce11 surface are uncoupled fiom the receptors in the endosomes and degraded in lysosomes (Pryer et al., 1992 ). Some protein toxins enter the ce11 by such mechanism and the enzymatic subunit dissociates fiom the binding subunit and translocates to the cytosol from endosomaUlysosomal compartrnents (Montecucco et al., 1994). The cytotoxicity of such toxins can be protected by dmgs which interfere with the maintenance of endosomal pH (Schapiro et al., 1998). However, some other toxins (such as CT, LT, ST and VT) escape from the endosomal compartment and enter the TGN via AP1-clathrin coated vesicles (Mallard et al., 1998), and undergo retrograde transport to Golgi cisternae and ER (Montecucco et al., 1994). TGN localization and cycling of proteins between TGN and endosomal compartrnents are mediated by targeting signals present in the cytoplasmic domain of the proteins (Rothman and Wieland, 1996). APl -clathrin coated vesicles recognize membrane proteins with cytoplasmic tyrosine rich dileucine signal or lumenal proteins, associated with such cytoplasmic signal-bearing integral membrane proteins. The mechanism of how protein toxins escape fiom endosomal compartment and enters TGN has not been defined. The association of toxin or toxin receptor with some unknown adaptors may facilitate the transport of the toxin to the TGN. The mechanism of retrograde transport of internaiized toxin beyond the TGN aiso has not been well characterized. In the biosynthetic pathway, proteins synthesized in ER are transported to the cis-Golgi for Merprocessing. Sorting of secretory proteins and lysosomal proteins takes place in the tram-Golgi (Griffiths and Simons, 1986). However, Golgi and ER resident proteins are retained at their restricted locations by specitic signais (Pelham and Munro, 1993, Munro, 1995). Golgi resident proteins are believed to be retained by transmembrane domain-mediated retention (Munro, 1995, Gleeson et al., 1994) or by formation of large hetero-oligomeric complexes by 'kin recognition' (Nilsson et al., 1993). Some ER resident proteins require Mer processing such as oligosaccharide modification. Such proteins are transported to Golgi compartments where the enzymes necessary for the protein modification reside but are reeieved fiom the secretory pathway and retumed to ER. Two types of ER retrieval signals have ken identified; a short KDEL motif (Munro and Pelham, 1987) and a KKXX motif (Jackson et al., 1990) at the C-terminus of the ER proteins. KDEL containing proteins are recognized and transported back to the ER by KDEL receptors on pst-ER compartrnents (Pelharn, 1991) (Jackson et ai., 1990). The mechanism of KKXX containing protein retrieval is less understood, although interaction between the KKXX motif and the coatomer; a polypeptide complex present in Golgi derived coated vesicles, has been proposed as the mechanism of KKXX retrieval (Cosson and Letourneur, 1994). The cholera toxin receptor, GM, ganglioside has been identified in the endocytic organelles and the tram-Golgi network (Parton, 1994). Both A and B subunits of CT could reach Golgi-like structures afier intemalization. However, only the subunit A, which contains the KDEL sequence undergoes retrograde transport to the ER while receptor the binding subunit B is translocated to the lysosomes (Majoul et al. 1996). Other toxins such as E-coli heat-labile toxin and Pseudomonas aeruginosa exotoxin A also contain a KDEL or a KDEL-like sequence for the retrograde transpon (Majoul et al. 1996 and Chaudhary et ai., 1990). Consequently, such protein toxins or their subunits may undergo retrograde transport to earlier compartments of Golgi and ER via KDE L/KDEL-receptor mediated transport. However, although Gb,-bound ligands have been reported to undergo retrograde transport to the ER and nuclear membrane (Sandvig et al., 1994 and Khine and Lingwood, 1994), the receptor binding subunits of VTl do not possess such signals and thus Gb, binding itself rnay be responsible for retrograde transport of Gbj-bound ligands to the ER and nuclear membrane. Both KDEL-containing VT-B subunit and non-iùnctional KDEL sequence (KDELGL)-containing VT-B subunit have been found to undergo retrograde transport to the ER, indicating that the KDEL motif is not necessary for the retrograde transport of VT/ST (Johannes et al., 1997). CD 19 also was internalized primaril y to the RER and nuclear membrane on1y in Gb,-positive cells (Figure. 25). Antibody-ligated CD19 is internalized by RME in other Gb,-positive ce11 lines such as MM-6and RklI (Pulczynski et al., 1993). In these studies, the internalization of gold-conjugated, antibody-labeled CD19 was followed by pre-embedding EM, and ultimate accumulation of gold-labeled CD19 was observed in lysosomes but not in the Golgi cistemae. This method could not determine whether gold particles accumulated in lysosomes were still associated with CD19 or not. The internalization of antibody crosslinked CD 19 could also be limited due to the bulky size of the crosslinked CD19 complex to which the gold particle (12 nm) was attached via the secondary antibody. In the present studies using pst embedding IEM, we have been unable to retain optimal Golgi morphology and hence cmot defuie whether CD19 transits the Golgi enroute to the ERhuclear membrane as is implied for the retrograde transport of verotoxin (Sandvig et al, 1994) (Sandvig et ai, 1992). However, labeling of internalized CD 19 in ER and nuclear membrane was observed only in Gb,-positive Daudi cells (Figure. 25). Due to the difficulty to preserve Iipid antigens for IEM processing, which could extract lipid from the ce11 during ethanolic dehydration, CO-localization of VTl and Gb, could not be confïrmed by double labeling IEM. Consequently, whether subunit B or Gb, is the responsible factor for the retrograde transport has not yet been clarified. However, in this study, the evidence for the retrograde transport of another potential Gb,-bound ligand; CD19, to ER and nuclear membrane mersupports the role of Gb, in the retrograde transport of Gb,-bound ligands. Thus by iderence, Gb, is responsible for the retrograde transport of VT from the plasma membrane to the GolgUER and nuclear membrane. Nuclear targeting of intemalized VT by retrograde transport is of primary importance in determining ce11 toxin sensitivity (Arab and Lingwood, 1998). Toxin targeting of the nuclear envelope by B the subunit or the holotoxin may result in the induction of apoptosis (Arab et al., 1998). Previous studies indicate that such targeting may be dependent on the Gb, fatty acid content (Sandvig et al., 1994) (Arab and Lingwood, 1998) and a mode1 to explain such Gb, sorting, based on the reduced dimensions of the GolgiER membranes has been proposed (Lingwood, 1996). Since the thickness of organelle membrane becomes thimer fiom the plasma membrane to the GolgiER, glycolipids with shorter fatty acid chains could be more easily accommodated in the membrane of organelles with smaller dimensions, such as the ER. An alternative concept is that the thickness of the bilayer membrane tends to decrease with vesicle formation and short chah lipids present on one side of the membrane can better accommodate the reduced membrane thickness required to induce invagination of the vesicle (Lingwood, 1999). Regarding verotoxin cytotoxicity, preferential targeting of VT to the ER, nuclear membrane and nucleus via shorter fatty acid containhg Gb, isoforms was found to be associated with induction of apoptosis (Arab and Lingwood, 1998). The finding of more significant CD19-mediated apoptosis in Gb,-positive ce11 may also be due to targeting of intemaiized CD19 to ER, nuclear membrane and nucleus. Since the lumen of the ER is continuous with the lumen of the nuclear membrane (Goldberg and Allen, 1995), toxins entering the ER could eventually be localized in the nuclear membrane as well. However, the mechanism of VT transport into the nucleus has not been understood yet. The nuc1ea.r membrane is an intenupted membrane with ever- open nuclear pores of -9nrn in diameter (Pante and Aebi, 1996). Small molecules can passively diffùse through the nuclear pore but larger molecules are selectively and act ively transported by a signal-mediated and ATP-dependent mechanism. Proteins smaller than 15 kDa can enter the pore without retardation, 67 kDa globular protein (BSA) cmslowly enter the pore and 450 kDa ferritin is totally excluded (Kalderon et al., 1984). The proteins with nuclear localization signals (NLS) bind to the cytosolic NLS- receptor/importin-a with the aid of the 97 kDa accessory protein, importin-B/p97. The whole complex binds to the nuclear pore and the small GTPase, WC4facilitates the translocation of the complex into the nucleus (Powers and Forbes, 1994). NLS sequences are either short basic sequences of 4 to 7 amino acids or longer bipartite sequences containing two stretches of basic amino acids separated by ten less conserved arnino acids (Powers and Forbes, 1994). Since the molecular weight of VT holotoxin is -70 kDa and the pentameric B subunit is -35 kDa, after the membrane translocation to the cytoplasm as a fiee toxin, the molecule may passively diffuse through the nuclear pore or may be selectively transported across the pore after binding to unknown NLS-bearing cytosolic proteins. On the other hand, the cytoplasmic domain of CD19 has a stretch of potential NLS sequence RIüCRKR (Tedder and Isaacs, 1989). However, the mechanism by which the integral membrane could be extracted to the cytosol before entry to the nucleus via the nuclear pore is still uncertain. During the development of B-cells, pre-B lymphocytes arise fiom bone marrow stem cells and contain cytoplasmic p heavy chains oniy. At the immature-B cell stage, IgM is assembled and expressed on the ce11 surface, and immature-8 cells migrate out of the bone marrow to the peripheraf circulation. Even in the absence of antigenic stimulation, the cells continue to develop into mature-B cells, on which both IgM and IgD are expressed (Abbas et al., 1994). Upon antigenic stimulation, intracellular signal transduction events take place and mature43 cells become activated B-cells. At the same time, protein antigens are internalized and processed by B-cells and presented to antigen- specific T cells. The physical contact between B and T-helper cells, via CD40KD40- ligand interaction, and T-cell-derived cytokines such as IL-2, IL-4 and IL-5, initiate the proliferation of B-cells (Abbas et al., 1994). During immune responses, some of the foci of B-lymphocytes develop into germinal centers in the follicles of secondary (peripheral) lymphoid organs and more activated B-cells migrate into GC (Figure. 30). The development of GC is a T-ce11 dependent process, triggered by B cell-T ce11 interaction. The major constituents of GC are activated B-cells, macrophages, T lymphocytes and follicular dendritic cells. FDCs are large non-lymphoid cells with elongated cytoplasrnic extensions that form the framework of the GC. FDCs take up antigens as antigen-antibody complexes or antigen- DARKWNE kliaLr dewmk cdb ebc) aa- --FDC T- T- - Lir-iiu c-#@x ammm of-- Figure. 30 The fate of B-lymphocytes in germinal center complement complexes and present them in immunogenic form for long pend of time (Lindhout and de Groot, 1995). Inside the GC, B-cells undergo rapid proliferation and somatic hypermutation at the Ig V genes. As a result, different V genes are produced and different proliferating-B- ce11 cIones expressing a broad spectnim of variant antibody molecules are generated (Lindhout and de Groot, 1995). However, upon interaction of the somatically hypermutated cells with the antigen displayed on FDCs, only the clone of B-cells, whose membrane Ig molecules are able to recognize the antigen with high afinity, is selected to survive by affurity maturation. Other unwanted clones of B-cells, which could not recognize the specific antigen die by apoptosis in the germinal center (Liu et al., 1989). Some of the afinity matured B-cells that exit GC develop into antibody-producing plasma cells and other high-afinity B-cells develop into memory B-cells, which may live for months or years without overt antigenic stimulation (Abbas et al., 1994). The germinal center B-ce11 stage is the only developmental stage of B-cells where both CD19 and Gb3/CD77 are CO-expressed(Figure. 29) and FDCs are the only type of cells in the immune system where CD19 and Gb, are expressed, other than B- lymphocytes. Therefore, if there is a functional relationship between CD19 and Gb,, it must take place in the GC, Binding of the GC B-cells to FDC is mainly mediated by the interactions between adhesion molecules, ICAM-I and VCAM-1 on the FDC and LFA-1 and VLA-4, respectively, on the B-cells (Koopman et ai., 1994). Isolated GC B-cells degenerate rapidly by apoptosis but can be rescued by crosslinking the B-ce11 antigen receptor with immobilized antibodies (Liu et al., 1989) or by crosslinking LFA- 1NLA-4 by immobilized ICAM-INCAM-1 (Koopman et al., 1994). There are other rescue mechanisms of FDC/GC B-ce11 interaction, such as interaction between CD40-ligand and CD23 on the FDC and CD40 and CD2 1, respectively on the GC B-cells (Foy et al., 1994, Liu et al., 1991 and Bonnefoy et al., 1993). The homotypic adhesion of CD 19-positive B- cells is found to be Gb, dependent (Maloney and Lingwood, 1994). In addition, specific binding of CD 19-positive Daudi Burkitt's lymphoma cells to Gb, containing oligosaccharide matrix indicates the potential CD19/Gb3 mediated cell-to-ce11 interaction, other than B-ce11 homotypic adhesion (Maloney and Lingwood, 1994). As VTl B binding to Gb,-positive Daudi cells can rescue CD 19-mediated apoptosis (Figure. 28), CD 19/Gb3 on FDC may also rescue GC B-cells fiom CD19/Gb,-mediated apoptosis. Therefore, specific interaction between CD19 and Gb, on the GC B-cells and FDC, followed by hornotypic adhesion of B-cells, could be the mechanism of B-cell homing to the germinal center or another rescue mechanism of affinity mature B-cells. On the other hand, GC is the place where massive apoptosis of somatically hypermutated B-cells, which are not selected for affinity maturation, takes place. Although the endogenous ligand for CD19 has not been identified yet, CD19 could mediate apoptosis in Burkitt's lymphoma ce11 lines (GC B-cells are the normal counter part of Burkitt's lyrnphoma cells) after antibody crosslinking (Chaouchi et al., 1995). Similarly, anti-Gb,, immobilized on the tissue culture dishes can also induce apoptosis of Burkitt's lymphoma ceIl lines, indicating another possible fùnctional relationship between CD19 and Gb, for GC B-ce11 apoptosis. However, in vitro, isolated GC B-cells spontaneously and rapidly undergo apoptosis (Liu et al.. 1989). Gb,-positive GC B-cells can be rescued from apoptosis in vitro in the presence of anti-CD40 monoclonal antibody and IL-4 (Manganey et al., 1995) and such cells no longer express Gb,. Similarly, antibody hyper-crosslinking or immobilized antibody crosslinking of mIg (BCR) can induce apoptosis of Burkitt's lyrnphoma ce11 lines (Chaouchi et al., 1995), but may also rescue apoptosis of isolated GC B-cells by immobilized antibody cross-linking (Jiu et al., 1989). In the present study, CD19 mediated apoptosis was found to be more efficient in Gb,-positive cells than in Gb,-negative cells (Figure 26 and 27), indicating a role for Gb, in CD1 9-mediated apoptosis (Chaouchi et al., 1995). However, unlike immortalized GC B-ce11 denved ce11 lines, fieshly isolated GC B-cells undergo spontaneous apoptosis and thus whether CD19/Gb3 interaction in GC is responsible for the GC B-ce11 apoptosis or the rescue of GC B-cells fkom entering apoptosis, is still not conclusive. Since CD19 and Gb, could be either laterally interacting on the B-ce11 or interacting between GC B-ce11 and FDCs, differential interaction depending on the type of CD19 ligation could be another factor determining GC B-cell survival or death. Lateral interaction of CD19 and Gb, may facilitate unknown CD 1 9-ligand induced GC B-ce11 apoptosis and intercellular interaction between CD19 and Gb, may be either the apoptotic signal itself or the rescue signal. One molecule could be the endogenous ligand for the other or the signal transduction events, mediated by one molecule couid be enhanced by the other and double crosslinking of both molecules may act synergistically. However, since CD19 itself is a member of a multimolecular signal transduction complex, apoptosis or rescue mediated by other associated molecules could not be excluded. CD21, which is a member of CD19 complex, is known to participate in the rescue of GC B-cells, via interaction with CD23 on the FDCs. Since CD19 is the signal transduction subunit of CD2 1, failure of CD 19 involvement in CD2 1omediated rescue mechanism, due to a stronger intercelular interaction between CD19 on the B-ce1 and Gb, on the FDC may also determine the fate of GC B-celts to undergo apoptosis. Similarly, since interaction of mIg and antigen presented on FDC is the resuce mechanism of affinity mature B-cells and CD19 is the CO-receptor of dg, the cornpetition between CDl9/mIg and CD19/Gb, may be another critical factor for GC B- ce11 apoptosis or rescue. In conclusion, the present findings correlate a potential stnicturai association of CD1 9 and Gb, with a fùnctional relationship. Mer the internalization of CD19 following antibody crosslinking, CD19 undergoes retrograde transport to the nuclear envelope and mediates apoptosis in a Gb,-dependent manner. Both features are absent in Gb,-negative cells. This correlation emphasizes the significance of nuclear membrane-localized CD1 9 for the subsequent signal transduction to induce apoptosis. 3.6 Future Directions The evidence for physical association and fiuictional relationship between CD19 and Gb, has been studied. However, the role for Gb, in the signal transduction of CD19 to modulate the CD19-mediated hornotypic adhesion or apoptosis has not been directly studied yet. Very recently, Katagin et al. 1999, reported the novel method of immunoprecipitation, using antibody against GSL (Gb,) to precipitate assoçiated protein (tyrosine kinase, Yes). This procedure couid be applied to examine whether CD19 aiso is physically associated with Gb, on the ce11 membrane. In addition, major signal transduction elements of CD 19, Lyn and Syk PTKs are pre-associated with CD19 at the cytoplasmic domain and consequently. PTKs also could be precipitated together with CD1 9 by using antibody to CD1 9 or by antibody to Gb, as well. Therefore, recruitment of PTKs, phosphorylation of PTKs and appropriate in vitro kinase assay of PTKs can be studied in CD19-positive B-cells before and after antibody ligation or crosslinking. To identiQ the involvement of Gb, in this process, the effect of VTlB subunit preko- treatment on antibody crosslinked-CD 19 induced PTK recruitment and activation should also be studied. The signaling through CD19 activates PTK, PLC and PKC and since CD19 is expressed on any B-cell, the studies have ken done on a wide variety of Bcell stages (Uckun et al., 1993). Gb, on the other hand is expressed ody on Burkitt's lyrnphoma ce11 Iines and GC B-cells. Although GC B-cells can be rescued from spontaneous apoptosis, the rescued cells do not express Gb3 anymore. Therefore, Burkitt's lymphoma cells are the only in vitro mode1 to study the relationship between CD19 and Gb,. The Gb,- mediated signal transdcution of B-cells for apoptosis induction is via CAMP-dependent PKA activation (Taga et al., 1997). Therefore, CD19 mediated signal transduction events should be re-investigated on CD19', Gb3- ce11 lines versus CD1 9', Gb,' ce11 lines, to determine whether additional signal transduction events, such as PKA, are activated in the presence of Gb,. Preliminary studies have shown that apoptosis can be induced in Daudi cells not only by immobilized antibody (Taga et al., 1997), but also by antibody crosslinking of either Gb3 or CD1 9. Unlike VTl-B induced apoptosis, Gb,-mediated apoptosis is stronger than CD 19-mediated apoptosis. Any synergistic or inhibitoy effect between apoptosis induced by two molecules should be investigated. Freshly isolated GC B-cells readily and rapidly undergo apoptosis. However, the cells can be rescued or delayed fiom entering apoptosis by adding a soluble form of CD23 (CD21-ligand) (Liu et al., 1991), anti-CD21 (Bonnefoy et al., 1993) or anti-dg crosslinking (Liu et al., 1989). The effect of anti-Gb, and anti-CD19 crosslinking on GC B-cells survival and on CD2Yanti-CD2Vanti-dg-mediated survival should be investigated to veriQ whether the role of CD19/Gb3 is for either GC B-ce11 survival or apoptosis. Chapter 4 Functional role for Gb, in interferon-a/ type 1 intederon receptor mediated anti viral activity 4.1 Abstract The extracellular domain of the type 1 IFN receptor, IFNARl subunit has hi& amino acid sequence similarity to the receptor bïnding subunit of VT1 (Lingwood and Yiu, 1992), which specifically binds to the cell surface receptor globotriaosylceramide, Gb, (Ga1 a 1->4 Gal P 1->4 Glc B 1->1 Cer). Gb, deficient, VT resistant cells are cross- resistant to IFN-a mediated antiproliferative activity (Cohen et al., 1987 and Ghislain et al., 1994). The association of eIFNARl with Ga1 a 1->4 Ga1 containing glycolipids has ken shown previously to be important for the receptor mediated IFN-a signal transduction for growth inhibition (Ghislain et al.1994). The essential role of Gb, for the signal transduction of IFN-a mediated anti viral activity has been investigated in the present study. IFN-a mediated anti viral activity, nuclear translocation of activated Statl (signal transducer and activator of transcription) and inçreased expression of PKR (IFN inducible double stranded RNA dependent protein kinase) were defective in Gb, deficient ver0 mutant cells, although the surface expression of IFNARI was unaitered. The VTlB subunit \vas found to inhibit IFN-a mediated anti viral activity, Statl nuclear translocation and PKR regulation. The differential role of Gb, fatty acid isoforms in anti viral activity was studied in MRC-5 cells which are resistant to IFN-a growth inhibition, but still remain Fully sensitive to IFN-a anti viral activity. MRC-5 cells predominantiy express long chain fatty acid containing Gb, isoforms. The study on MRC-5 cells and two astrocytoma cell lines, which express different Gb, isoforms indicated that long chain fatty acid containing Gb, isoforms, which were less effective to mediate VT1 cytotoxicity, were found to correlate with higher IFN-a mediated anti viral activity. 4.2 Introduction The human type 1 IFN family consists of IFN-a, IFN-B and 1FN-o (PfeKer, 1997) which elicit a variety of biological activities such as antiproliferative and anti viral activities afier initial high afinity interaction with the ce11 surface receptor (Pestka, 1987). All members of type 1 IFN bind to the same receptor; type 1 IFN receptor. A functional type 1 IFN receptor mainly consists of two transrnembrane subunits; IFN-a R1 (IFNARI) and IFN-a R2 (IFNARS) and two Janus famiIy tyrosine kinases; Tyk2 and Jakl. constitutively associated with the cytoplasmic domain of IFNARl and IFNAR.2, respectively (Williams and Haque, 1997). Both IFNARl and IFNARZ are important for ligand binding as well as signal transduction. Upon ligand binding, tyrosine kinases associated at the cytoplasmic end of the receptor are auto- andor tram-phosphorylated and subsequently phosphorylate and activate two cytosolic signal transducers and activators of transcription; Stat 1 and Sut2 (Williams and Haque, 1997). Activated Stat 1, StaQ and a third cytosolic protein; p48, form a complete transcription factor ISGF3 (IFN- cc stimulated gene factor 3) (Schindler, 1992). ISGF3 undergoes nuclear translocation and binds to the conserved ISRE (IFN stimulated responsive element) of ISG (IFN stimulated genes) for the transcriptional activation of ISGs (Williams, 1991) such as 2'4' oligoadenylate synthetase (2-SA Synthetase), double stranded RNA-dependent protein kinase (PKR), Mx protein etc (Williams and Haque, 1997). ISGF3-dependent ISG activation has been well characterized as a principal pathway of IFN-a-mediated signal transduction. However, ISGF3-independent alternative pathways, mediated by other Stat factors, also cm activate ISG without ISRE, such as GAS (interferon-y activation site) element containing ISGs (Yang et al., 1996, Zhong et ai., 1994 and Fasler-Kan et al., 1998). IFNARI is a 63 kDa glycoprotein with an N-terminal extracellular domain, a short transmembrane segment and a C-terminal cytoplasmic domain (Pestka, 1997). However, due to the highly glycosylated nature of IFNARI, the apparent molecular weight may Vary fiom 105 to 135 kDa (Contstantinescu et al., 1995). Verotoxin resistant Gb, deficient Daudi mutant cells are cross-resistant to a-interferon- mediated growth inhibition (Cohen et al, 1987). ïhe extracellular domain of IFNARl (eIFNAR1) surprisingly has high arnino acid sequence similarity to the binding subunit B of verotoxin 1 (VT1 B) (Figure. 14)(lingwood and Yui, 1992). More irnportantly, the amino acids of VTlB which are important for receptor binding are shared by IFNARI, indicating that Gb, can be laterally associated with the eIFNAR1 domain and modulates the fiinction of IFNARl (Lingwood, 1996). A fusion protein of eIFNARl linked to the Fc portion of IgGl specifically binds to the terminal Ga1 a 1->4 Ga1 containing glycolipids (Ghislain et al., 1994), suggesting a lateral association between IFNARl and GbJGb; at the ceil surface. Although the heterogeneity of the IFN-a-receptor interaction and ce11 surface expression of IFNARI are unaltered in Gb, deficient Daudi mutant cells, the IFN- u binding capacity of the mutant cells is significantly reduced (Ghislain et al., 1994). In addition VTlB binding inhibits IFNa binding to the receptor (Ghislain et ai., 1994). reflecting that association with Gb, maintains the appropriate presentation of the receptor to IFN-a (Ghislain et al., 1994). ISGF3 binding to ISEof the 2-SAS gene, following IFN-a treatment also has been found to be defective in Gb, deficient cells (Ghislain et al., 1994), indicating the fûnctional role of Gb, as a component of type 1 interferon receptor multimoIecular signal transduction cornplex. The MRC-5 ce11 line (Ghislain et ai., 1995) and some rend cancer ce11 lines (Pfeffer et al, 1996) are resistant to IFN-a induced antiproliferation but are still sensitive to IFN-a mediated anti viral activity, separating the signaling pathways for the two major biological functions of type 1 IFN. In both studies, an additional factor or biological event was found to be required for the antiproliferative activity despite the comparable level of receptor structural cornponents, receptor binding capacity, anti viral activity and induction of ISGs such as ISGlS and ISG54 in both IFNa induced antiproliferation sensitive and resistant cells. In the present study, a functional role for Gb, in IFN-a mediated anti viral activity against Echo 11 virus and vesicular stomatitis virus (VSV) has been demonstrated. The intracellular traficking and cytotoxic activity of VT is determined by G4 isoforms rather than the total Gb, expression (Arab and Lingwood, 1998). Short chah fatty acid containing Gb, isoforms have ken found to be more significant for the targeting of the tosin to ER and nuclear membrane and subsequent cytotoxicity, while long chab fatty acid Gb, isoforms target the toxin mainly to the Golgi. In the present study, IFN-a induced anti virai activity has been investigated in cells containing different Gb, isofoms. Despite the lesser role of long chah fatty acid containing Gb, isoforms in VTl cytotoxicity, such isoforms were found to be suficient to modulate IFN-a mediated anti viral activity, suggesting that Gb3 isoforms mediate the different biological functions of IFN-cc. 4.3 Materials and Methods 4.3.1 Materials Chemica 1s: PPMP (1 -pheny l-2-hexadecanoylamino-3-morpholino propanol) was fiom Matreya Inc., Pleasant Gap, PA. Sodium butyrate and alkaline phosphatase substrate BCNPNBT (5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazoliurn) tablets, -oelatin, bovine senim albumin, aprotinin, sodium orthovanadate, PMSF (phenylmethylsulfonyl fluoride) and FITC-avidin were fiom Sigrna St. Louis, MO and FITC fiom Molecular Probe, Eugene, OR. IFN-a2b was kindly supplied by Schering Canada, Pointe-Claire, QC. Verotoxin: As described in 3.3.1. Immunoreagents: Monoclonal antibody to eIFNARl was a gif? fiom Amgen, Thousand Oaks. CA. Monoclonal antibody to IFNARI, polyclonal anti-IFNARI, anti-PKR, anti- Statl p84/p91 and protein A agarose were fiom Santa Cruz Biotechnology Inc., Santa Cruz, CA. Biotinylated goat anti mouse secondary antibody was fiom Jackson Immuno Research Laboratories Inc., West Grove, PA. Peroxidase conjugated goat anti-mouse antibody was from Sigma. Cell Culture: Vero cells and the Gb, deficient, VT resistant ver0 mutant VRP ceil line (Pudymaitis et al., 1991) were grown in a-MEM with 5% fetal calf serum and 40 pg/ml Gentamycin at 370C in the presence of 5% C02. The human astrocytoma ce11 line SF- 539 (Arab et aI., 1998) and MW-5 were grown in a-MEM with 10% fetal calf Sem, and XF-398 (Arab et al., 1998) in RPMI-1640 with 10% fetal calf serum. For the indicated experiments, XF-498 cells were pretreated with 2.5 pM PPMP or with 2mM sodium butyrate for 6 days. 4.3.2 Methods 4 3.2.1 FITC-VT1B cell surface Iabeling VTlB subunit was labeled with FITC (Molecular Probe, Eugene, OR). FITC to VT 1 B 11 :1 (w/w) J was added directly to the toxin in OSM carbonate/bicarbonate bufZer pH 9.5 and the mixture was gentIy rotated for 2 hours at room temperature (Johnson and Holborow, 1986 ). Free FITC was removed by dialysis against 10 mM PBS pH 7.4. Vero and VRP cells grown on cover slips were kept on ice and labeled with 1 pg/ml of FITC- VTlB at 40C for 30 min. After thorough washïng to remove unbound FITC-VTIB with ice cold PBS, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 minutes and mounted on the slide for fluorescence microscopy (Khine and Lingwood, 1994). The preparations were examined and photographed under incident UV ilhmination using a Polyvar fluorescence microscope on EL 135, 400 ASA film at rnagnification x 100. 4.3.2.2 eIFNAR1 indirect immunofluorescence surface labeling Vero and VRP cells, kept on ice were labeled with monoclonal antibody to eIFNARl (Ghislain et al., 1994) or with mouse IgG as a control, for 30 min on ice. Afier thorough washing (5 min, 3 times) with ice-cold PBS, cells were fûrther incubated with biotinylated goat anti mouse secondary antibody and FITC-avidin for 30 min each on ice with thorough washing at each step. The ceIls were finally fixed with 4% paraformaldehyde in PBS as described in 4 3.2.1. 4.3.2.3 Immunoprecipitation Vero and VRP cells (1x10') were lysed and scraped in 1 ml of RIPA buffer (1% sodium deoxycholate, 1% NP-40, 0.1% SDS, 2 rnM EDTA, 150 mM NaCl in lOrnM sodium phosphate buffer, pH 7.0). 0.1 mg/ml PMSF, 1 mM sodium orthovanadate (phosphatase inhibitor) and 1 pg/mI aprotinin (protease inhibitor) final concentration were added to RIPA bufTer just prior to use. The ce11 tysates were incubated on ice for 45 min in 1.5 ml eppendorf tubes. The ce11 lysates were spun at 15,000g at 4°C for 30 min and the clear supernatant was transferred to a new tube. lmg of total protein was used for each imrnunoprecipitation. IFNARl was irnrnunoprecipitated by using monoclonal antibody to IFNARl 5 pg (25 pl in volume) and 100 pl of IO% protein A-agarose. The immunoprecipitate bond to protein A-agarose was pelleted by spinning at 14,000 g for 10 min at 4°C and the pellet was washed 3 times with RIPA buffer. As a control, ce11 lysates were irnrnunoprecipitated with mouse IgG in place of prirnary antibody 40 pl of SDS-PAGE sample buffer was added to the pellet and the sample was boiled for 90 seconds. 15 pl of each sample was nui on SDS-PAGE. 1.3.2.4 Western immunoblotting At the end of the indicated experirnents, ce11 lysates were subjected to separation on 7.5% SDS-PAGE and the protein was electro-transferred to a nitrocellulose membrane. The blot was blocked with 5% milk powder and 0.5% Tween-20 in 50 mM TBS at 37°C for 30 min, followed by imrnunoblotting with polyclonal anti-IFNAR1 or anti-PKR (1 :IO00 in blocking buffer) at 37°C for 2hr, followed by GAR-alkaline phosphatase ( 12000 in blocking buffer) with thorough washing at each step with 50 mM TBS. The immunoblot was visualized using an alkaline phosphatase detection system. 4.3.2.5 VT1 cytotoxicity assay Cells grown to confluency were trypsinized and 100~1of a l.j?t105/rnl ce11 suspension was seeded in 96 well ce11 culture plates for 24 hr at 370C prior to the experiment. Then, 10 pl of serial dilutions of VT1 were added and ce11 culture plates were Merincubated at 370C for another 48 hr. At the end of the incubation period, the cells were fixed with 2% formaldehyde in PBS and stained with crystal violet as described (Petric et al., 1987). The percentage of live cells was calculated fiom absorbance of destined cells read at 570 nm using a microtiter plate reader (Dynatech Laboratories, Chantilly, VA). 4.3.2.6 IFN-a anti viral assay 100pl of 15x104/ml cells were seeded in a 96 well tissue culture plate and grown for 24 hr. Cells were pretreated with serial two fold dilutions of IFN-a2b for 24 hr. Then the cells were incubated with 104 phi of Echol 1 virus or 10' phof VSV per well (kindly supplied by Drs M. Petric and R. Tellier, Dept Pediatric Lab. Medicine, HSC.) for another 24 hr (Ghislain et al., 1995). The cells were fmed and stained with crystal violet as described above and the percentage of protection fÎom viral cytopathic effect was calculated. 4.3.2.7 IFN-a growth inhibition assay 100~1of 1Sxl ~'/rnl cells were seeded in a 96 well tissue culture plate and grown for 24 hr. Then, 2-fold dilutions of IFN-a2b was added to the cells and the plates were incubated at 37"C, 5% CO2 for 96 hr. At the end of the experiment, the cells were fixed uith 2% formaldehyde in PBS and stained with crystal violet. The absorbance of the destined cells was measured as described abve and % of growth inhibition was calculated. 4.3.2.8 Statl immunofluorescence Vero and VRP cells grown on 8-well chamber slides (Nalge NUNC International, Rochester, NY) were treated with 104 IU/ml IFN-u2b for 30 min at 370C before fixation as described above. Then cells were permeabilized with 0.1% TritonX-100 in PBS at room temperature for 10 min, and then blocked with 1% BSA and 0.1% TritonX-100 in PBS at 37°C for 30 min. The immunolabeling was performed by using rabbit polyclonal antibody to Statl p84/p91 (1 :100 in blocking solution) and FITC labeled goat anti rabbit secondary antibody (1:100 in blocking solution) with thorough washing for 5 min, 3 times at each step. 4.3.2.9 Glycolipid extraction and thin layer cbromatography (tlc) overlay of VT1 The glycolipids fiom vero, VRP, MRC-5 and astrocytoma ce11 lines were extracted as described (Cohen et al., 1987). 2x1 O' trypsinized cells were pelleted by centrïfbgation at 800 rpm for 10 min and washed one more time with PBS (0.1M phosphate buffer saline, pH 7.4). The total GSL was extracted and separated by tlc as described in 3 -3-2.1. The tlc plates were blocked with 1.5% gelatin in 50 mM TBS (Tris buEer saline, pH 7.4) at 370C overnight, incubated with 0.1 pg/ml VT1 and immunodetected using anti-VT1 monoclonal antibody (1:1000) and peroxidase conjugated goat anti-mouse antibody (1 2000) at 37°C for 2 hr each. ïhe piates were washed with 50 mM TBS for 5 minutes. 3 times at each step. The bound antibody was visualized with the peroxidase substrate 4-chloro- 1-naphth01 (Lingwood et al., 1987). 4.4 Resu lts 1.4.1 Cell sudace Gb, facilitates the 1FN-a mediated anti viral activity against Echo 11 virus and VSV In order to investigate any possible role of Gb, in IFN-a mediated anti viral activity, Echo 1 I and VSV anti virai assays were perforrned on Gb,-positive ver0 cells and the Gb,-negative ver0 mutant VRP ce11 line. Prior to the anti viral assay, the level of Gb; expression in ver0 and VRP cells \vas determined by tlc of glycosphingolipid extracts followed by VTI overlay (Figure.3l.A). Total Gb, expression was reduced by >80% in VRP as compared to vero cells (Figure.3l.A). The surface expression of Gb, also was examined on vero and VRP cells by staining with FITC-VTlB subunit at 40C (Figure. 32 panel a and b). No surface VTl B staining of VRP cells was found (Figure. 32, panel b). The total expression of IFNARl was compared by immunoprecipitation of the ce11 lysate, followed by western immunoblotting (Figure. 31.B). In contrat to Gb, expression, comparable levels of total IFNARl expression were observed in ver0 and VRP cells (Figure. 31.B). Due to the variation in glycosylation, the apparent molecular weight of IFNARl varies fiom IO5 to 135 kDa (Constantinescu et ai., 1995). In ver0 and VRP cells, a -130 kDa band was observed as the major species of IFNARl and other minor bands between 60-130 kDa also were detected (Figure. 31.B). The surface expression of IFNARl was examined by indirect irnmunofluorescence of eIFNARl (Figure. 32 panel c and d) and the surface expression of IFNARl also was found to be comparable in vero (Figure. 32 panel c) and VRP (Figure. 32 panel d) cells. A fûnctionai assay for Gb, was conipared on vero and VRP cells by VTI cytotoxicity assay (Figure. 33). Consistent with the Gb, content, ver0 cells were very sensitive to VTl cytotoxicity with a CD50 <5 pg/ml while VRP cells were totaily resistant to VTl cytotoxicity (Figure. 33.A). Functional assays for type 1 IFNAR were orcinol VT1 overlay Cr - ? std vero VRP std ver0 VRP Figure. 31A Total cellular Gb3 on ver0 and VRP celis. TLC of celiular glycolipid content by orcinol spray and VT1 overlay. The totai glycolipid extracted from lxlO(7) ceils was loaded in each lane. ver0 VRP Figure. 31B Total cellular IFNARl on ver0 and VRP ceiis. lmmunoprecipitation with monodonal anti-IFN.4R1 followed by rx-estern blotting. Lmmunoprecipitate of 1 mg total ceil ly sate was loaded in each 1ane.A~a control, ceil lysate was imunoprecipitated with mouse IgG (data not shown) TTC- TIB Figure. 32 Surface expression of Gb3 and elFNARl on vero cells and VRP celis. Surface buidmg of FITC conjugated VTiB to verda) and VRP@) ceiis and indirect immunofluorescen<~surface labeling of elFNARl on vero(c) and VRP(d) cek. Mouse IgG was wdas a control instead of anti-eIFNAR1 (data not shown). VT1 log nghl Figure. 33.A Cornparison of VTl cytotoxicity on ver0 and VRP cells. (n = 4) IFN log IUIml Figure. 33.B Cornparison of 1FN-a-mediated anti viral activity on vero and VRP cells. (n = 4) compared on ver0 and VRP cells by IFN-a-mediated antiWal assays, using Echo 11 virus and VSV. In contrast, despite the comparable expression of IFNAR1, while 1FN-a compfetely protected vero cells fiorn Echo 1 1 virus and VSV at the concentration at 1000 IU/ml, only partial protection was observed in Gb, deficient VRP cells (Figure. 33.B). Since the maximum protection was less than 50%, VRP cells could be regarded as resistant to IFN-a-mediated anti viral activity. To Merinvestigate the requirement of surfàce Gb, for IFN-a mediated antiviral activity, ver0 cells were preincubated with 50 pg/ml of VTIB subunit pnor to the VT1 cytotoxicity or anti viral assay. VTIB subunit alone has the same binding affinity to ce11 surface Gbj as the holotoxin (Ramoter et al., 1990), and can enter the ce11 by receptor rnediated endocytosis (Khhe and Lingwood, 1994). It was hypothesized that the co- incubation/intemalization of VTlB would utilize the surface availab!e Gb,, and compromise the ability of Gb, to modulate its potentially ce11 surface associated receptor, IFNARI. In some susceptible ce11 lines such as Daudi Burkitt's lymphoma cells, high dose of VT18 alone (1 pg/mi) can induce apoptosis (Mangeney et al., 1993) but such activity was not observed in vero cells. Due to the cornpetition between VT 1 and VTl B subunit for Gb, binding and internalization. VTl cytotoxicity was found to be 100-fold reduced following pretreatment with VTlB (Figure.34.A). IRJ-a anti viral activity against both Echo Il and VSV also was attenuated following pretreatment with VTlB (Figure 34.B), indicating the involvement of cell surface Gb3 for IFN-a-mediated biological function. 4.1.2 Functional role of Gb, as an accessory molecule for receptor mediated IFN-asignal transduction Following IFN-a binding to the type 1 IFN receptor, the cascade of signal transduction is initiated by interaction with and activation of TyW tyrosine kinase by IFNMI and Jakl by IMARî (Pestka, 1997) (Figure. 13). The signal transduction event is very rapid and activation of IFNARl was found as early as 1 min, Stat activation within 5 min (Constantinescu et al., 1994) and ISGF3 activation at 15 min after IFN-a - ver0 - vero+ 50 ug/ml VTlB Vïï log nglml Figure. 34.A Effect of VTl B CO-incubationon VT1 cytotoxicity. Vero celIs were preincubated with excess (50pg/ml) VTlB at 370C for 30 min and CO-incubatedwith VTI for cytotoxicity assay. (n = 4) IFN log IUfml Figure. 34.B Effect of VTlB CO-incubation on IïNa for anti viral activity. Vero cells were preincubated with excess (50pg/ml) VTl B at 370C for 30 min and CO-incubatedwith IFN-a for anti viral activity against Echo 11 virus and VSV. (n = 4) vem VRP Figure. 35 Gb3 dependency of IFN-alpha induced Statl nudear translocation. Indirect immunofluorescence of Statl in conbol vero(a) and VRP(c) cells and &ter the treatment with 10(4) IU / ml IFN-alpha at 370C for 30 min in ver-) and VRP(d) cells. Vero cells were pretreated 6thSOug/ ml VTlB at 370C for 30 min(e) and treated with IFN-alpha after VTlB pretreatment(f). treatment (Ghislain et al., 1994). Activated ISGF3 translocates into the nucleus and transcriptionally activates the ISGs (Williams, 1991). To investigate a role for Gb3 in IFNAR 1-mediated signal transduction, the nuclear translocation of activated ISGF3 was examined by irnmunofluorescence labeling of Statl in ver0 and VRP cells (Figure. 35). In untreated control cells, cytoplasmic distribution of Statl was observed (Figure 353, c and e). The nuclear translocation of Statl was detected 30 min aller IFN-a treatment and was found to be more significant in Gb,-positive vero cells (Figure. 35. panel b). Only minimal nuclear translocation of Statl was observed in IFN-atreated Gb,-negative VRP cells (Figure. 3 5. panel d) and vero cells pretreated with VT 1B subunit (Figure. 35. panel f). After IFN-a binding to IFNAR, activated Statl and Sut2 associate as hetrodirners and further regulate the gene transcription of ISRE containing ISGs. In addition, Statl can also form homodimers and regulate GAS containhg ISGs by different signal transduction mechanism (Haque and Williams, 1994). Therefore, weaker nuclear translocation of Statl observed in VRP and VTlB-pretreated ver0 cells could be due to Gb3-independent activation of Statl, not related to ISGF3 formation. Activated ISGF3 binds to ISE of ISGs such as 2-5A synthetase and PKR, and regulates gene transcription in response to IFN-a. ISGF3 binding to ISRE of 2-5A synthetase was obsewed only in Gb3-positive ceils in contrast to Gb3-negative cells (Ghislain et al., 1994). However, 2-5 A synthetase specifically protects the host cells against picoma viruses only and in contrast, PKR is responsible for the anti viral activity against a wide variety of viruses. IFN-a dependent regulation of PKR in Gb,-positive and -negative cells was examined by western imrnunoblotting (Figure. 36) after treatment of the cells with 1041~/mlIFN-a for 30 min to 48 hr. In ver0 cells, increased expression of PKR (-70-80-ma) was observed at 24 hr but a lower molecular weight species (40- Da), likely a degraded product, was observed at 48 hr (Figure. 36.A). In VRP cells, instead of increased PKR expression, the lower molecular weight product was already present at 18 hr and complete degradation was found at 24 hr (Figure. 36.A). Earlier degradation of PKR was also observed in ver0 cells pretreated with VTlB subunit followed by IFN-a stimulation for 24 hr (Figure. 34.B), indicating that Gb3-dependent IFN-a signal transduction is necessary for the regulation of PKR expression. A KDa Ver0 VRP time(hr) O 18 24 48 O 18 24 48 IFN-alpha treatment KDa Vero Figure. 36 Western irnrnunoblotting analysis of PKR expression in control and IFN-alpha treated ver0 and VRP cek. A: Vero and VRP cells were incubated with 1q4)IU/ ml IFN-alpha2b at 370C for indicated time. B: Vero ceiis with and without pretreatment with 50 ug/ml VTlB were treated as in panel A. 3.4.3 Difierential rote of Gb, isoforms for VTl cytotoxicity and IFNu mediated anti viral activity On thin layer chromatography Gb, can be separated into an upper (preferentially containing longer chah fatty acid Gb, isoforms) and a lower band (containing shorter chain fatty acid species). The shorter chah fatty acid Gb, isoforms are particularly critical for VTl retrograde transport to ER and nucleus, and VTI cytotoxicity (Arab and Lingwood, 1998). While MRC-5 cells are resistant to IFN-a mediated growth inhibition, these cells are still sensitive to IFN-a mediated anti viral activity (Ghis1ai.n et al,, 1995)- To determine whether different Gb, isoforms might be necessary for IFN-a mediated anti viral activity, as opposed to growth inhibition, the Gb, content of MRC-5 cells was exarnined. A VT1 overlay of the glycolipid extract showed that control MRC-5 cells mainly expressed the upper band Gb,, compared to ver0 ce11 extract, which expressed both upper and lower bands (Figure. 37). Consistent with the difference in Gb, isoform composition, MRC-5 cells were over 100 fold less sensitive than vero cells to VTI cytotoxicity (Figure. 38.A). Although MRC-5 cells were resistant to IFN-a growth inhibition with ~50%maximum inhibition (Figure 38.B), IFN-a mediated protection of viral cytopathic effect against Echol 1 virus was still up to 100% at 1000 IU/ml of IFN- a2b (Figure 38.C), suggesting that the expression of long chah fatty acid Gb, was sufficient to modulate the type I IFN receptor-mediated IFN-a induced anti viral activity. Treatment of MRC-5 cells with the Gb, synthesis inhibitor; PPMP significantly reduced the IFN-a mediated anti viral activity in MRC-5 ceIls (Figure 38.D), indicating the requirement of Gb, in such activity. To fbrther investigate the preferential involvement of the upper band Gb, in IFN- cc anti viral activity, a cornparison of IFN-a anti viral activity in two astrocytoma cells was performed. As previously reported (Arab and Lingwood, 1998), the lower band Gb, was restricted to SF-539 cells and this correlated with increased susceptibility to VT cytotoxicity. VTl binding to Gb, isoforrns of astrocytoma cells glycolipid extract on tlc is shown in Figure. 39.A. SF-539 and butyrate treated XF-498 had more short chah fatty acid Gb, (lower band) and XF-498 had more long chah fatty acid Gb, (upper band) as orcinol VT1 overlay w - - Std vero MRC Std ver0 MRC-5 Figure. 37 Total cellular Gb3 expression and VT1 tlc overlay of vero and MRC-5 ceils. Total glycolipid extracted from 1x10(7) celis was loaded in each lane and separated by tlc. - vero - MRGS - 8 - 6 - 4 - 2 O 2 4 VTI log nglml 0 Figure. 38.A Cornparison of VTl cytotoxicity on ver0 and MRC-5 cells. (n = 4) IFN log IU/ml Figure. 38.8 Cornparison of IFN-aantiproliferative assay on vero and MRC-5 cells- (n = 4) IFN log IU/ml Figure. 38.C Cornparison of 1FN-a anti viral assays against Echol 1 virus and VSV on ver0 and MRC-5 cells. (n = 4) 500 1 O0 IFN unitslml Figure. 38.D Effect of inhibition of glycolipid synthesis with PPMP on antiviral activity in MRC-5 cells. (n = 4) previously described (Arab and Lingwood, 1998). XF-498 cells îreated with the G4 synthesis inhibitor, PPMP, expressed a reduced level of Gb, overall. VTl cytotoxicity for these astrocytoma ce11 lines is shown in Figure. 39B. While butyrate treatment sensitized XF-498 cells to VTI cytotoxicity, treatment of the cells with PPMP depleted cellular Gb, and abolished VTI cytotoxicity in XF-498 cells (Figure. 39.8). In contrast to VT1 cytotoxicity, XF-498 cells were found to be more sensitive to IFN-a mediated anti viral activity than SF-539 cells (Figure. 39.C) as predicted. The reduction of anti viral activity in PPMP-treated XF-498 correlated with the fùnctionai roie of total cellular Gb, in anti viral activity. Butymte treatment of XF-498 cells induced a significant selective increase in short chah fatty acid containing G4(Figure. 39.A) but an increase in sensitivity to anti viral activity was not observed (Figure. 39.C). The Gb, dependent anti viral activity observed in butyrate treated XF-498 cells was likely due to the presence of the basal level of long chah fatty acid Gb3. Unfortunately, conditions to selectively induce the long chah fatty acid containing Gb, have not been identified to more definitely demonstrate the role of these Gb, isoforms in the anti viral response. 4.4.1 Relative requirement of IFNdtype 1 IFN receptor internalization for 1FN-a-rnediated signal transduction and biological activities According to the proposed mode1 of Gb, sorting, based on the fatty acid chain length of Gb, and the reduced dimension of the GolgiER membrane (Lingwood, 1996), longer fatty acid chah containing G4 isoforms tend to remain in the plasma membrane ancilor compartments, closer to the plasma membrane. On the other hand, the shorter fatty acid isoforms could more easily accommodate vesicle formation during the trafficking towards the organelles with smaller membrane dimensions. Therefore, if longer fatty acid Gb, isoforms play a more significant role for IFN-a-rnediated anti viral activity, signal transduction events required for the anti viral activity should be more restricted to the plasma membrane and less dependent on the ligand/receptor internalization. In order to orcinol VT1 overlay Std SF XF XF+ XF- Std SF XF XF+ XF- Figure. 39.-4 Total celiular Gb3 eqxesion and VT1 tlc overlay on astrocytoma ceiis. Total glycolipid ewtracted from lxlO(7) cells was loaded in each lane and separated by tlc. Std = GSL standards, SF = SF539, XF = XF498, XF+ = butyrate treated XF498, XF- = PPMP treated XF498. SF-539 XF498 butyrate-XF PPMP-XF IFN IU/ml Figure. 39.C Effect of modification of G4 cellular content on IFN-a anti viral assay against Echo 11 virus (on SF-539, XF-498 and butyrate or PPMP modified XF-498 cells). (n = 4) venfy the relative requirement of IFN-a/ type 1 IFNR internalization for 1FN-a-mediated biological activities, IFN-a mediated Statl nuclear translocation, antiproliferative activity and anti viral activity were exarnined while receptor mediated endocytosis was inhibited. Cytosolic acidification of ver0 cells by different concentration of acetic acid was applied to inhibit receptor-mediated endocytosis (RME). Reducing the intracellular pH prevents pinching of the coated vesicles fiom the plasma membrane and inhibits RME (Heuser et al., 1989). Both IFN-a (Zoon et al., 1983) and VTl B subunit (Khine and Lingwood, 1994) have been known to enter the cells by RME. Since there are only low number of type 1 IFNR on the cells, it has been very difficult to visualize the internalization of type 1 IFNR (Branca et al., 1982). Therefore, internalization of FITC- conjuçated VTlB internalization was exarnined to confirm the effectiveness of acetic acid-induced cytosolic acidification as an inhibitor of RME (Figuredo). FITC-VTlB entered vero cells after incubation at 37°C for 30 min (Figure.40 pane1 a) and this entry was eficiently prevented in the presence of 20 mM acetic acid (Figure.40 panel b). Mowever. acetic acid treatment had no obvious effect on the cytosolic distribution of Statl in control ver0 cells (Figure.40 panel c and d) and nuclear translocation of Statl in IFN-a treated cells (F-igure.40 panel e and f). Therefore, IFN-a/type 1 IFN receptor mediated signai transduction for Statl activation is likely a ce11 surface event which does not require iFN-odreceptor internalization. The effects of RME inhibition on IM-a mediated antiproliferative activity and anti viral activity were compared. As a control experiment to confIrm the effectiveness of acetic acid treatment on RME, dose dependent effect of acetic acid on VTl cytotoxicity was Brst examined. A dose dependent protection effect was observed and 20 rnM acetic acid completely protected vero cells fiom VT1-induced cytotoxicity (Figure.41). IFN-a- mediated antiproliferative activity also was prevented with about the sarne eficiency (Figure.42.A). However, Im-a-mediated anti viral activity was found to be less inhibited by acetic acid treatment (Figure.42.B), indicating that IïN-a-mediated anti viral activity was more independent of IFN-cd type 1 IFNR intemaiization, compared to antiproliferative activity. - acetic acid + acetic acid Figure. 40 Efted of cytosolic acidification on FITC-conjugated VTlB subunit internaiization and Statl nuclear translocation. FITC-VTIB internalization in vero celis (a and b),imrnuno fluorescence of Statl in vero cells (c and d) and IFN-alpha [10(3) IU/ml at 37oC for 30 min] treated vero celis (e and f), in the absence (acre)and presence (b,d,D of 20m.M acetic acid. - VT1 oniy - + 10mM Acetida Y +15mM VTl log nglml Figure. 41 Effect of cytosolic acidification on VT1 cytotoxicity (vero cells). (n = 4) - IFN-alpha2b only - +10mMAcetic/a Y +15rnM +20mM 0 IFN log IUlml Figure. 42.A Effect of cytosolic acidification on iFN-a-mediated antiproliferative activity (on ver0 cells). (n = 4) IFN-alpha2b only ---fx-- Y clOmM acetida - +15mM +20mM IFN log IUIml Figure. 42.B Effect of cytosolic acidification on IFN-a-mediated anti viral activity (on vero cells). (n = 4) 4.5 Discussion The type 1 IFN receptor is a multimolecular complex which mainly consists of two essentiai transmembrane subunits (IFNARI and IFNARS) (Figure. 13) and two members of Janus family non-receptor tyrosine kinases (Tyk2 and Jakl) which are recruited to the membrane receptor after ligand binding. However, the complete composition of a fùnctional type 1 IFN receptor is not yet fully understood (Uze et al., 1995). Not oniy protein accessory components, but also Ga1 a 1->4 Gai containing glycolipids such as Gb2 and Gb, are the candidates for cornponents of the type 1 IFN receptor complex (Ghislain et al., 1994). Since the type 1 IFN receptor is a comrnon receptor for many subtypes of type 1 IFNs, various patterns of ligand/receptor interaction exhibit different patterns of signal transduction and various outcornes of biological activities. The role of different components of the receptor involved may also Vary for different IFNs. The identification of additional components of type 1 IFN receptor complex is still a focus of IFN research. At least one additional accessory factor encoded on chromosome 21 has been proposed to be necessary for the hi& affinity ligand receptor interaction and IFN-a mediated growth inhibition based on the separation of growth inhibition and antiviral activity in MRC-5 cells (Ghislain et al., 1995). The present study provides an alternative basis for this separation. Ho! Iand et al., 1997 have also reported a novel factor designated as ISF2 1, the fiction of which is independent of receptor binding but necessary for 2-SAS activity and anti viral response. The gene encoding this factor also is located on the chromosome 21 but still distinct fiom the genes encoding other major components of type 1 IFN signaling such as IFNARI, IFNAR2 and Mx genes, which also are located on chromosome 21. ISF21 is found to be necessary for the type I IFN signal transduction and transcriptional activation of ISGs. ISF2l is not only necessary for the ISGF3-dependent transcriptional activation of ISRE containing ISGs such as 2-SAS, but also for the activation of the 6-16 gene which contains both ISRE and GAS (Holland et al., 1997), and guanylate-binding protein (GBP)gene, which contains GAS only (Lew et al., 1991). Physicai and functional associations between Gb, and its ptentiaily associated ce11 surface receptor, IFNARl have been reported. The fusion protein containing eIFNARl and Fc portion of IgGl binds to terminal Ga1 al4Ga1 containing Gb, and Gb, on tlc (Ghislain et al., 1994). Functionally, Gb, is found to be required for IFN-a induced antiproliferative activity (Cohen et al., 1987, Ghislain et al., 1994). The study on Daudi Burkitt's lymphoma ceils by Cohen et ai., 1987 has show that there are two components of IFN-a binding, high afinity components with Kd=4.9x10-1 lM, 400 molecules/cell and low affinity components with Kd=4.2x10-9M, 10,000 molecules/cell. Gb; deficient VT resistant cells which are also cross-resistant to IFN-a, have low affinity components only. In contrast, Ghislain et al., 1994 reported that Gb, deficient cells also maintained both high and low affinity components but the total binding capacity of the cells for EN- a was 4-fold reduced in Gb, deficient cells, compared to Gb,-positive Daudi cells. Despite the discrepancy, both studies indicated that Gbj is required for the proper presentation of the receptor on the ce11 surface for the high affinity interaction between IFN-a and type 1 IFNR, in order to induce the antiproliferative activity (Cohen et al., 1987) (Gtiislain et al., 1994). Gb, itself can function as a signal transduction molecule. The cross-linking of surface Gb, by plate-bound antibodies can induce activation of protein kinase A and ceramide dependent apoptosis in Burkitt's lymphoma cells (Taga et al., 1997). Mer IFN- cc binding to the type I IFNR, Jakl and Tyk2 tyrosine kinases are activated and fiuther activate the transcriptional activators, Statl and Stat2. Together with p48, Statl and Stat2 fom a gene-regulating factor ISGF3 and the complex translocates to the nucleus to activate ISRE containing ISGs. The nuclear translocation of activated Statl (Figure. 35) and the binding of transcription factor ISGF3 to ISRE of 2'-5' oligoadenylate synthetase eene (Ghislain et al., 1994) were found to be Gb, dependent. These findings emphasize a C differential role of Gb, as an accessory factor for signal transduction, in addition to the classical role as a receptor for toxin endocytosis. There are two components of StatI, 84- kDa Stat 1a and 9 1-kDa Stat 1P. Upon phosphorylated activation, hetrodimers of Stat2 and either Statla or Statl P are preferentially formed. However, Statl can also fom Statla-Statla homodimers (Haque and Williams, 1994) (Figure. 14). In contrast to Statl-Stat2 containing ISGF3, Statl homodimers bind to GAS elernent and activate ISGs without ISRE (Lew et al., 1989 and Haque and Williams, 1994). The significance of Gb, in 1FN-a-mediated signai transduction other than ISGF3 has not been excluded yet. Statl hornodimerization is the principal pathway of IFN-y-mediated signal transduction and which is activated by Jak2, not by Tyk2 (Pestka, 1997). Since Tyk2 is the PTK associated with 1FNARl and Statt cm be activated independent of Tyk2, by inference, Statl homodimer formation may be IFNARITTyk2 independent and Gb, rnay not play a role in this event, Type 1 IFNs mediate important biological functions such as antiproliferative activity, anti virai activity, immunornodulatory activity etc. Type 1 IFNs are more potent for the first two activities and Type II IFN, IFN-y is more efficient for the irnrnunomodulatory activity (Stites and Terr, 1991). However, the studies on some ce11 iines such as MRC-5 (Ghislain et al., 1995) and rend carcinoma cell lines (Pfeffer et al., 1996) showed that IFN-a-mediated growth inhibition-resistant cells are still sensitive to IFN-a-mediated anti viral activity, indicating the diverse signaling for the two major biological fùnctions of type 1 IFN. Since the role of Gb, for the IFN-a-mediated antiproliferation has been established, any potential role in anti viral activity was investigated in the present study. 1FN-a mediated anti viral activity was found to be more significant in Gb,-positive cells (Figure. 31.B) and was attenuated by VTlB intemalization, associated with ce11 surface Gb, 'utilization' (Khine et al., 1998), indicating the significance of Gb, for IFN-a-mediated anti viral activity. Some IFN-induced enzymedproteins such as 2-SAS, PKR and Mx protein have been known to play important roles in IFN-a-mediated anti viral activity but selective activation of enzyme/protein is virus specific (Samuel, 1991). The specificity of enzyme induction may be due to viral specific single andor double stranded RNA, required for the enzyme activation. Human Mx protein expression is induced in response to adenovirus and vesicular stomatitis virus only (Pavlovic et al., 1990). 2'-5' oligoadenylate synthetase mediated anti viral activity is selecdve for picomavinws ody (Samuel, 1991). 2'4' oligoadenyiate synthetase is involved in both the growth inhibitory and antiviral activity of IFN-a (Hassel et al., 1993). ISGF3 binding to ISRE of 2'4' oligoadenylate synthetase gene in response to IFN-a \vas observed only in Gb,-positive Daudi cells but not in Gb,-negative Daudi mutant and PPMP-treated Daudi cells (Ghislain et al., 1994). PKR, which inhibits both viral and celldar protein synthesis by phosphorylation of eiF2-a, is induced in response to a wide varïety of vinises such as adenovirus, reovirus, encephalomyocarditis virus, mengo virus and poliovirus (Samuel, 1991). In the present study, a role of Gb, in regdation of PKR synthesis following IFN-a addition was investigated. Interferons induce the expression of PKR and double stranded RNA derived fiom viruses or host celis stimulate the autophosphorylation of PKR (Williams, 1991). Increased expression of PKR was found after 24 hr treatment with IFN-a. Degradation of PKR was observed afier 48 hr in vero cells (Figure. 36A). In VRP cells early degradation of PKR was observed at 18 hr and complete degradation of PKR was found at 24 hr (Figure. 36.A). To perform an IFN-a anti viral assay, cells were pretreated with IFN-a for 24 hr prior to the addition of virus. The earlier degradation of PKR might explain the deficiency of protection against viral cytopathic effect in VRP cells. The faster degradation of PKR was also observed in ver0 cells pretreated with VTlB subunit (Figure. 36.B). In Daudi Burkitt's Lymphoma cells, prolonged activation of Jakl, Tyk2 kinases, Stat 1, Stat2 phosphorylation, ISGF3 formation and transcription of ISGs have been found to be correlated with high sensitivity to antiproliferative and anti viral activities of IFN-a (Lee et al., 1997). In Daudi cells sensitive to 1FN-a-mediated growth inhibition, Statl phosphorylation was found to be maximal at 16-24 hr after IFN-a treatment and declined der 36 hr. In resistant cells, the maximai Statl phosphorylation was at 4 hr and negligible at 8 hr after IFN-a treatment (Grimley et al., 1998). ISGF3 binding to ISRE of ISGlS also was observed as early as 2 hr and maximum at 16 to 32 hr and persisted for 48 hr after IFN-a treatment in sensitive cells. In contrast, only minimal ISGF3-DNA binding was observed in resistant cells, which was negligible afier 8 hr (Grimley et al., 1998). The increased expression of PKR for longer duration and later degradation of PKR in vero cells in the present study also could be due to the prolonged maintenance of signal transduction for the sustained biologicd functions. Therefore, association of signal transduction subunit of type 1 IFN receptor; IFNARI, to Gb, may be an important factor for the maintenance of prolonged signal transduction. Type 1 IFNs induce 50 to more than 100 IFN-induced proteins and the functions of many of them have not been identified yet (Sen and Ransohoff, 1997). Signal transduction, followùlg type 1 IFN binding is not restricted to Statt -Stat2 activation only. Other mernbers of the Stat family such as Stat3, Stat4, StatS and Stat6 also are activated and the duration of activation for other Stats also is longer in IFN-a-sensitive cells (Grimley et al., 1998). Stat3 activation is independent of lak kinase and occurs through a direct interaction between Stat3 and tyrosine-phosphorylated IFNARI. Stat3 can be phosphorylated by EGF and IL-6 and activated Stat3, as a homodimer or Statl-Stat3 hetrodimer can bind to GAS-like promoter element of DNA, which could be a small sequence variation around a consensus GAS element (Zhong et al., 1994). The roles of StaO in both antiproliferative and anti viral activities via NikB DNA-binding activity have also been identified (Yang et ai., 1998). StatS is constitutively associated with Tyk2 at the cytoplasmic domain of IFNARl (Fish et al., 1999). CrkL (Crk-like) is a celluiar homolog of viraKrk oncogene (v-Crk). Crk family proteins are adaptor molecules which consist of SH2 and SH3 domains. Various receptors and large multi-site docking proteins are the upstream components and several protein kinases and guanine nucleotide release proteins (GNRPs) are the downstream components of Crk and CrkL proteins (Feller et al., 1998). In response to IFN-a and IFN- p stimulation, Tyk2-dependent phosphorylated form of StatS provides a docking site for the SH2 domain of CrkL, and CrkL-StatS complex translocates into the nucleus and transcriptionally activates 1SGs via GAS elements (Fish et al., 1999). ïhe role of this novel signaling pathway in type 1 IFN-mediated antiproliferative activity has ken reported (Fish et al., 1999). Since IFN inhibits both host ce11 proliferation and viral replication, CrkL pathway may also play a role in anti viral activity. Due to the very wide diversity of type 1 IFN-mediated signal transduction pathways and a large variety of ISGs, the role of Gb, may be restricted only to some specific pathways and partial sensitivity to IFN-a-mediated antiviral activity in Gb3-negative cells (Figure. 33.B) could be likely due to other Gb3-independent mechanisms. The glycolipids of rnost cells are separated by tlc into doublets correspondhg to differences in acyl chain length and hydroxylation. Previous studies in our laboratory have shown differential properties of Gb, isoforms (Arab and Lingwood, 1998). Gb, expression and VTI sensitivity of a number of human astrocytoma ce11 lines were tested. Despite the comparable level of Gb, expression, cells with higher level of shorter faîty acids (C 16:0, C 18:2) containing Gb, (SF-539 cells) were significantly more sensitive to VTI compared to the ce11 line with higher level of longer fatty acid (C22:0, C24:O) Gb, content (XF-498 cells) (Arab and Lingwood 1998). In spite of an equal efficiency of internalization, VT1 was directed only to the Golgi region in XF-498 cells while retrograde transport of VTI to ER, nuclear membrane and nucleus was observed in SF- 539 cells (Arab and Lingwood. 1998). The mechanisms of differential targeting based on Gb, isoforms have been proposed (Lingwood, 1996 and Lingwood, 1999). The ability of the shorter fatty acid Gb, isoforms to accommodate into the smaller dimensional vesicles or reduced thickness of the bilayer membrane may determine the retrograde transport of such isoform-bound ligand to the compartments with thimer membrane such as ER and nuclear membrane. In contrast, longer fatty acid Gb, isoforms may remain restricted to the ce11 surface plasma membrane and the compartments closer to the ce11 surface such as Golgi. MRC-5 cells are less susceptible to VTl cytotoxicity and also resistant to IFN-a- mediated antiproliferative activity but still sensitive to 1fN-a-mediated anti viral activity (Ghislain et al., 1995). In order to verie the relative importance of Gb, isoforms in IFN-a mediated anti viral activity, Gb, species expressed in MRC-5 cells were examined and compared with the Gbj extract of vero cells (Figure. 37). Since MRC-5 cells expressed long chain fatty acid Gb, mainly and IFN-a anti viral activity was still efficient (Figure. 38.C), these isoforms of Gb, must be sufficient to modulate IFN-a anti viral activity. Treatment of MRC 5 cells with the inhibitor of glycosphingolipid synthesis; PPMP (Abe et al., 1992), blocked the anti viral sensitivity (Figure. 38.D). Inhibition of glucosylcerarnide synthesis by PPMP couid increase ceramide generation which may affect the response of the cells to IFN-a.However, involvement of ceramide is mainly associated with biological actions of IFN-y than with 1FN-a (Wakita et al., 1996) and is more ce11 type specific than is generally assumed (Veldman R. J. et al., 1998). The studies on astrocytoma cells also showed that XF-498 cells with long chain fatty acid containing Gb, isofons had more significant 1FN-a mediated anti viral activity (Figure. 39.C). To Meremphasize this finding, the anti viral activity was studied in butyrate stimdated XF-498 cells. Butyric acid is a ce11 differentiation agent which induces an overall increase in cellular Gb, synthesis in several ce11 types (Arab and Lingwood, 1998, Sandvig and van Deurs, 1996, Louise et al., 1995, Keusch et al., 1996). In the astrocytoma ce11 line XF-498, shorter fatty acid containing Gb, was preferentially elevated after sodium butyrate treatment (Arab and Lingwood, 1998). Consequently, butyrate treated XF-498 cells were more sensitive to VT1 compared to wild type XF-498 cells (Figure. 39.B). However, the absence of a comparable increase in IFN-a mediated anti virai activity following butyrate treatment (Figure. 39.C) could be due to the less important role of shorter fatty acid containing Gb, in this process. PPMP-treated XF-498 cells expressed only minimal Gb, (Figure. 39.A) and were resistant to VT1 cytotoxicity (Figure. 39.B). IFN-a mediated anti viral activity against Echo 11 virus aiso was significantly reduced by PPMP treatment in these cells (Figure. 39.Q supporting a role for long chain fatty acid containing Gb, isoforms in IFN-a mediated anti viral activity. IFN receptor mediated signal transduction is a very fast event in which type 1 IFN-induced tyrosine phosphorylation of IFNARl was observed within 1 minute and Tyk2 phosphorylation within 5 min (Constantinescu et al., 1994). ISGF3 binding to ISRE of 2-SAS also is complete within 15 minutes afier IFN-a treatment (Ghislain et al., 1994). Very fast kinetics of IFN-a-mediated signaling events rnay indicate that the induction of signal transduction may be a ce11 surface event and is independent of ligand- receptor internalization. Since long chain fatty acid Gb, isoforms are expected to be preferentially localized in the ce11 surface plasma membrane, role of Gb,, at least in signal transduction for IFN-a-mediated anti viral activity should also be the ce11 surface event. The prevention of IFN-a internalization by inhibition of clathrin dependent receptor mediated endocytosis, using cytosolic acidification did not affect the nuclear translocation of Statl (Figure. 40) and had a smaller effect on anti viral activity as compared to growth inhibition (Figure. 42). The reduction of surface available Gb, during the early events of signai transduction may attenuate the IFN-a-mediated signal transduction and anti viral activity in VTlB treated ver0 cells (Figure. 33 and 34B). Previous studies have identified that intedization of 1FN-a is essential for both antiproliferative activity (Killion et al., 1994) and anti viral activity (Anderson et al., 198 1 and Yonehara et al., 1983). The intracellular delivery of interferon by liposomes alone is not suficient to mediate 1FN-u mediated antiproliferative activity and internalization of 1FN-a is necessary for this hction (Killion et al., 1994). The anti viral activity cannot be induced when the receptor-mediated endocytosis of IFN-a is prevented by temperature block or NaF (Yonehara et al., 1983) or by treatment of the cells with primary amines (Anderson et al., 1981). A study on mouse L cells expressing non- secreted human type 1 IFN has shown that intracellular IFN also is capable of activating 2-5AS gene and inducing anti viral activity but with a lesser extent than the events following extracellular IFNI type 1 IFNR interaction, followed by intemalization. Therefore, internalized IFN, following high aflkïty ligandheceptor interaction may Mercontribute to the biological activities via surface receptor-independent mechanism (Rutherford et al., 1996). However, microinjection of type 1 IFN into the cytoplasm (Huez et ai., 1982) or into the nucleus (Riviere and de Maeyer-Guignard, 1990) could not induce an anti Wal state, indicating that an initial ligandheceptor interaction at the ce11 surface is critical for the anti viral activity. The comparison of the dose dependent effect of RME inhibition by cytosolic acidification on IFN-a-mediated antiproliferative activity and anti viral activity (Figure. 42) indicated that anti viral activity is less dependent on the ligand/receptor internalization. This finding also is consistent with the proposed role of ce11 surface-restricted long chab fatty acid Gb, isoforms in anti viral activity rather than the antiproliferative activity. In conclusion, the G4 dependency of the IFNARI mediated anti viral activity primarily involves Gb, with longer fatty acid species. The Gb, dependency of VTl cytotoxicity (and 1FN-a growth inhibition) is primarïly a function of short chain fatty acid containing Gb, species involving intemalization and intracellular traficking. These data thus, for the fïrst the, suggea functional differences for the lipid heteromers of glycolipids. 4.6 Future directions Although the activation of Stat proteins can also be achieved afier iFN-a treatment of IFN-resistant cells, high affrnity receptor binding may induce a sufficiently prolonged activation of Stats for sustained biological activities. Gb, is likely involved in this process by facilitating the proper ligand/receptor interaction at the ce11 surface. However, due to the diverse nature of type 1 IFN subspecies, multi-components of the type 1 IFNR, the diversity of transcnptional factors and ISGs, and multiple fiinctions of type 1 IFN, the identification of specific rote for Gb, in type I IFN/receptor-mediated biological activities will be very complex. The interferon-inducible enzymedproteins, critical for IFN-a-induced biological activities are usually the products of ISRE-containing ISGs and such genes are speci fically activated by the Stat 1-Stat2-containing multi-component gene transcription factor, ISGF3. Other Stat proteins bind to the GAS element or the GAS-like element of ISGs with no ISRE. The role of Gb, in ISGF3-dependent activation of ISE-containing ISGs has been identified. IFNARl is constitutively associated with Tyk2 at the cytoplasmic end and activated Tyk2 serves as a docking site for Stat2, which mer recruits and activates Statl. Stat2-containhg ISGF3 only has ken found to activate ISRE-containing ISGs and either homo- or hetro-dimers of al1 other Stat proteins bind to promoter elements other than ISE. Since the potential association between type 1 IFNR and Gb, is through IFNARI, the role of Gb, in IFN-a signaling is very likely through IFNARlRyWStatS-dependent ISGF3 formation. However, the significance of Gb, in the activation of other Stat proteins and DNA-binding to non-ISRE elements should also be identified to focus the specific signal transduction mechanism facilitated by Gb,. IFN-a-induced Stat3 activation is via direct interaction with phosphorylated IFNARl and independent of Tyk2. Stat3 may form a homodimer or a hetrodimer with Statl and binds to GAS-like elements with small sequence variations around a consensus GAS element (Zhong et al., 1994). The role of Gb, in Tyk2-independent activation of non-ISRE-containing ISGs should be studied by examining the 1FN-a-mediated Stat3 binding to the nuclear extracts of Gb,-positive and -negative cells (Wiiliams and Haque, 1997). Another Stat protein, StatS is constitutively associated with Tyk2 and activated StatS serves as a docking site for an adaptor protein CrkL. The resulting CrkL-StatS translocâtes to the nucleus and activates GAS-containhg ISGs. The role of Gb, in Tyk2- dependent activation of GAS-containing ISGs should be studied by StatS-CrkL binding to the nuciear extracts of Gb,-positive and -negative cells (Fish et ai., 1999). To further emphasize the role of different chain length Gb, isofoms in 1FN-a mediated antiviral activity, the studies can be dme in Gb, negative cells reconstituted with either long chain fatty acid containing isoforms or short chah fatty acid containing isoforms only. Cbapter 5 Gb, dependent intracellular transport mechanisms of verotoxin 1 Abstract Afier binding to its glycosphingolipid receptor; globotriaosyl cerarnide (Gb3), verotoxin enters the cells primarily by clathrin dependent receptar mediated endocytosis. Unlike many other protein toxins, which dissociate into subunits and translocate fiom the membrane to the cytosol from the endocytic pathway, VT undergoes retrograde transport to the biosynthetic/secretory pathway and the membrane translocation of the subunith to the cytosol is presumed to take place fiom Golgi or ER. Filipin, an inhibitor of clathrin- independent, caveolar-mediated endocytosis, partially inhibits the intemaiization of VTlB subunit. The functional significance of clathrin-dependent endocytosis and caveolar-mediated endocytosis in VTl B intemalization and VTI induced cytotoxicity have been correlated in the present study. Clathrin mediated endocytosis is found to be the principal route of VT1 intemalization and functionally significant for VT1-induced cytotoxicity. In addition, VTI can be intemalized by caveolar-mediated endocytosis as an alternative pathway. VTI undergoes retrograde transport to ER and nuclear membrane in a Golgi dependent manner. However, the targeting of the caveolar associated protein, caveolin, directly to the ER fiom the plasma membrane upon cholesterol oxidation (Conard et al., 1995) and vascular EGF-induced transport of caveolin to the nucleus (Feng et ai., 1999) has ken reported, indicating the Golgi-independent caveolar-mediated transport to ER and the nucleus. The significance of the Golgi dependent and the caveolar-mediated, Golgi-indcpendent VT1 intracellular targeting for VTl cytotoxicity has been studied. The relative importance of the two intemaiization routes varies as a fhction of both time and VT1 concentration, At a concentration of VTI lower than 50 ng/ml, the cytotoxicity of VTI is absolutely dependent on the intact Golgi structure. However, at 24 hr incubation with VTl higher than 50 ng/ml, VTI intemalization and cytotoxicity mediated by caveolar-dependent endocytosis becomes significant. BFA, which prevents Golgi dependent retrograde traffic, protects cells from low VTI concentrations but not following prolonged toxin exposure or at higher VTI concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to protect cells against VTI cytotoxicity. A novel alternative pathway for targeting VTl and its receptor glycolipid, Gb, directly to the ER and nucIear membrane via caveolar-mediated Golgi-independent transport is now reported. Introduction In eukqotic cells, protein molecules are transported fiom one cornpartment to another via vesicular-mediated transport (Pryer et al., 1992). In the biosynthetic or secretory pathway, newly synthesized proteins are folded in ER and transported from ER to Golgi compartments via vesicle-mediated trafEcking. Further proteolytic processing and oligosaccharide modification of the proteins takes place in Golgi compartments where appropriate enzymes reside (Pryer et al., 1992). The trans-Golgi network serves as a sorting station for plasma membrane proteins, secretory proteins and lysosorna1 enzymes (Pelham and Munro, 1993). Afier the necessary protein modifications, ER resident proteins bearing a C terminal tetrapeptide sequence KDEL or HDEL are recognized and retrieved by KDEL receptors in the Golgi apparatus and remto the ER by a retrograde transport pathway (Townsley et al., 1993, Munro., 1995). On the other hand, a variety of proteins such as LDL, transfemn, insulin and EGF are taken up into the cells by the endocytic pathway. Certain protein toxins utilize the endocytic pathway to gain access into cells (Monteccuco et al., 1994). Endosornes fûnction as sorting organelles for the incoming proteins in which ligand-receptor dissociation, due to endosomal acidification takes place (Schwartz, 1995). Some proteins are recycled to the plasma membrane and some are degraded in the lysosomes. The active subunits of some protein toxins translocate to the cytosol from the acidic endosomal compartments (Mukhejee et al., 1997). However, some protein toxins escape fiom the endosomal degradation and enter the biosynthetid secretory pathway such as the Golgi and ER in a retrograde manner (review Lord and Roberts, 1998) via Apt-clahin-coated vesicle- rnediated transport from early endosornes to TGN (Mallard et al, 1998). Afier binding to either glycoprotein or glycolipid receptors, protein toxins enter the susceptible cells by clathrin-dependent receptor mediated endocytosis or clathrin- independent endocytosis or both (Montecucco et al., 1994). For most glycoprotein receptor binding ligands, ligand-receptor complexes are sequestered into clathrin coated pits. The cytoplasmic domains of the receptors are important for recruiting adaptor proteins on the cytoplasmic side of the plasma membrane, and sorting and aggregation of ligand-receptor complexes into the coated pits (Schwartz, 1995). Heparin-like growth factor binding DT and a2-macroglobulin binding ETA (Montecucco et al., 1994) are the exarnples of glycoprotein receptor binding toxins which enter the cells by clathrin- dependent endocytosis (Montecucco et al., 1994). In contrast, glycolipid receptor binding toxins such as TT and CT, enter the cells via clathrin-independent endocytosis (Mmtecucco et al., 1994). Ricin, which binds to the terminal galactose of glycoproteins and glycolipids enters the cells by both clathrin dependent and independent mechanisms (Sandvig and van Deurs, 1996). Caveolae are flask-shaped membrane invaginations of the plasma membrane and caveolar mediated endocytosis is one of the examples of clathrin independent endocytosis (van Deurs et al., 1989). The lipid-based microdomain structure of caveolae is made up of caveolin and speci fic lipids such as gl ycosphingolipids and cholesterol (Rothberg et al., 1992). A study on a mouse transfonned ce11 line, PAM 212 showed that GPI-anchored proteins (ïhy- 1 -2,f32-microglobulin), GSL (lactosyIceramide, Gb, and Forssman antigen) and sphingomyelin, randomly distributed on the ce11 surface became concentrated in the caveolae after antibody crosslinking (Fujimoto, 1996). Therefore, caveolar mediated cIathrin independent endocytosis seems to be a favorable mechanism for the endocytosis of lipid receptor binding toxins. Despite the different nature of the endocytic mechanisms, ligands intemalized by either clathrin dependent or independent pathway are delivered to the endosornes (Tran et al., 1987). However, upon cholesterol oxidation, the caveolar associated protein; caveolin, is redistributed directly to ER from the plasma membrane, bypassing the Golgi (Smart et al., 1994). Verotoxins are a farnily of subunit protein toxins produced by some strains of Escherichia coli and are known to be associated with hemolytic uremic syndrome and haemorrhagic colitis (Karrnali, 1989). VT comprises a single enzymatic subunit A and a non-covalently associated receptor binding B subunit pentamer. The subunit A inhibits pratein synthesis by removal of a single adenine residue from 28s RNA of the 60s ribosomal subunit (Sandvig and van Deurs, 1996). The subunit B binds to the ce11 surface glycolipid receptor; Gb, (Lingwood, 1993). VT is the first descnbed lipid receptor binding protein toxin, which enters the cells by clathrin dependent receptor mediated endocytosis (Sandvig et al., 1989 and Khine & Lingwood, 1994). It has been reported recently that caveolar mediated endocytosis inhibitor, filipin partially inhibits VT binding subunit B internalization (Schapiro et al., 1998), suggesting an alternative route of VT internalization. The number of caveolae and type of lipid microdomains associated with caveolae Vary fiom ce11 to cell (Mukhe jee et al., 1997). A study on ACHN rend tubular endothelial ce11 line showed that Gb, is already pre-associated with detergent-insoluble domains, suggested to be caveolae (Katagin et al, 1999). To intoxicate the susceptible cells, at least the enzymatic subunits of the toxins need to translocate the membrane of intracellular organelles to gain access to their specific targets in the cytoplasm or cytoplasmic side of the membrane. Some protein tosins such as DT and TT, dissociate in the acidic endosomal cornpartment and catalytic subunits translocate the membrane fkom endosornes (Montecucco et al., 1994). Therefore, cytotoxicity induced by such toxins cm be protected by the dnigs, which interfere with the endosomal pH (eg. Bafïlomycin; the inhibitor of vacuolar ATPase H+ pump) (Schapiro et al., 1998). In contrast, other protein toxins such as CT, LT, ETA, ST, VT and ricin escape fiom the degradation in the endosomal system and enter the biosynthetic/secretory pathway in a retrograde manner and the toxins translocate the membrane from either Golgi complex or ER (Mallard et ai., 1998). Consequently, the dmçs which reversibly destroy the Golgi structure such as brefeldin A protect the cells from toxin induced cytotoxicity (Donta et al., 1993, Yoshida et al., 1991, Garred et al., 1997). The retrograde transport of some toxins such as CT, ETA and LT can be explained by the presence of ER retention signal, KDEL in CT and LT (Majoul et al., 1996) or KDEL like sequence; REDLK in ETA (Chaudhary et al., 1990). However, the bais of retrograde transport of VT, which does not contain an ER retention signal. is still unknown. CD19 is a B-ce11 specific antigen, the extracellular domain of which has high amino acid sequence homology to VTlB subunit, suggesting a possible physical association between CD19 and G4 on the ce11 surface (Maloney and Lingwood, 1594). Evidence of physical associations between these two molecules has been reported (Maloney and Lingwood, 1994 and Khine et al., 1998). After antibody crosslinking, CD 19 is internalized and targeted to the ER and nuclear membrane in the same manner as VT1 retrograde transport in Gb,-positive cells (Khine et al., 1998), indicating that Gb, itself may be the factor responsible for the retrograde transport of Gb,-bound ligands. High VT sensitivity correlates with intracellular targeting of the toxid receptor complex to the ER/ nuclear membrane, rather than the Golgi, which in turn correlates with the presence of shorter fatiy acid containing Gb, isoforms (Arab and Lingwood, 1998b). It has been hypothesized that Gb, isofonns with shorter fatty acid chahs could be easily accomrnodated into smaller dimensional vesicles or vesicles with less bilayer thickness and transported closer toward ER and nuclear membrane (Lingwood, 1996 and L ingwood, 1999). In the present study, the hctional significance of different mechanisms of VT 1 endocytosis and intracelluar transport pathways has been correlated with VTI intracellular targeting and VT I induced cytotoxicity. 5.3 Materials and Metbods Materials Chemicals: Brefeldin A, filipin, nocodazole, BSA, tritonX-100 and streptolysin O were from Sigma, St. Louis, MO. 'H-leucine was from Amersham. Acridine orange, ethidium bromide, and FITC were from Molecular Probe, Eugene, OR. Verotoxin: As described in 3.3.1. Immunoreagents: VTlB subunit was purified and antibody to VTlB was raised in Lingwood laboratory. Antibody to Rab6 was fiom Santa Cruz Biotechnology Inc., Santa Cu,CA. Mouse IgG 1 and FITC-conjugated goat-anti-mouse secondary anti body were from Sigma. Texas red-labeled wheat germ agglutinin and FITC-labeled transfenn were from Molecular Probe. Cell Culture: Vero cells fiom Amencan Type Ce11 Culture (Rockville, MD) were grown in a-MEM with 5% fetal caif senim and 40 pghl gentamycin at 370C in the presence of 5% CO-. Fetal calf senun, gentamycin, trypsin-EDTA and a-MEM (minimum essential medium) were from Gibco-BRL. 5.3.2 Methods 5.3.2.1 VT1 cytotoxicity assay Vero cells grown to confluency were trypsinized and 100p1 of 1.5xl0~/mlce11 was seeded in 96 well ce11 culture plates for 24 hr at 37OC prior to the experiment. Then, serial dilutions of VTl were added and ceIl culture plates were Further incubated at 370C for another 48 hr. In some experiments, cells were pretreated with indicated concentration (as described in respective expenments) of BFA, filipin, nocodazole, sucrose or acetic acid containing medium for 30 min at 370C followed by incubation with the toxin in the presence of the inhibiton. At the end of the incubation period, the cells were fixed with 2% formaldehyde in PBS and stained with crystal violet as described (Petric et al., 1987). The percentage of live cells was calculated fiom absorbance of destained cells read at 570 nrn using a microtiter plate reader (Dynatech Laboratories). 5 3.2.2 Cytochemical staining of apoptotic cells To examine the morphological changes in the nuclear chromatin, cells undergoing apoptosis were identified by acridine orange/ethidiurn bromide staining of nuciei (Vasconcelos, 1994). Afier incubation of ver0 cells with VTI in the presence or absence of BFA for 24 hr at 370C, cells in the incubation medium and trlpsinized ceils were pelleted. After washing with PBS, cells were resuspended in the nuclear stain (100 pg/ml acridine orange and 100 pg/ml ethidiurn bromide) and examined under UV illumination. Cells with condensed chromatin and apoptotic bodies were scored as apoptotic cells. At least 200 cells were counted for each experiment. 5.3.2.3 Protein synthesis inhibition assay Vero cells were prepared in 96 well plate for the experiment. At the end of the indicated experiment, medium was rernoved and cells were incubated in 200 pl of the same medium containing 1 mCi/rnl 3~-leucine(Amersham) for 10 min at 37OC. After washing, cells remained in the plate were washed twice with 100 pl of 5% trichloroacetic acid and precipitated protein in the wells was solubilized with 100 pl of 0.1M KOH.Acid precipitable radioactivity was measured by using Bechmann LC 3800 C counter. FITC labeled VTlB subunit internalization for fluorescence microscopy Vero cells grown on coverslips were incubated with medium containing 0.5 &mi FITC-VT1B at 370C for 3hr or 24 hr and fixed at room temperature with 4% paraformaldehyde in PBS for 15 minutes. The coverslips were mounted on the microscope slide, using fluorescent mounting medium (DAKO) and the cells were examined using a Polyvar fluorescence microscope. In some experirnents, cells were pretreated with indicated concentration of BFA, filipin, nocodazole, sucrose or acetic acid containing medium for 30 min at 370C followed by incubation with the toxin in the presence of the inhibitors for indicated duration. 5.3.2.5 Indirect immunofluorescence labeling At the end of indicated experiments, cells were fixed as described above and permeabilized using 0.1 % Triton X-100 in PBS at room temperature for 10 min. Afier blocking of non-specific protein binding by using 1% %SA and 0.1 % Triton X-100 in PBS, the cells were immunostained by using polyclonal primary antibody to Rab6 (SantaCnrz Biotechnology Inc.) or monoclonal antibody to VTlB subunit followed by FITC conjugated secondary antibodies. As control studies, rabbit pre-immune semor mouse IgGl was used in place of pnmary antibody incubation. For organelle staining, cells were incubated with 5 pg/ml Texas red conjugated wheat germ agglutinin or 50 pg/ml FITC conjugated transfemn 370C for 45 min. For double labeling with VTlB immunofluorescence, Texas red labeled WGA was added during incubation with secondary antibody. 5.3.2.6 Streptoiysin O permea bilization SLO 50 IU/rnl in SLO buffer (1 15 mM KAc, 25 mM HEPES, 2.5mM MgC12, ImM dithiothrietol) was preactivated by incubating at 370C for 30 min and kept on ice until use. At the end of indicated experiments, cells grown on coverslips were kept on ice and washed twice with cold PBS containing 0.5 mM CaC12, 1 mM MgCl2 and once with cold SLO buffer. Activated SLO was added to the cells and incubation was continued on ice for 10 min. Unbound SLO was washed twice with SLO buffer and the cells were incubated at 37% for 30 min in 37oC prewarmed SLO buffer to allow SLO permeabilization (Andersson et al., 1997). Afier washing with PBS, cells were fixed with 4% paraformaldehyde and immunostained by using anti-VTlB or mouse IgGl as a contro!, and FITC Iabeled secondary antibody. Results Effect of BFA on VTI induced cytotoxicity BFA is a fimgal metabolite, which eliminates the Golgi complex as a morphologically distinct organelle. In the presence of BFA, the cis, medial and trans Golgi redistribute to the ER (Lippincott-Schwartz et al., 1990) and the tram-Golgi network fuses with early endosornes (Wood et al., 1991). The significance of VT retrograde transport to Golgi and ER for VT induced protein synthesis inhibition has been studied. BFA completely protects ver0 cells fiom VT induced protein synthesis inhibition afier 3 hr treatment with VT (Sandvig et ai., 1991, Garred et al., 1997). However, 10 to 20 times higher dose of BFA was required to protect against VT cytotoxicity than to protect against ricin cytotoxicity (Sandvig et al., 1992, Garred et al., 1997). Donta et al., 1995 reported that BFA protects protein synthesis inhibition induced by lpg/ml of VT1 but the protection is not sustained after 6 hr toxin treatment. Therefore, the effect of BFA induced Golgi disruption on VTl mediated total ce11 death, apoptosis and protein synthesis inhibition afier short-term (3 hr) and long-term (24 hr) VTl treatment on ver0 cells were investigated in the present study. Since VTl cytotoxicity assays for ce11 killing and apoptosis took at least 24 hr, the effective dose of BFA in ver0 cells which was sustained until24 hr was identified by immunofluorescence staining of the Golgi with the tram and media1 Golgi marker, Rab6 (Figure. 43A). Loss of distinct Golgi staining was observed in the presence of BFA after incubation at 370C (Figure. 43A, panel b). Golgi disruption was found as early as 30 min but a dose dependent effect sustained for 24 hr was identified by counting at least 200 cells irnrnunolabeled with Rab6 in Figure. 43A (Figure. 43B). The effective dose; 0.1-1 pg/ml was used for the following experiments. The effect of BFA on VT1 induced total ce11 death was examined aîter treating ver0 cells with VTI in the absence or presence of BFA for 24 hr. At concentrations of VT1 lower than 50 ng/ml, protection of VTI cytotoxicity by BFA was found to be very efficient after 24 hr incubation but was only partial at >50 ng/ml (Figure. 44.A). control + 0.5 ug/ml BFA Figure. 43A Effect of BFA on Golgi complex organization. Indirect imrnunofluorescence labeling of trans- and medial Golgi protein Rab6 in conhol vem(a) and BFA treated vero(b) cells. BFA (uglml) Figure. 43.B Dose dependent effect of BFA on Golgi disruption. (n = 4) Similar partial protection only was observed for VTI induced apoptosis of ver0 cells after 24 hr treatment with >50 ng/ml (Figure. 44.B). The incomplete protection by BFA at higher dose of VTI (in term of total ce11 killing) was discrepant from an earlier report that BFA completely protects vero cells from ST induced protein synthesis inhibition afier short term treatrnent (Sandvig et al., 1991 and Garred et al., 1997). Therefore, effect of BFA on VTI induced protein synthesis inhibition at short tenn (3 hr) and long term (24 hr) treatment with VTI was examined. Consistent with the previous reports (Sandvig et al., 1991, Garred et al., 1997 and Donta et al., 1992) and our present finding, although the protection was complete for short term VTl treatment (Figure. 49, it was only partial for higher dose of VT1 at long term treatment (Figure. 45). 5.4.2 Effect of BFA on internalization and intracellular targeting of VTlB in ver0 cells Despite the sustained BFA-induced Golgi disruption (Figure. 43 A and B), VTl cytotoxicity and apoptosis were not protected at VT1 dose >50 @ml (Figure. 44 and 45). The effect of BFA treatment on intracellular distribution of VTI was Merexamined. To avoid the complication of cytotoxicity induced by holotoxin, VTlB subunit was used for the internalization study. Afier 3 hr incubation with FITC labeled VTlB at 370C, internalized VTl B was found mainly in the Golgi (Figure. 46.A panel a) as described by Kim et al., 1996. Mer 24 hr treatment, VTlB distribution was fond more intensely around the perinuclear ER like structure (Figure. 46A panel c). In the presence of BFA, loss of distinct Golgi labeling by FITC-VTIB was observed at short tem incubation. Difhsed FITC-VT1 B labeling was found in most cells but some cells with more concentrated staining at one intracellular compartrnent also were observed (Figure. 46A panel b). Such restricted labeling became more intense afier 24 hr treatment with FITC- VTl B (Figure. 46A panel d). The effect of BFA on VT 1 intemalization was also studied by indirect immunofluorescence labeling of internalized VTl B subunit (Figure. 46B). - VTIonly - + 0.1 ugml BFA - + 0.5 uqlml BFA VTï log @ml Figure. 44.A Effect of BFA on VTl induced total ce11 kiliing. (n = 4) Figure. 44.B Effect of BFA on VTl induced apoptosis. (n = 4) VTI log nglml Figure. 45 Effkct of BFA on VTl induccd protein synthesis inhibition. (n = 4) - BFA + 0.5 ug/ml BFA Figure. 46A FlTC labeled VTlB înternalization in vero cds. At 3hr(a and b) and 24hr(c and d), in the absencda and c) and in the presence(b and d) of BFA. - BFA + 0.5 ug/ml BFA Figure. 468 Indirect immuno fluorescence of internalized VTlB in vero cds. At 3hr(a and b) and 2&(c and d), in the abeence(a and c) and in the presence(b and d) of BFA. Similar compartments with restricted intracellular staining was also observed and gradua1 inhibition of VTlB entry at the ce11 surface was better noted by this method, in BFA treated cells (Figure. 46B panel b and d). To identiQ the cornpartment where VTI accumulated in BFA treated cells, double labeling studies of intemalized FITC-VTIB and organelle marken were done (Figure. 47). The distinct pattern of TGN labeling by Texas-red conjugated WGA in control cells (Figure. 47A panel a) became difhed at 3 hr afier BFA treatment (Figure. 47A panel b) and collapsed into a tightly fluorescent labeled cornpartment afier 24 hr BFA treatment (Figure. 47A panel c). FITC-VTIB was fouad to be CO-localizedwith collapsed TGN (Figure. 47A panel c and d). Labeling of early endosornes with FITC labeled transfemn (Figure. 47B panel a) was not affected by BFA treatrnent at 3 hr the point (Figure. 47B panel b). However, early endosomes (Figure. 47B panel c) were found to be iüsed with TGN (Figure. 47B panel d) afier 24 hr BFA treatment. In summary, in the presence of BFA, intemalized VTlB (5pg/ml) was mainly accumulated in collapsed TGN, which later became fbsed with early endosomes. Therefore, incomplete protection effect of BFA for VTI cytotoxicity during BFA-induced Golgi disruption could possibly be due to membrane translocation of VT fiom collapsed TGN or due to a Golgi independent alternative transport of VT1. 5.4.3 Identification of an alternative route of VT1 iaternalization Depending on the ce11 type, Gb, binding toxins; ST and VT, enter the cells by clathrin dependent receptor mediated endocytosis (Sandvig et al., 1989 and Khine and Lingwood, 1994) as well as by caveolar mediated endocytosis (Schapiro et al., 1998). The hctional significance of different endocytosis mechanisms for VTI induced cytotoxicity was correlated in this study. Effect of clathrin dependent or caveolar dependent endocytosis inhibitors on FITC-VTl B internalization and VT1 induced total ce11 death was examined. Hypertonie media containing 0.45 M sucrose inhibits receptor TxR-WGA TS-WGA control vero + 0.5 ug/ml BFA for 3hI + 0.5 ug/ml BFA for 24hr + 0.5 ug/ml BFA for 24hr FITC-den tran Ta-WGA Figure. 47A mect of BFA on FiI%-VTlB internalization in vero cells (double labehg with TGN rnarker). Texas-red conjugated WGA staining in conhl cells(a) and after 24 hr trea tment wi th BFA(b). Double labeling of internalized FITC-VTlB(c) and texas-red MrGA(d), at 24 hr treatment with SFA. FITC-des- FITC-dextrui control vero + 0.5 ug/ml BFA for 3hr + 0.5 ug/ml BFA for 24hr + 0.5 uglml BFA for 24hr TxR-WGA FITC-dertrur Figure. 478 Wect of BFA on TGN and early endosorne markers in ver0 cells (double labeling). FITC conjugated dextran (early endosorne marker) stallUng in control ceiis(a) and after 24hr treatment with BFA(b). Double labeling of texas-red WGA (TGN marker)(c) and FITC dextran(d), at 24 hr treatment with BFA. mediated endocytosis by preventing clathrin coated pit formation (Heuser et ai., 1989) and reducing intemal pH of the cells by acetic acid prevents coated vesicles pinching off from the plasma membrane (Heuser., 1989). Both procedures completely prevented VTlB intemalization (Figure. 48A panel b and c) and protected vero cells fiom VTl induced cytotoxicity (Figure. 48B). Firipin; a sterol binding drugo inhibits caveolar mediated endocytosis by disassembling caveolar organization and preventing invagination of caveolae (Rothberg et al., 1992). Treatment of the cells with 10 ughl filipin partially prevented VTIB intemalization (Figure. 48A panel d) and partially protected the cells from VTI cytotoxicity (Figure. 48B) but the effect of filipin was minimal, compared to the effect of other clathrin-rnediated endocytosis inhibitors. To confirm the significance of caveolar mediated endocytosis in VTl intemalization and VT 1Oinduced cytotoxicity, dose dependent effect of filipin on VTl cytotoxicity was examined. Inhibition of VT1 cytotoxicity by filipin was found to be more significant at higher dose but due to the cellular toxicity of filipin itself, the effect of filipin higher than 20 &ml couid not be determined (Figure. 49A). In addition. combination effect of BFA and filipin on VTl cytotoxicity was also observed especially at higher dose of VTl, at which BFA alone could not protect the cells (Figure. 49B), suggesting that caveolar mediated endocytosis could still be operating or up-regulated while Golgi structure was disrupted and Golgi-dependent retrograde transport was inhibited. Caveolin can be distributed directly to the ER fiom the ce11 surface upon cholesterol oxidation (Smart et al., 1994) and microtubules fhction as highways for the membrane trafficking (Lippincott-Schwartz and Cole, 1995). Combination effect of microtubule disrupting agent; nocodazole, and filipin on VTl induced cytotoxicity was examined (Figure. 49C). The additional protection against VTl cytotoxicity by filipin and nocodazole together was observed and it was Merenhanced in the presence of BFA, indicating the ongoing microtubule dependent caveolar mediated VT 1 trafficking while Golgi dependent retrograde transport was suppressed. control vem cells + 0.4SM sucrose + 30mM acetic acid + 10uglml filipin Figure. 48.A Effect of endocytosis inhibitors on FITC-VT I B intemalization (30 min at 37dZ)- Figure. 48.B Effect of endocytosis inhibitors on VTI cytotoxicity. (n = 4) 1000 1 O0 10 1 o. 1 0.01 VTl ng/ml Figure. 49.A Dose dependent effect of filipin on VTI cytotoxicity. (n = 4) VTI oniy + BFA OSug/ml + Filipin 20 ug/ml C/,1 +B+F Figure. 49.B Effect of BFA and filipin on VTl cytotoxicity. (n = 4) Figure. 49.C Effect of BFA, filipin and nocodazole on VT 1 c ytotoxicity. (n = 4) 5.4.4 Membrane translocation of VTlB into the cytoplasm Streptolysin O permeabilization method was used for VTl B immunofluoresence to examine the toxin translocated into the cytoplasm. Since SLO selectively perforates the plasma membrane, sparing the membrane of intracellular organelles (Andersson et al., 1997), the irnmunofluorescence labeling following SLO treatment detected the VTlB in the cytoplasm or on the cytoplasmic side of the membranous structures. The cytosolic distribution of VTlB was observed at 3 hr treatment with VTlB (Figure. 50A panel a) and both dified labeling in the cytoplasm and more intense perinuclear staining was observed at 24 hr incubation (Figure. SOA panel c). Cytosolic distribution Iikely represents fiee vesicles in the cytoplasm and perinuclear labeling could be the toxin on the cytoplasmic side of RER. In the presence of BFA at 3 hr treatment, cytosolic translocation of VTI B was prevented (Figure. 50A panel b) but &er 24 hr, perinuclear or nuclear membrane labeling of VTlB imrnunofluorescence was observed in BFA treated cells (Figure. 50A panel d). The inhibition of VTlB entry in BFA treated cells was also found as in Figure. 44B. To investigate the transport mechanism of VTiB to the perinuclear region in BFA treated cells, the cells were pretreated with BFA and filipin together (Figure. 50B). In the presence of filipin, nuclear membrane labeling of VTlB in BFA treated cells (Figure. 50B panel a) was totally abolished (Figure. 50B panel b), clearly indicating the Golgi independent transport of caveolar mediated VT 1 internalization directly to the perinuclear regàon or the nuclear membrane. - BFA + 0.5 ug/ml BFA Figure. 50.A Effect of BFA on cytosolic translocation of internalized VT lB (indirect immuno fluorescence in SLO permeabilized vero ceIl$ At 37oC for 3 hr(a and b) and 24 hr(c and d).In the absence of BFA(a and c) and presence of BFA(b and d). + 0.5 uglml BFA + 0.5 ug/ml BFA and 10 ug/ml fiiipin Figure. 50.B Effect of BFA and filipin on cytosolic translocation of intemalized VTl B (indirect imrnuno fluorescence in SLO penneabilizcd vero cetl$ In the presence of BFA(a) and BFA+filipin(b). 5.5 Discussion Protein toxins usually enter the cells after binding to their specific receptors on the ce11 surface (Montecucco et al., 1994). Depending on the nature of the receptors, the mechanisms of endocytosis vary. Clathrin dependent endocytosis is usually found to be associated with receptor mediated endocytosis (Schwartz, 1995). It was generalized that this pathway was the only efficient way of RME and as a result, the terms, clathrin- mediated endocytosis and RME had been used interchangeably (Lamaze and Schmid, 1995). Receptors could be already pre-associated with coated pits or recruited into the coated area after specific ligand-receptor interaction. The movement of ligand-receptor complex into the coated pits is mediated by interactions of adaptor molecules in the cytoplasm and the cytoplasmic tails of protein receptors (Schwartz, 1995). Therefore, clathrin dependent receptor mediated endocytosis becomes the favorable mechanism for the internalization of protein receptor binding toxins such as DT and ETA (Montecucco et al., 1994). In contrast, flask-shaped, non-clathnn coated membrane invaginations, caveolae, are assembled with caveolin protein and lipid microdomains (Rothberg et al., 1992). Consequently lipid receptors of the toxins could aiso be already pre-associated with caveolae. Pre-association of Gb, with caveolae ACHN rend tubular epithelial cells has been reported recently (Katagiri et al, 1999). Mer antibody crosslinking, randomly distributed GSL become concentrated in caveolae (Mayor et al., 1994), indicating that caveolar-mediated endocytosis also is an another mechanism of RME. Therefore, caveolar-mediated, clathrin-independent endocytosis seems to be an appropriate mechanism for lipid receptor binding toxin intemdization. Gb, receptor binding ST and VT are the only lipid receptor binding protein toxins which have been known to enter the cells by clathrin-dependent receptor mediated endocytosis (Sandvig et al., 1989 and Khine and Lingwood, 1994). The endocytosis of ST and VT is prevented by protocols, which influence the clathnn coat formation and invagination (Sandvig et al., 1989). Despite the lipid nature of the receptor, VT internalization was found to be inhibited by the dmgs such as monodansyl cadavenne (Khine and Lingwood, 1994) which inhibits transglutaminase enzyme, involved in the clustering of ligand receptor complexes into the coated pits (Haigler et ai., 1979 and Davies et al., 1980). This finding indicated that Gb, could be recmited into the coated pits through association with some transmembrane ce11 surface proteins. So far, two transmembrane proteins have ken identified to be possibly associated with Gb, on the surface plane of the cell. B lymphocyte ce11 differentiation antigen CD19 (Maloney & Lingwood, 1994) and the IFNARI chah of interferon alpha receptor (Lingwood and Yui, 1992) are the candidate proteins which could be associated with Gb, since the extracellular domains of both have very hi& amino acid sequence homology to VTlB subunit. Possible physical association and functional relationships between Gb, and CD1 9 (Maloney & Lingwood, 1994 and Khine et al., 1998) or IFNARl (Ghislain et al., 1994 and Chapter 4) also have been identified. Gb, co-caps with antibody crosslinked CD 19 (Maloney & Lingwood, 1994) and extracellular domain of IFNARI -1gGl fusion protein binds to Gb, and Gb, on the thin layer chromatography plate (Ghislain et al., 1994), indicating the potential phy sical association between Gb3 and CD1 9flFNARI. Afier the endocytosis, STNT subunit B escapes fiom the degradation in the endosomd system and enters TGN fiom early endosornes via clathrin coated vesicles (Mallard et al, 1998). From TGN, the toxin enten the cornpartment of biosynthetic pathway such as Golgi complex and ER in a retrograde marner (Mallard et al, 1998). Since drugs such as BFA (Sandvig et al., 1991, Garred et al., 1997), nocodazole (Johannes et al., 1997) and ilirnaquinone (Nambiar & Wu, 1995), which disrupt the Golgi structure, protect cells fiom VTl induced protein synthesis inhibition, Golgi and/or ER are believed to be the site of toxin translocation. In the present study, although the disruption of Golgi structure was sustained throughout the 24 hr penod of VTI cytotoxicity assay, BFA did not completely protect the cells fiom VT1 induced cytotoxicity. especially at higher dose of VT1. It has also been reported recently that caveolar mediated endocytosis could be partly responsible for VTl internalization (Schapiro et ai., 1998). Due to the lipid nature of the receptor Gb,, intemalization via this mechanism was well expected. Recent finding by Katagiri et al., 1999, also indicated that Gb3 could be pre-associated in the caveolae. Despite the different mechanisms of endocytosis, intracellular transport of proteins via both clathrin dependent and independent endocytoses can be targeted to endosomes (Tran et al., 1987). However, the protein component of caveolar structure; caveolin. is transported directly from the plasma membrane to ER (Smart et al., 1994) and to the nucleus (Feng et al., 1999), indicating that caveolar mediated endocytosis could be the mechanism of VT1 intracellular targeting in a Golgi-independent mamer. Synergistic protective effect of BFA and filipin against VTl cytotoxicity (Figure. 49.B) also indicated the presence of Golgi-independent VTl ûanspon while the Golgi structure was disrupted. Vesicle-mediated intracellular transport requires the tdc guided by microtubules (Lippincott-Schwartz & Cole, 1995). Partial protection of VTI cytotoxicity by filipin and nocodazole, and the additional protection observed in the presence of BFA induced Golgi disruption (Figure. 49C) indicated the existence and/or upregulation of caveolar mediated microtubule dependent targeting for VT 1 induced cytotoxicity , when Golgi dependent retrograde transport was disturbed or down-regulated. The correlation between mechanism of endocytosis and VTl cytotoxicity in Figure. 48 and 49 showed that clathrin dependent endocytosis is the dominant route of VT1 intemalization responsible for VTI induced cytotoxicity while filipin-sensitive caveolar mediated endocytosis could still be an alternative pathway for VT1 induced cytotoxicity. Since the number of caveolae, on the ce11 surface may Vary from ce11 to ce11 and consequently, the role of caveolar-mediated VTI transport for VTI cytotoxicity may Vary accordingly. During the course of retrograde transport through Golgi and ER, VT may translocate the membrane to reach the ribosomes in the cytoplasm or cytoplasmic side of ER for inhibition of protein synthesis. However, the exact site of membrane translocation is still unknown. The protection of VT1 cytotoxicity by BFA indirectly suggests that Golgi and ER could be the sites of VTI membrane translocation (Donta et al., 1993, Yoshida et al., 1991, Garred et al., 1997). In the presence of intact hctional Golgi cornplex. VTlB was mainly found in the Golgi at 3hr tirne point and progressed to a perinuclear ER like structure and nucleus at longer incubation (Figure. 46A and B). Since SLO selectively permeabilizes the plasma membrane only, VT 1B immunofluorescence observed in Figure. 50 was VTlB present in the cytoplasrn. Membrane translocation of VTlB to fiee isolated cytosolic units, likely vesicles, was observed at 3 hr and to perinuclear targets at 24 hr (Figure. 50). Such pe~uclearstaining could be targeting of VTl B to the cytoplasmic side of ER and nuclear membrane. Although the Golgi disruption was still sustained and most of the intemalized VTl was accumulated in collapsed TGN fwd with early endosomes until 24 hr treatment with BFA (Figure. 46 and 47), VTl was eventually transported directly to the perinuclear region in a Golgi independent manner (Figure. 5O.A panel d). Loss of SLO-permeabilized perinuclear staining in cells treated with both BFA and filipin confmned that perinuclear VTlB targeting in Figure. 50B was due to filipin-sensitive caveolar-mediated VTlB transport. A gradua1 increase in ce11 surface immunofluorescence labeling of VTlB in BFA-treated cells (Figure 46.B panel b and d, Figure 50.A panel b and d. and Figure. 50B) indicated that endocytosis of VTlB from the ce11 surface was eventually perturbed when the Golgi structure was disrupted. The ce11 membrane labeling of the toxin was better observed in indirect immunofluoresence labeling of VTl (Figure 46.B panel b and d, Figure 5O.A panel b and d. and Figure. 50B) compared to FITC conjugated VTlB labeling (Figure. 46A). This could be due to the higher sensitivity of indirect immunofluorescence method. This finding suggested the major role of Golgi-dependent intracellular transport for VT 1 targeting in ver0 cells. Since both clathrin-dependent and caveolar-dependent endocytic pathways may unite at endosomes, the toxin which enters the cells via caveolar-mediated endocytosis also may undergo Golgi-dependent retrograde transport. The plant protein toxin, ricin enters the cells via clathrin-independent, caveoiar- independent endocytosis, in addition to clathrin-dependent and caveolar-dependent pathways (Lord and Roberts, 1998) and VTl intemalization via such pathway has not been excluded. In conclusion, clathrin dependent receptor mediated endocytosis of VTl, and retrograde transport to Golgi complex and ER is the principal mechanism of VTl targeting for the cytotoxic action. However, a novel alternative mechanism of VTl targeting to the ER and nuclear rnembraiie via caveolar-mediated Golgi-independent transport, which also is partly responsible for W1 cytotoxicity, has ken identified in the present study. Future directions Intemalization of ST and VTI by clathrin-dependent endocytosis has ken studied by immuno-electronmicroscopy. However, due to the lipid nature of Gb, receptor, senal dehydration of the cells during preparation for EM, rnay extract Gb, fiom the cells. Consequently, association of Gb, and VTl during internalization has never been studied. Based on the method descnbed by Katagiri et al., 1999, Gb, associated with detergent insoluble microdomain @IM) (caveolae) and Gb, associated with detergent soluble fraction (DS) can be differentiated. Since antibody to Gb, can be used to co- immunoprecipitate Gb3-associated proteins, Gb3-bound VTI can be identified by immunoprecipitation with anti-Gb,, followed by immunoblotting with antibody to VT. Then, ce11 surface Gb,-bound VTl at 4°C incubation in DIM and DS fiactions and the course incubation of the cells at 37°C at early time point can be examined to identiQ the accumulation of VTI associated with DIM (caveolae) or DS (likeiy to be clathrin). Co- immunoprecipitation of Gb,, VTI and other caveolar associated proteins cm be done to examine whether any caveolar associated protein also undergoes retrograde transport together with VTI . By using the similar concept, the kinetics of Gb,-VTl association and dissociation, the kinetics of Gb, recycling etc, during VT1 intemalization cm be studied. By using antibodies, directed to VT subunits A and B, whether VTlA subunit dissociates from Gb,-VTlB, prior to VTlB dissociation from Gb, can also be determined. In addition, Gb, in the nuclear extract of control and VT-treated cells and Gb3-bound VT1 in nuclear extract can also be examined to confirm the Gb,-dependent VTI nuclear transport. Inhibitors of clathrin-dependent or caveolar-dependent endocytosis can also be used to eliminate one pathway or the other. However, the number of caveolae and Gb, expression may vary fiom ce11 to ce11 and thus, the seiection of ce11 type will be cntical. Caveolar-mediated endocytosis can be Golgi-dependent afier uniting with endosomes or Golgi-independent via direct transport to ER or nucleus. Golgi-independent pathway may not require successive budding of membrane vesicles for the retrograde transport to ER and nuclear membrane. Therefore, such transport pathway may be suitable for Gb, isoforms with longer chah fatty acid isofoms, which are expected to be difficult to accommodate into smaller dimension vesicles or thimer membrane of earlier compartments of Golgi and ER However, not only the increased expression of longer fatty acid Gb, isofoms, the abundance of caveolae in the cells also is equally important for this kind of study. Chapter 6 The comparison of VT1 and VTZ internakation and iotracellular targeting Abstract VTI and VT2 are closely related members of Escherichia coli-derived verotoxin family, both of which specifically bind to the Gb, receptor. VTl has been known to enter the cells pnmarily via clathrindependent receptor mediated endocytosis and undergo retrograde transport to the ER and the nuclear membrane via the Golgi-dependent mechanism. Moreover, VTI can be targeted to the pen-nuclear region via Golgi- independent, caveolar-mediated endocytosis and this pathway also is partially responsible for VTl cytotoxicity. The difference in protection fiom VTI and VT2-induced cytotoxicity by the Golgi-disrupting anti-fimgal agent; brefeldin A (BFA) suggested a possible variation in intracellular -cking of VTl and VT2. The effect of BFA and the caveolar-mediated endocytosis inhibitor, filipin on VT1 and VT2-induced cytotoxicity has been examined in the present study. VT2-cytotoxicity was found to be particularly more sensitive to the BFA protective effect whiie VT1-induced cytotoxicity was protected by both BFA and filipin, indicating the presence of an alternative intemalization pathway especialiy for VTl. The intracellular targeting of VTI and VT2 have been examined by immunofluorescence and immuno-electronmicroscopy. VT2 was found to be more restricted to the Golgi while VTI was also found at the peri-nuclear region, in addition to the Golgi localization. The higher affinity Gb, binding of VT1 could be the responsible factor for the alternative internalization pathway of VTI. Introduction Certain serotypes of Escherichia coli such as 0 157:H7, O26:H 11, 0 11 1:NM etc produce one or more types of cytotoxins, al1 of which are reiated to the clinical syndromes of diarrhea hemorrhagic colitis and hemolytic uremic syndrome (Riely, 1987 and Karmali, 1989). These toxins are cytotoxic for gastrointestinal tract epithelium by inducing diarrhea and hemorrhagic colitis. In addition, the toluns are absorbed into the blood Stream and compromise the fùnctions of extra-intestinal organs such as kidney and central nervous system by damaging the vascular endotheliurn of the target organs (review, Tesh & O'Brien, 1991). The first described member of the toxins was named as verotoxin due to the distinctive cytotoxic effect on ver0 cells which was distinguishable fiom E.coli heat labile toxin (LT) induced ver0 ce11 response (Konowalchuk et al., 1977). Since this toxin has the same subunit composition, isoelectric point and biological activities as Shigeiia dysentriue 1-derived Shiga toxin, it has ken described as E-coli Shiga toxin or Shiga-like toxin (O'Brien et al., 1983). E-coli 0157:H7 strain 933 produces more than one kind of cytotoxins but only one cm be neutralized by anti-Shiga toxin antibody. The toxin, neutralized by anti-shiga toxin was described as Shiga-like toxin 1, and the other as Shiga-like toxin II (Strockbine et al., 1986). E-coli 0.1 57:H-strain E32511 also produced a ver0 cytotoxin, the cytotoxicity of which could not be neutralized by antisenun to Shiga toxin. However, the antiserum to E32511 toxin could neutralize strain 933 induced cytotoxicity and thus, E325 1 1-derived toxin was termed VT2 and generally assurned to be synonymous with SLT II (Scotland et ai., 1985). However, it was identified later that SLT II and VT2 also were antigenically different. SLT II induced cytotoxicity \vas completely neutralized by anti-VT2 but VT2 induced cytotoxicity was only partially neutralized by anti-SLT II (Head et al., 1988). E. coli strains such as 0138,0139 , 0141 which cause edema disease in pig also were found to produce a cytotoxin which could be neutralized by ad-SLT II and thus it was named as a variant of SLT II (SLT IIv) (Marques et d., 1987). According to the intemationally standadized toxin nomenclature, VTdSLTs can be classified as class 1, which is identical to ST and others as class II. VTl/SLT 1 is in class 1 and the members of class II are VWSLT II (former SLT II), VT2c/SLT IIc (former VT2) and VTZdSLT IIe (review, Gyles CA, 1992 and Lingwood C-A, 1993). Both VTl and VT2 are 1A5B subunit toxins and the molecular weight of VT2 subunits are slightly larger than VTI subunits. There is 55% amino acid sequence homology between A subunits and 57% between B subunits of VT1 and VT2. The isoelectric points of VT 1 and VT2 A subunits are 11.1 and 9.83 respectively and the B subunits have almost identicai isoelectric points (Jackson et al., 1987). The subunit A of both toxins has the same enzyrnatic activity to inhibit protein synthesis by cieaving N- glycosidic bond at A424 in 28s ribosomal RNA of the 60s ribosomal subunits (Igarashi et al., 1987). The subunit B binds to the glycosphingolipid receptor Gb, of the sensitive cells (Lingwood, 1993). The cytotoxicities induced by VTl or VT2 may Vary with ce11 type. The cytotoxicity of VT1 on ver0 cells is 6 times more toxic than VT2 (Noda et al., 1987 and Yutsudo et al., 1987). Similar cytotoxicity was also reported on human renal cortical epithelial cells (HRCEC) (Kiyokawa et al., 1998). The cytotoxicity of VT can be infïuenced by many factors including the difTerence in intracellular targeting of the toxin. Astrocytorna cells in which VTI targets to the ER and nucleus are more susceptible to VTI cytotoxicity than those with VT1 targeting to the Golgi (Arab et ai., 1998b). Afier Gb, receptor binding, VTl enters the sensitive cells mainly via clathrin dependent receptor mediated endocytosis and partly via caveolar dependent endocytosis (Chapter 5). Subsequently, VTI undergoes retrograde transport to the ER, nuclear membrane or nucleus, pnmarily in a Golgi-dependent manner and partially by a Golgi-independent caveolar-mediated transport (Chapter 5). A fimgal met abolite, BFA, which disrupts the Golgi structure, prevents the retrograde transport of VT1 to the ER and nuclear membrane, and protects the cells fiom VTl induced cytotoxicity (Sandvig et ai., 1991). Since VT2 shares the receptor Gb, with VTl, and VT2 cytotoxicity also is sensitive to BFA (Donta et al., 1999, it has ken postulated that the mechanisms of intemalization are the same. However, the protective effect of BFA against VT2 cytotoxicity has ken found to be stronger and more sustained (Donta et al., 1999, suggesting a potential difference in intracellular transport pathways of the toxins. The differential role of Golgidependent retrograde transport and Golgi-independent caveolar mediated transport in VTI and VT2 intracellular targeting and cytotoxicity has been investigated in the present study. While VTI codd be targeted to the ER and nuciear membrane via both Golgi-dependent and Golgi-independent caveolar mediated transport. VT2 was found to be localized mainly in Golgi and the intracellular transport pathway is more restricted to the Golgi-dependent mechanism. Despite the similar receptor binding specificity of the toxins, the binding afhity of VTI to Gb, is 50 fold stronger than that of VT2 (Jacewicz et al., 1999). Higher affinity Gb, binding of VTl could be the important factor for the aiternate pathway of VTI targeting via caveolar dependent endocytosis. Materials and Methods 6-3-1 Materials Chemicals: Brefeldin A, filipin, BSA, sodium metaperiodate, gelatin fiom cold water fish skin, and Triton X-100 were from Sigma (St. Louis, MO). Fcrmaldehyde was fiom Fisher scientific (Pittsburgh, PA). Glutaraldehyde, paraformaldehyde, osmium tetroxide, uranyl acetate and Epon 8 12 were fiom Polysciences Inc. (Warrington, PA ). Verotoxin: As described in 3.3.1. Immunoreagents: Monoclonal antibody to VTlB and polyclonal antibody to VT2B were raised in the Lingwood laboratory. Mouse IgG 1, FITCconjugated GAM and FITC- conjugated GAR secondary antibodies were from Sigma. 15 nm gold-conjugated GAM and FITC-conjugated GAR secondary antibodies were fiom Zymed Laboratories Inc. (South San Francisco, CA). Cell Culture: Vero cells fiom Amencan Type Ce11 Culture (Rockville, MD) and vero mutant, Gb3 deficient VRP ceIls (Pudymaitis et al., 1991) were grown in a-MEM with 5% fetal calf semand 40 &ml Gentamycin at 370C in the presence of 5% COZ. a- MEM, fetal calf serum, trypsin-EDTA and gentamycin were fiom Gibco-BRL. 6.3.2 Methods 6.3.2.1 VT1 cytotoxicity assay Cells grown to confluency were trypsinized and lOOpl of 1.5xl0j/ml ce11 suspension was seeded in 96 well ce11 culture plates for 24 hr at 37% prior to the experiment. Then, 10 pl of serial dilutions of VT1 were added and ce11 culture plates were fûrther incubated at 370C for another 48 hr. In some experiments, cells were pretreated with indicated concentration of BFA orhd filipin for 30 min at 370C before addition of toxin. At the end of the incubation period, the cells were fixed with 2% formddehyde in PBS and stained with crystal violet as described (Peûïc et al., 1987). The percentage of live cells was calculated fiom absorbante of destained cells read at 570 nm using a microtiter plate reader (Dynatech Laboratories). 6.3.2.2 Indirect immunofluorescence labeling At the end of indicated experiments, immunofluorescence labelling was done as described in 5.3.2.5. The cells were fixed with 4%fonnaldehyde in PBS for 15 min and permeabilized using 0.1% Triton X-100 in PBS at room temperature for 10 min. Afier blocking of non-specific protein binding by using 1% BSA and 0.1% Triton X-100 in PBS, the cells were imrnunostained by using monoclonal primary antibody to VTlB subunit or polyclonal pnmary antibody to VTZB subunit followed by FITC conjugated secondary antibodies. As control studies, mouse IgGl or rabbit pre-immune serum was used in place of primary antibody incubation. 6.3.2.3 Post-embedding immuno-electron microscopy 6.3.2.3.1 Preparation of cells for electron microscopy To study the intracellular targeting of internalized VTI and VT2, vero cells were grown to confiuency in 6-well tissue culture plates. At the end of the indicated experiments, the cells were processed as described in 3.3.2.4. 6.3.2.3.2 Immuno-gold labeling of the sections Afier sectioning of the polymerized blocks using an ultramicrotome, the sections for study of intemalization were irnmunogold labelled as described in 3.3.2.5.The irnmunolabeling was done by incubation with 1:25 dilution of anti-VT1B or 1 50dilution of anti-VRB primary antibody followed by 150 dilution of 15 nm gold-conjugated GAM or GAR secondary antibodies, respectively, for 1 hr each at room temperature with thorough washing at each step. As controls, some sections were treated with unrelated primary antibody (anti-FITC antibody) followed by GAM-gold secondary antibody conjugate or with secondary antibody alone. 6.3.2.3 Total cellular glycosphingolipid extraction and thin layer chromatography overlay of VT. The glycolipids fiom ver0 and VRP cells were extracted and TLC overlay was done as described in as described in 4.3.2.9. The plates were blocked with 1.5% gelatin in 50 rnM TBS (Tris buffer saline, pH 7.4) at 370C for 2hr to ovemight, incubated with 0.1 pg/ml VTl or VT2 and irnmunodetected using anti-VT1B monoclonal antibody or anti- VTZB polyclonal antibody, followed by peroxidase conjugated secondary antibodies at 37°C for 2 hr each. 6.4 Results 6.4.1 Golgi dependent retrograde transport of VT1 and VT2 The intemalization routes of VTI and VT2 were studied in ver0 Afncan green monkey kidney ce11 line which is known to be very sensitive to verotoxins. The cytotoxicity induced by VTl and VT2 varies with different host cells. In ver0 cells VTI was found to be -10 fold more toxic than VT2 (Figure.51). A hgal metabolite BFA which reversibly disrupts the Golgi structure protects the celis fiom the cytotoxic effect induced by the toxins which undergo Golgi-dependent retrograde transport to the compartments where subunit dissociation and translocation take place (Mallard et al., 1998). BFA protected vero cells from VT1 and VT2 induced cytotoxicity (Figure.52). Although BFA protection \vas significant at the dose of VT 5-10 ng/d or lower, the protection was onIy partial at the dose higher than 5-10 ng/ml (Chapter 5 and FigureS2A). The incomplete protection fiom VT1 cytotoxicity was found to be associated with the presence or up-regulation of Golgi-independent caveolar mediated internalization while Golgi-dependent retrograde transport was inhibited (Chapter 5). Although the cytotoxicity induced by VT1 and VT2 was very similar at the dose range of 10-1000 @ml, the protective effect of BFA was more effective against VT2 induced cytotoxicity (Figure. 52B), indicating the differential dependence on Golgi for the retrograde transport of the toxins. 6.4.2 Alternative pathway of VT1 internalizatioa VTI -induced cytotoxicity was found to be mainly dependent on Golgi-dependent retrograde transport. However, VTl could be transported to the perinuclear region via Golgi-independent caveolar mediated pathway and this alternative pathway also was partially responsible for the VTI -induced cytotoxicity (Chapter 5). The inhibitor of caveolar-mediated endocytosis, filipin could partially protect vero cells from VTI - VT log @ml Figure. 51 VTI and VT2 cytotoxicity assay on ver0 cells. (n = 4) VT nglml Figure. 52.A Effect of BFA and filipin on VT1-induced ver0 ce11 cytotoxicity. (n = 4) +os ug/rni BFA +10 ughl Filipin Figure. 52.B Effect of BFA and filipin on VT2-induced vero ce11 cytotoxicity. (n = 4) induced cytotoxicity (Figure. 52A). In addition, BFA and filipin synergistically protect vero cells fiom VT1-induced cytotoxicity (Figure. 52A), indicating that the Golgi- independent caveolar-mediated pathway was still functioning, while the Golgi structure was disrupted. Since VTXnduced cytotoxicity was more reIated to Golgi-dependent retrograde transport, the role of Golgi-independent caveolar-mediated transport for VT2- induced cytotoxicity was examined. Despite the partial protection against VTl- cytotoxicity (Figure. 52A), filipin had no protective effect on VT2-cytotoxicity (Figure. 52B) and a combination effect of BFA and filipin was not observed (Figure. 52B), indicating that VTXnduced cytotoxicity \vas rnainly mediated by the Golgi-dependent retrograde transport. 6.4.3 Intracellular targets of VT1 and VT2 Due to the difference in protection effect by BFA and filipin on VTI and VT2 cytotoxicity, intmcellular Iocalization of internaiized VT1 and VT2 and the effect of BFA and filipin treatment on the targeting of VT1 and VT2 were examined. Immunofluorescence labeling of intemalized VTI and VT2 indicated a difference in targeting of the toxins. VTI was found as both Golgi-pattern staining and perinuclear labeling, likely to be ER and nuclear membrane (Figure. 53, panel a). In contrast, VT2 irnrnunofluorescence was found predominantly as Golgi-pattern staining (Figure. 53, panel b).The differentiai intracellular targeting of VTI and VT2 was confirmed by post- embedding irnrnuno-electronmicroscopy (Figure. 54). Both VTI and VT2 were found in Golgi, nuclear membrane and the nucleus. However, VT1 was found to be more associated with nuclear membrane while VT2 was more Iocalized in the Golgi. In the presence of BFA, the characteristic targeting pattern of internalized VTI at 37°C for 3 hr (Figure 55B, compared to 55A) was lost and VTI was fond to be accumulated at a strongly fluorescent-labeled cornpartment (Figure. SB). This cornpartment had ken identified as collapsed TGN, fùsed with endosornes (Chapter 5). Accumulation of the toxin in the collapsed TGN and the inhibition of intemalization was Figure. 53 Indirect irnmuno fluorescence of intemalized VT 1 and VTî in vero cells. (370C at 3hr) Figure. 54 Immuno electron microscopy of internalized VT 1 and VT2. (370C for 3 hr). mtrol vero ce Figure. 55 Effect of BFA and filipin on intedized VT 1 in vero celis. (Indirect imrnuno fluorescence)VT1 was internalized at 370C for 3hr in contml cells(a) and in the presence of BFA(b) or filipin(c). control vem cells + 0.5 ug/ml BFA Figure. 56 Effect of BFA and fdipin on intenialid VT2 in ver0 cells. (Indirect immuno fluorescence)VT2 was internalized at 370C for 3hr in control cells(a) and in the presence of BFA(b) or filipin(c). found to be more distinct eventually afier 24 hr internalization by using VTl B subunit (Chapter 5). However, in the present study, since VT2B subunit was unavailable in our laboratory, VTI and VT2 holotoxins were compared. Consequently, due to the cytotoxicity of the toxins at the dose range, required for the study, 24 hr incubation with toxin could not be done. Accumulation of VT2 in collapsed TGN was found to be more prominent afier BFA treatment (Figure. 56B) compared to VT1 (Figure. SB), which still had sorne peri-nuclear labeling afier BFA treatment- In the presence of caveolar-mediated endocytosis inhibitor, filipin, overall internalization of VTI was reduced (Figure. SC), cornpared to Figure. SA, indicating the role of caveolar-mediated endocytosis in VTI internalization. In contrast, there was no apparent change in VT2 internalization and VT2 intracellular targeting after filipin treatment (Figure. 56C). In sumrnary, irnmuno-labeling studies indicated that internalization and intracellular targeting of VT2 was more restricted to Golgi-dependent paùiway and that of VTl was via both GoIgidependent and Golgi-independent caveolar- mediated pathways. Differential Gb, binding and cytotoxicity of VT1 and VT2 on low-level G b, expressing cells VTI has 10-50 times higher binding affinity to Gb, compared to VT2 and may preferentially bind to the cells with lower Gb, expression (Tesh et al, 1993 and Jacewicz et al., 1999). In order to identiQ whether the advantage of VTI over VT2, for the internalization via different pathways, was due to the difference in binding affinity of the tosins, VTI and VT2 binding to Gb, extract of Gb,-positive ver0 cells and Gb,-deficient VRP cells was examined. The relative preference of VTl and VT2 to upper and lower band Gb, of vero ceIl was difficult to determine by tlc overlay (Figure. 57). Although the total expression of Gb, was -80% reduced in VRP cells compared to ver0 cells, the residual expression of upper band was still observed in VRP cells (Figure. 57) and that was recognized only by VT1 not by VT2 (Figure. 57), indicating the higher affimity binding of VTl to low-level Gb,. The cytotoxicity assay also showed that VRP cells were still partially sensitive to VTI cytotoxicity while totally resistant to VT2 (Figure. 58), and this residual VTI cytotoxicity was still partially sensitive to filipin, indicating the importance of high afhity G4 binding of VTl for alternative pathway of VTl internalization. orcinol VT1 overlay VT2 overlay Figure. 57 Total Gb3 extraction of vero and VRP cells [lx10(7)] and VT 1 and VT2 overlay. Lane 1, GSL standards, lane 2, Gb3 extract of vero cells, lane 3, Gb3 extract of VRP cells, lane 4, Gb3 extract of butyrate treated VRP cells. 237 VT log nglml Figure. 58 VT 1 and VT2 cytotoxicity assay on vero and VRP cells. 6.4 Discussion VTl and VT2 are very closely related toxins which could be produced from different strains of E.coli or fiom the same organism (Scotland et al., 1985). Both toxins are associated with diarrhea and complications such as hemorrhagic colitis and hemolytic uremic syndrome (Riley, 1987 and Karmali, 1 989). Differential intracellular targeting of VTl was found to be important to determine VT1-induced cytotoxicity (Arab and Lingwood, 1998). Despite the comparable level of total Gb, expression, VTI cytotoxicity was found to be >5000-fold increased in astrocytoma ce11 line SF-539, in which VTI was targeted to the ER and nuclear membrane, compared to XF-498, in which VTl was targeted to the Golgi only (Arab and Lingwood, 1998). ST and VTI have ken known to enter the cells by clathrin-dependent, receptor mediated endocytosis and undergo retrograde transport to the ER and nuclear membrane (Sandvig et al., 1989 and Khine and Lingwood, 1994) via direct transport of the toxin fiom early/recycling endosomes to TGN (Mallard et ai., 1998). Therefore, the cytotoxicity of the toxins could be prevented by the chernical agents, which disrupt the Golgi structure such as BFA (Donta et al., 1993 and Sandvig et al., 1991). VTI could also partially enter the cells via clathrin-independent, caveolar- mediated endocytosis (Schapiro et al., 1998 and Chapter 5). Both clathrin-dependent endocytosis and caveolar-mediated endocytosis may enter the same endocytic cornpartment (Tran et al., 1987). Consequently, the Golgi-dependent retrograde transport has been found to be associated with the toxins, intemalized by either clathrin-dependent endocytosis (Sandvig et al., 1991) or caveolar-mediated endocytosis (Orlandi et al., 1993). In addition, caveolar-mediated endocytosis could directly target to the ER or the nucleus in a Golgi-independent manner (Conard et al., 1995 and Feng et al., 1999). The direct targeting of VTlB subunit to a peri-nuclear region, likely the ER and nuclear membrane via a Golgi-independent, caveolar-mediated endocytosis has been observed in ver0 cells (Chapter 5). The cornparison of the funciional significance of Golgi-dependent and Golgi-independent pathways for VTl cytotoxicity indicated that the Golgi-dependent pathway is pnmarily responsible for the VT1-induced cytotoxicity while Golgi- independent caveolar-mediated endocytosis could be an alternative pathway (Chapter 5). Since the Golgi-disrupting agent BFA could prevent VT2-induced cytotoxicity, VT2 also had been expected to undergo Golgi-dependent retrograde transport to the ER for membrane translocation @onta et al., 1995). However, the protective effect of BFA on VTZ was found to be more sustained than on VT1-induced cytotoxicity (Donta et al., 1995). Therefore. the role of Golgi-dependent and Golgi-independent pathways for the cytotoxicity induced by VTl and VT2 has been compared in the present study. The intracel lular targeting of VT2 was found to be more localùed to the Golgi (Figure. 53 and 54) and more restricted to the Golgi-dependent pathway (Figure. 52, 56B). Increased cytotoxicity of VTI could be due to the Golgi-independent alternative pathway to the peri-nuclear region via caveolar-mediated endocytosis. Despite the sarne receptor binding specificity of VTI and VT2, the binding affinity of VTI to Gb, is 10-50 times higher than that of VT2 (Tesh et al., 1993 and Iacewicz et al., 1999). However, in animal models such as moue and rat. hemorrhagic lesions (Tashiro et al., 1994). acute rend tubular necrosis (Wadolkowski et al., 1990) and lethality of the animal (Wadolkowski et al., 1990 and Tesh et al., 1993) have been found to be more significant in animals injected with VT2 than with VT1. Due to the higher binding affinity. VTl may bind to host tissues with lower expression of Gb3 such as intestinal epithelium and VT1-induced pathology may be confined to the colonic epithelium and microvasculature suppiying the colon. On the other hand, VT2 with lower Gb, binding affinity may be more readily disseminated to the blood circulation and preferentially bind to the higher Gb3-expressing tissues of the other systemic organs and their microvasculature, resulting in more extensive systemic damages (Tesh et al., 1993). The difference in binding affi~nitymay also be the determining factor for the differential intra-cellular pathway and targeting of the toxins. The relative cytotoxicity of VTl and VT2 has ken found to vary wvith ce11 types. In some ce11 lines such as ver0 (FigureSl), HeLa (Donta et al., 1993) and HRCEC (Kiyokawa et al., 1998) VTI cytotoxicity was 6 to 10 folds higher than VT2 cytotoxicity. However, on endothelial cells of the microvasculature such as KIMEC (human intestinal microvâscular endotheliai cells) and HMVEC (human microvascular endotheliai cells), VT2 \vas 10 to 100 times more toxic than VTI (Jacewicz et al., 1999 and Ohmi et al., 1998). The comparative study on VT1 and VT2c has shown that the toxins preferentially recognize different fatty acid containing Gb, homologues. VTI prcferentially binds to C 320 and C 22: 1-containing Gb, and VT2c to C 18:O and C 18: 1-containing Gb,. VTl cytotoxicity has been found most toxic on the Gb,-deficient cells, reconstituted with C 22: 1-containing Gb, and VT2c cytotoxicity on the cells, reconstituted with C 18:l- containing Gb, (Kiarash et al., 1994). The differential Gb3 binding afinity of VT2 to Gb, homologoues has not been studied yet. However, VT2 and VT2c are 97% identical in arnino acid sequence and thus, differential cytotoxicity induced by VTl and VT2 on different ce11 lines could also be due to the difference in expression of Gb, isoforms. The cellular extract of Gb, cm be separated on the thin-layer chromatography as two closely migrating bands. Fast atom bombardment mass spectrometry (FAB MS) analysis has identified that the lower band is composed of either C 18:O based with Cl 6: 1 or C 18: 1 based with C 16: 1 fatty acid. The higher band is composed of C 18:l based with C 24: 1/ C 24:O fatty acids ( Sandvig et al., 1994). Based on the preferential binding of VTI and VT2c to Gb3 isoforms (Kiarash et al., 1994), differential binding of VTI and VENT2c to either lower band or higher band Gb, could not be easily detected. The study on astrocytoma ce11 lines has shown that lower band Gb, preferentially targets VTI to the ER and nuclear membrane while upper band Gb, to the Golgi (Arab and Lingwood, 1998). The differential targeting of VTl by Gb3 isofoms has been postulated due to the ability of the shorter fatty acid Gb, isoforms to accommodate easily into smaller dimension budding vesicles targeting to the thimer membranous cornpartment such as ER (Lingwood, 1996) or into the vesicles with thinner bilayer dimension (Lingwood, 1999). Since caveolar-mediated internalization may target to Golgi and ER via both Golgi-dependent and Golgi-independent mechanisms, the transport via this pathway could be suitable for both longer and shorter fatty acid containing Gb,. In contrast, clathrin-dependent RME via Golgi dependent retrograde transport to ER and nuclear membrane is more appropnate for shorter fatty acid Gb3 isoforms. Therefore, based on the preferentiai binding of VTI and VT2c to Gb3 isoforms (Kiarash et al., 1994), VTI may have more advantage to be transported by a Golgi-independent mechanism, in addititon to the Golgi-dependent pathway. However, the significance of different pathways may Vary with ce11 types, depending on the content of Gb, isoforms and the abundance of caveolae. VRP cells are ver0 mutant VT-resistant cells (Pudymaitis et al., 1991). Total Gb, expression of VRP cells was found to be -80% reduced compared to ver0 cells but the residual Gb, present in VRP was found to be upper band Gb3. Although the relative binding of VT1 and VT2 to upper and lower band Gb, was difficult to differentiate, VTl binding to residual upper band Gb, of VRP cells could be still observed, while VT2 did not bind to Gb, of VRP cells (Figure. 57). Consistent with Gb, binding, VRP cells were still partially sensitive to VTl while totally resistant to VT2-induced cytotoxicity (Figure. 58). This residual VTl-cytotoxicity on VRP cells was still found to be partialIy protected by either BFA and filipin, indicating the advantage of VT1, with higher affinity to Gb, (Tesh et al., 1993), for intemalization via different pathways, even in cells with low level Gb,. Therefore, the presence of alternative intracellular targeting rnechanism for VT1 couId also be partly due to the high affinity VTI binding to Gb, receptor. In conclusion, despite the sarne receptor binding of VTI and VT2 to Gb,, differential intracellular transport pathways and targeting of the toxins have been reported for the first time. Future directions Although VTI and VTWVT2c may preferentially bind to different fatty acid Gb, homologues, due to the mixture of Gb, isoforms in different migrating bands of Gb3, the role of upper and lower band Gb, for the intemaiization of VTI vs. VT2, and for the different pathways of internalization have been difficuit to correlate. Butyrate treatment on astrocytoma ce11 line with predominant upper band Gb, showed that not only the relative ratio of short chain fatty acid Gb, isoforms(lower band), but aiso the total Gb, expression was elevated (Arab and Lingwood, 1998). In order to identiS. the significance of Gb, isoforms, VT cytotoxicity and protection effect of BFA and filipin can be determined on Gb,-deficient cells, reconstituted with long chain fatty acid such as C 24:O- containing Gb, (preferred by VTI) or short chain such as C 18:O-containing Gb, (presumably preferred by VT2). Since microvasculahue endothelial cells are particularly more sensitive to VT2 cytotoxicity, Gb, analysis of these cells should be done to see whether the cytotoxicity of VT2 is related to certain Gb, isoforms. The intemalization pathways of VT2 also should be detemined on these cells to identifj. whether the mechanism of VT2 intemalization varies with the expression of particular Gb, isoforms and the affinity binding of VT2 to such Gb, isoforrns. Amino acid sequences of VTlB and VT2B subunits have 60% similarity to each other (Jackson et al., 1987). The structurai differences in Gb, binding sites due to different amino acid sequences may determine the differential Gb, binding and intracellular transport pathways. For example, Glu65 of VTlB and the corresponding Glu64 of VT2 have been known to be involved in Gb3 binding (Tyrell et al., 1992). in VT1, Glu65 is partially buried and interacts with Thrl. Lack of Thrl and Pro2 in VT2 may be an important factor for the exposure of Glu64 for the receptor binding (Nyholm et al., 1996). Substitution of amino acid sequences by site directed mutagenesis, and receptor binding and intemalization studies of such mutant toxins may explain the differential targeting of the two toxins. Chapter 7 Conclusion In a manner similar to VT, ce11 surface CD19 also has been found to undergo retrograde transport to the ER and nuclear membrane, providing evidence of a role for Gb, in the retrograde transport of Gb3-bound ligands. Antibody-crosslinking induced CD19-mediated apoptosis has been found to be more significant in Gb,-positive cells, indicating a modulatory role for Gb, in CD19-mediated biological functions. Competitive inhibition of VTl -B subunit binding on CD 19 internalization and CD 19-mediated apoptosis, and inhibition of VT1-B intemalization by CD19 crosslinking fùrther confirmed the role of ce11 surface Gb, in the biological activities of CDl9. As for IFN-a-mediated antiproliferative activity, anti viral activity is enhanced by the presence of ce11 surface Gb,. In the present study, the role of Gb, in maintaining the proper presentation of IFNAR for IFN-a binding (Cohen et ai., 1987 and Ghislain et al., 1994), was found to be important for the subsequent IFN-a-mediated signal transduction events. Activation of KGF-3, detected by nuclear translocation of Statl, and prolonged maintenance of signal transduction events for the subsequent expression and activation of PKR enzyme (which is critical for the anti viral activity), was found to be dependent on ce11 surface Gb,. In addition, long-chah fatty acid containing-Gb,, the localization of which has been postulated to be more restricted to the ce11 surface, was found to be more important for the IFN-a-mediated antiviral activity, in contrast to the role of short-chah fatty acid containing-Gb, in VT 1 retrograde transport and VT 1-induced cytotoxicity . The Golgi-dependent retrograde transport of intemalized VT1 is the principal pathway of VTI targeting for the VT-1-induced cytotoxicity, while Golgi-independent, caveolar-mediated endocytosis also was also found as an alternative route. Golgi- independent targeting of VTl to the perinuclear region via caveolar-mediated endocytosis also has been detected. Despite the structural and receptor binding similarities, closely related VTl and VT2 were found to undergo intracellular trafEcking via different transport pathways, which may explain the difference in intracellular localization. VT2 internalization was found to be more dependent on the Golgi-dependent pathway and consequently, intracellular targeting is more restricted to Golgi. In contrast, VTI may be internalized via Golgi-independent caveolar-rnediated alternative route, in addition to Golgi- dependent retrograde transport. The peri-nuclear region (likely to be ER and nuclear membrane) targeting, in addition to Golgi localization, has been found to be more significant for VTI internalization. List of Publications The study on functional role of Gb, in antibody crosslinked CD19 internalization and apoptosis (chapter 3) has been published as "CD77 (Globotriaosylceramide) detemines the intracellular target of CD 19 and regulates CD 19 mediated endocytosis: Functional relationship between CD77 and CD19 in germinal center B-ce11 apoptosis". Khine, A.A., M. Firtel and C.A. Lingwood (1998), J. Cellular Physiology, 176: 281-292. The study on functional role of Gb, in interferon-ahype 1 interferon receptor rnediated anti viral activity (chapter 4) has been accepted for publication in J-Cellular Physiology as "Functional significance of globouiaosylcerarnide in alpha-2 interferod type I interferon receptor mediated antiviral activity". Khine, A.A. and C.A. Lingwood (1 999). The study on Gb, dependent intracellular transport mechanisms of verotoxin (chapter 5) has ken prepared for the publication as "An alternative pathway of verotoxinl internalization; Caveolar mediated, Golgi independent VTI transport to the nuclear membrane". Khine, A.A. and C.A. Lingwood (1 999). The study on comparison of VTI and VT2 internalization and intracellular targeting (chapter 6) has been prepared for the publication as "Different internalization mechanisms of two closely related verotoxins; VTI and VT2". Khine, A.A. and C.A. Lingwood (1 999). References 1. Abbas, A. K.. A. H. Lichfman, and J. S. Pober. In "Cellular and Molecular immunology". Second Edition, 1 994. 2. Abe, A., J. Inokuchi, J., M. Jimbo, H. Shirneno, A. Nagamatsu, J. A. Shayman, G. S. Shukla, and N. S. Radin. Improved inhibitors of glucosylceramide synthetase. J. Biochem. 1 1 1 (2), 19 1-196, 1992. 3. AdoIf, G. R., B. Fmhbeis, R Hauptmann, 1. Kalsner, 1. Mauerer-Fogy, E. Ostermann, E. Patzelt, R. Schwendenwein, W. Sommergruber, and A. Zophel. Human interferon ml: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein. Biochem. Biophys. Acta., 1089, 167- 174, 199 1. 4. Adolf, G. R. Human interferon omega, a review. Multiple Sclerosis. 1, Suppl 1, S44-7, 1995. S. Andersson. A. M., L. Melin, R Persson, E. Raschperger. L. Wikstrom, and R. F. Petterson. Processing and membrane topology of the spike proteins G I and GZ of Uukunierni virus. J. Virology, 7 1 (1), 21 8-225, 1 997. 6. Anderson, P., B. Tycko, F. Maxfield, and J. Vilcek. Effect of primary amines on interferon action. Virology, 1 17, 5 10-5 15, 1982. 7. Anderson, R. G. W., M. S. Brown, U. Beisiegel, and J. L. Goldstein. Surface distribution and recycling of the low density Iipoprotein receptor as visualized with antireceptor antibodies. J. Cell. Biol. 93, 523-53 1, 1982. 8. Anderson, R. G. B., B. A. Kamen, K. G. Rothberg, and S. W. Lacey. Potocytosis: sequestration and transport of small molecules by caveolae. Science, 255,4 10-4 1 1, 1992. 9. Aniento, F.. F. Gu, R. G. Parton, and J. Gruenberg. An endosomal f3-COP is involved in the pH-dependent formation of transport vesicles destined for late endosornes. 1. Cell. Biol., 130,294 1, 1996. 10. Arab, S, E. Russsel, W. B. Chapman, B. Rosen, and C. A. Lingwood. Expression of the verotoxin receptor slycolipid, globotriaosylcerarnide, in ovarian hyperplasias. Oncology Research, 9,553-563, 1997. 1 1. Arab, S., and C. A. LinWood. Influence of phospholipid chah length on verotoxin/globotriaosyl cerarnide binding in mode1 membranes: comparision of a supponed bilayer film and liposomes. Glycoconjugate Journal, 13, 159-166, 1996. 13. Arab, S., M. Murakami, P. Dirks, B. Boyd, S. L. Hubbard, C. A. Lingwood, and J. T. Rutka Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells. J. of Neuro-Oncology, 40, 137-150, 1 W8a. 13. Arab, S., and C. A. Lingwood. intracellular targeting of the endoplasmic reticulum/nuclear envefope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl cerarnide fatty acid isoform trafic. J. of Ceil. Physiology, 177,646-660, 1998b. 14. Arab, S., J. Rutka, and C. Lingwood. Verotoxin induces apoptosis and the complete, rapid, Ion,-O term elimination of human astrocytoma xenografis in nude rnice. Oncology Research, 1 1,33-39, 1999. 15. Baron, S., G. J. Stanton, W. R. Fleischmann, Jr., and F. Dianzani. Introduction: General considerations of the interferon systern. In "the interferon system", 1987. 16. Bergelson. L. D. Serurn gangliosides as endogenous imrnunomodulaton. Immunol. Today, 16 (1 O), 483- 486. 1995. 17. Bonnefoy, J. Y., S. henchoz, D. Hardie, M. J, Holder, and J. Gordon. A subset of anti-CD21 antibodies prornote the rescue of germinal center B cells fkom apoptosis. Eur. J. Immunol. 23 94), 969-972, 1993. 1 8. Boyd, B., and C. A. Lingwood. Verotoxin receptor glycolipid in human renal tissue. Nephron, 5 1,207-2 10, 1989. 19. Bradbury. L. E., V. S. Goldrnacher, and T. F. Tedder. The CD19 signal transduction complex of B lymphocytes. Deletion of the CD19 cytoplasrnic dornain alters signal transduction but not complex formation with TAPA-2 and Leu 13. J. Immunol., 15 1 (1 6), 291 5-2927, 1993. 20. Braell,W. A., D. M. Schlossman, S. L. Schrnid, and J. E. Rothrnan. Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase. J. Cell. Biol., 99,734-74 1, 1984. 21. Brama, A. A., S. B. D'alessandro, and C. Baglioni. Intemalization and degradation of human alpha-A interferon bound to bovine MDBK cells: Regulation of the decay and resynthesis of receptors., J. Interferon Research, 3 (4), 465-47 1, 1983. 22. Bras, A., C. Martinez-A, and E. Baixeras. B ceIl receptor cross-linking prevents Fas-induced ceIl death by inactivating the IL- 1wonverthg enzyme protease and regulathg Bd-uBcl-x expression. J. Immunol., 3 168-3 177, 1997. 33. Bretscher, M. S.. Directed lipid flow in ce11 membranes. Nature, 260,21-23, 1976. 34- Brown, V. l., and M. 1. Greene. Molecular and ceHular mechanisms of receptor-mediated endocytosis. DNA and Cell Biology, 10 (6), 399-409, 199 1. 25. Caldenvood, S. B. Overview of toxin-structure-function,receptors and ce11 biology. In Recent advances in verocytotoxin-producing Escherichia coli infections. Edited by M. A. Karmali, and A. G. Goglio. 1994. 26. Cambier, J. C., C. M. Pleiman, and M. R Clark. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Imrnunol., 12,457-486, 1994. 27. Carter, R. H., and D. T. Fearon. CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science, 256, 105-1 07, 1992. 2s. Chaouchi, N., A. Vaquez, P. Galanaud, and C. Leprince. B ceIl antigen receptor-mediated apoptosis. Importance of accessory rnolecules CD19 and CD22, and of surface IgM cross-linking. J. Immunol., 3096- 3 103, 1995 29. Chatte jee, S. Oxidized low density Iipoproteins and lactosylceramide both stimulate the expression of proliferating ce11 nuclear antigen and the proliferation of aortic smooth muscle cells. Indian J. Biochem. Biophys. 34 (1 -2)- 5660, 1997. 30. Chattejee, S. A.K. Bhunia, A. Snowden. and H. Han Oxidized low density lipoproteins stimulate galactosyltransferase activity, ras activation, p44 mitogen activated protein kinase and c-fos expression in aortic srnooth muscle cells. Glycobiology, 7 (9,703-7 10, 1997. 3 1. Chaudhary, V. K., Y. Jinno, D. Fitzgerald, and 1. Pastan. Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl. Acad. Sci. USA. 87, 308- 3 12, IWO. 33. Clark. C et al., and J. L. Brunton. Phenylalanine 30 plays an important role in receptor binding of verotoxin- 1. Mol. Microbiol., 19, 891-899, 1996. 33. Cohen, A.. G. E. Hannigan, B. R. G. Williams, and C. A. Lingwood. Roles of globotriosyl- and galabiosylceramide in verotoxin binding and hi& affinity interferon receptor. J. Biol. Chem. 262 (35), 1 7088-9 1, 1987. 34. Cohen. B., D. Novick, S. Barak, and M. Rubinstein. Ligand-induced association of the type I interferon receptor components. Mol. And Cell. Biology, 15 (8), 4208-42 14, 1995. 35. Conard, P. A., E. J. Smart, Y-S Ying, R. G. W. Anderson, and G. S. Bloom. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule- independent steps. J. Cell. Biol., 13 1 (6),142 1-1433, 1995. 36. Constantinescu, S. N, E. Croze, C. Wang, A. Murti, L. Basu, J. E. Mullersman, and L. M. Pfeffer. Role of interferon a/p receptor chain 1 in the structure and transmembrane signaling of the interferon a@ receptor complex. Proc. Natl. Sci. Acad. USA., 9 1,9602-9606, 1994. 37. Constantinescu, S. N, E. Croze, A. Murti, C. Wang, L. Basu, D. Hollander, D- Russell-Harde, M. Betts, V. Garcia-maninez, J. E. Mullersman, and L. M. Pfeffer. Expression and signalhg specificity of the IFNAR chain of the type I interferon receptor complex. Proc. Natl. Acad. Sci. USA., 92, 10487-91, 1995. 38. Cooling, L. L. W., K. E. Walker, T. Gille, and T. A. W. Koerner. Shiga toxin binds hurnan platelets via globotriaosylceramide (pk antigen) and a novel platelet glycosphingoiipid. Infection and Immunity, 66 (9), 43554364, 1998. 39. Cosson, P., and F. Letourneur. Coatomer interaction with dilysine endoplasrnic retention motifs. Science, 263. 1629-1631, 1994. 40. Cupers, P., A. Veithen, A. Kiss, P. Baudhuin, and P.J. Courtoy. Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts. J. Cell. Biol., 127 (3), 725-735, 1994. 4 1. Damke, H., T. Baba., A. M. Van Der Bliek, and S. L. Schmid. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin. J. Cell. Biol., 13 1,69-80, 1995. 43. Darnke, H. Dynarnin and receptor-rnediated endocytosis. FEBS Leners, 389,48-5 1, 1996. 43. Davies, P. J. A., D. R Davies, A. Levitrki, F. R- Maxfield, P. Milhaud, M. C. Willingharn, and I. H. Pastan. Transglutarninase is essential in receptor-mediated endocytosis of a2-macroglobulin and polypeptide hormones, Nature, 28, 162- 167, 1 980. 44. DeGrandis, S. H. Law, J. Bntnton, C. Gyles, and C. A. LinPooci. Globotetraosylceramide is recognized by the pig edema disease toxin. J. Biol, Chem., 264 (2 l), 12520-25, 1989. 35. Donaldson, J. G., and R D. Klausner. ARF: a key regulatory switchin membrane trafic and organelle structure. Current Opinion in Cell Biology, 6,5279532, 1994. 46. Donnenberg, M. C., and J. B. Kaper. Enteropathogenic Escherichia coli. Infection and Imrnunity, 60, 3953-396 1, 1992. 47. Donohue-Rolfe, A., G. T. Keusch, C. Edson, D. Thoeley-Lawson, and M. Jacewicz Pathogenesis of chigella diarrhea IX. Simplified high yield purification of shigella toxin and characterization of subunit composition and hction by the use of subunit-specific monocIona1 and polyclonal antibodies. J. Exp. Med. , 160, 1767- 178 1, 1984. 48. Donta, S. T., S. Beristain, and T. K. Tomicic. Inhibition of heat-labile Cholem and Escherichia coli enterotoxins by brefeldin A. Infection and Immunity. 61 (8), 3282-3286, 1993. 49. Donta, S. T., T. K. Tomicic, and A Donohue-Rolfe. Inhibition of Shiga-like toxins by brefeldin A. J. In fectious Diseases, 17 1, 72 1-724, 1995. 50. Doody, G. M., P. W. Dempsey, and D. T. Fearon. Activation of B lymphocytes: integrating signals from CD 19, CD32 and FcyRilb 1. Current Opinion in Immunology, 8,378-382, 1996. 5 1. Downes, F. P., T. J. Barrett, and J. H. Green. Affhity purification and characterization of shiga-like toxin II and production of toxin-specific monoclonal antibodies. Infection and immunity, 56, 1926- 1933, 1988. 52. Doxsey, S. J., F. M. Brodsky, G. S. Blank, and A. Helenius. Inhibition of endocytosis by anti-clathrin antibodies. Cell, 50, 453463, 1987. 53- Dunn. W. A., and L. Hubbard. Receptor-rnediated endocytosisof epidermal growth factor by hepatocytes in the perfbsed rat liver: ligand and receptor dynamics. J. Cell. Biol., 98,2148-21 59, 1984. 53. Dytoc, M., L. Fedorko, and P. M. Sherman. Signal transduction in hurnan epithelial cells infected with attaching and effacing Escherichia coli in vitro. Gastroenterology, 106 (5), 1150- 1 16 1, 1994. 55. Farkas-Himsley, H., R. Hill, B. Rosen, S. Arab, and C. A. Lingwood. The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1. Proc. Natl- Acad. Sci. USA. 92,6996-7000, 1995. 56. Fasler-Kan, E., A. Pansky, M. Wiederkehr, M. Battegay, and M. H. Heim. Interferonsr activates signal transducers and activators of transcription 5 and 6 in Daudi cells. Eur. J. Biochem. 254,5 14-5 19, 1998. 57. Fearon. D., and R, Carter. The CD19/CRî/TAPA-1 cornplex of B-lymphocytes: Linking natural to acquired immunity. Ann. Rev. Immunol., 13, 127-149, 1995. 58. Feller, S. M., G. Posern, J. Voss, C. Kardinal, D. Sakkab, J. Zheng, and B. S. Knudsen. Physiological signals and oncogenesis mediated through Crk family adaptor proteins. J. Cell. Physiol., 177 (4). 535-552, 1998. 59. Fenderson, B. A., E. M. Eddy, and S. Hakomori. Glycoconjugate expression during ernbryogenesis and iis biological significance. Bioessays, 12 (4) 173-1 79, 1990. 60. Feng, Y., V. J. Venema, R C. Venema, N. Tsai, and R B. Caldwell. VEGF induces nuclear translocation of flk- 1KDR, endothelial nitric oxide synthase, and caveolin- l in vascular endothelial cells. Biochem Biophys Res Commun, 5; 256 (1)- 192- 197, 1999. 61. Fischer, K., K. Tedford, and S. M. Penninger. Vav links antigen-receptor signaling to the actin cytoskeleton. Seminars in Immunology, 10,3 17-327, 1998. 62. Fish, E. N., S. Uddin, M. Korkmaq B. Majchrzak, B. J. Dniker, and L.C. Plantanias. Activation of a CrkL- StatS signaling complex by type 1 interferons. J. Biol. Chem., 274 (2), 571-573, 1999. 63. Flati, V., S. J. Haque, B. R. Williams. lnterferon-alpha induced phosphorylation and activation of cytosolic phospholipase A2 is required for the formation of interferon-stimulated gene factor three. EMBO J., 15 97), 1560- 157 1, 1996. 64. Foy, T. M., J. D. Larnan, J. A. Ledbetter, A. Aniffo, E. Claassen, and R J. Noelle. Gp39-CD40 interactions are essential for germinal center formation and the development of B ce11 rnernory. J. Exp. Med., 180 (l), 157-163, 1994. 65. Fraser, M. E., M. M. Chernaia, Y. V. Kozlov and M. N. G. James. Crystal structure of the holotoxin fiom Shigella dysentriae at 2.5Ao resolution. Structural Biology, 1 (1 ), 59-64, 1994. 66. Fredman, P. Glycosphingolipid turnor antigens. Adv. In Lipid Research. 25,213-234, 1993. 67. Fredman, P., and A. Lekman. Glycosphingolipids as potential diagnostic markers andior antigens in neurological disorders. Neurochemical Research, 22 (S), 107 1-83, 1997. 68. Fujimoto, T. GPI-anchored proteins, glycosphingolopidsm and sphingomyelin are sequestered to caveolae only after crosslinking, J. Histochem- Cytochem., 44 (8), 929-94 1, 1996. 69. Futerman, A. H. Distinct roles for sphingolipids and glycosphingolipids at different stages of neuronal deveiopment. Acta. Biochirn. Pol., 45 (2), 469478, 1998. 70. Gahmberg, C. G., and S. Hakomori. Extemal labeling of cell surface galactose and galactosarnine in glycolipid and glycoprotein of human eryduocytes. J. Biol. Chem., 248,43 1 1-43 17, 1973. 7 1. Garred, O., E. Dubinina, A- Polesskaya, S. Olsnes, J. Kozlov, and K. Sandvig. Role of the disulfide bond in Shiga toxin A-chain for toxin entry into cells. J. Biol. Chem., 272 (17), 11414- 19, 1997. 72. Ghetie, M-A., L. J. Picker, J. A. Richardson, K. Tucker, J. W. Uhr, and E. S. Vitetta Anti-CD19 khibits the growth of hurnan B-ce11 tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest. Blood, 83 (5), 1329- 1336, 1994. 73. Ghislain, J., C. A. LinWood, and E. Fish. Evidence for glycosphingolipid modification of the type 1 IFN receptor. J. Immunol. 153,3655-3663, 1994. 73. Ghislain, J., G. Susman, S. Goelz, L. E. Ling, and E. N. Fish. Configuration of the interferon-ab receptor cornplex determines the context of the biological response. J. Biol. Chern., 270 (37), 2 1785-92, 1995. 75. Ghosh, R- N., and F. R, Maxfield. Evidence for nonvectorial, retrogade transfemn traficking in the early endosomes of Hep2 cells. J. Cell. Biol., 128,549-56 1. 1995. 76. Gleeson, P. A., R D. Teasdale, and J. Burke. Targeting of proteins to the Golgiapparatus. Glycoconj. J., 1 1, 38 1-394, 1994. 77. Goldberg, M. W., and T. D. Allen, Structural and functiona1 organiaion of the nuclear envelope. Current Opinion in Cell Biology, 7, 301-309, 1995. 75. Grayson. G. and S. Ladisch. Immunosuppression by human gangliosides. II. Carbohydrate structure and inhibition of human NK activity. Cell Immunol, 139 (l), 18-29, 1992. 79. Gregory, C. D., T. Tursz, C. F. Edwards, C. Tetaud, M. Talbot, B. Caillou, A. B. Rickinson, and M. Lipinski. Identification of a subset of normal B cells with a Burkitt's lymphoma (BL)-like phenotype. J. Immunol.. 139, 3 13-3 18, 1987. 80. Griffïths. G., and K. Simons. The trans Golgi network: Soning at the exit site of the Golgi comlex. Science. 234.438443, 1986. 8 1. Grirnley, P. M., H. Fang, H. Rui, E.F. petriocin III, S. Ray, F. Dong, K. H. Fields, R HU, K. C. Zoon, S. Audet, and J. Beeler. Prolonged STATl activation related to the growth amest of malignant lymphoma cells by interferon-a. BIood, 91 (8), 30 17-3027, 1998. 82. Gurr, M. I., and J. L. Harwood. In "Lipid Biochemistry: an introduction", 4h edition, 1991. 83. Gyles, C. L. Escherichia coli cytotoxins and enterotoxins., Can. J. Microbiol., 38, 734-746, 1992. 84. Haddad, J. E., and M. P. Jackson. Identification of the Shiga toxin A subunit residues required for holotoxin assembly. J. Bacteriology, 175 (23), 7652-7657, 1993. 85. Haigler, H. T., F. R. Maxfield, M. C. Willingham. and 1. Pastan. Dansylcadaverine inhibits internalization of "S1-epidermal growth factor in BLAB 3ï3 cells. J. Biol. Chem., 255 (4), 1239-1241, 1979. 86. Hakomori, S., and Y. Zhang. Glycosphingolipid antigens and cancer therapy. Chem. And Biol., 4 (2), 97- 104, 1997. 87. Hakomori, S., and Y. Igarashi. Functional role of glycosphingolipid in cell recognition and signaling. J. Biochem. (Tokyo), 118 (6), 1091-1 103, 1995. 88. Hakomori, S. Turnor malignancy defined by aberrant glycosylation and sphingo(g1yco)lipid metabolism. Cancer Research, 56 (23), 5309-53 18, 1996. 89. Hassel, B., A. Zhou, C. Sotomayor, A. Maran, and R. Silverman. A dominant negative mutant of 2-SA- dependent Rnase suppresses antiproliferative and antiviral effects of interferon. EMBO J., 12, 3297-3304, 1993. 90. Haque, S. J., and B. R-Williams. Identification and characterization of an interferon (IFN) stimulated response elernent-lm-stimulated gene factor 3-independent signaling pathway for interferon-alpha. J. Biol. Chem., 269 (3 1 ), 19523-29, 1994. 91. Haque, S. J., and B. R. G. Williams. Signal transduction in the interferon systern. Seminars in Oncology, 25 (l), Suppl 1, 14-22, 1998. 92. Hay, J. C., and R. H. Scheller. Current Opinion in Cell Biology. 9,505-512, 1997. 93. Hazes, B. and R J. Read. Accumulating evidence suggests that several AB-toxins subvert the endoplasmic reticulum-assocoated protein degradation pathway to enter target cells.Biochemistry, 36 937), IlOS 1- 1 1054, 1997. 94. Head, S. C., M. A. Karmali, M. E. Roscoe, M. Petric, N. A. Strokbine, and 1. K. Wachsmuth. Serological differences between verocytotoxin 2 and shiga-like toxin II, The Lancet, 75 1, 1988a. 95. Head, S. C., M. Pebic, S. Richardson, M. Roscoe, and M. A. Kamali. Purification and characterization of verocytotoxin 2. FEMS Microbiology Leners, 5 1,21 1-2 16, 1988b. 96. Head, S., M. A. KarmaIi, and C. A. Lingwood- Preparation of VTI and VT2 hybrid toxins from their purified dissociated subunits: Evidence for B subunit mlxMation of A subunit fiinction. J. Biot. Chem., 266 (6),36 17-362 1. 199 1. 97. Henley, J. R., E. W. A. Krueger, B. J. Oswald, and M. A. McNiven. Dynamin-mediated internalization of caveolae. J. Cell. Biol. 141 (l), 85-99, 1998. 98. Heuser, J. Effects of cytoplasmic acidification on clathrin lattice morphology. J. Vell. Biol., 1 OS, 401 -4 1 1, 1989. 99. Heuser. J. E., and R G. W. Anderson. Hypertonie media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. 3. Cell. Biol., 108, 389-400, 1989. IOO.Holland, K. A., C. M. Owczarek, S. Y. Hwang, M. J. Tymms, S. N. Constantinescu, L. M. Pfeffer, 1. Kola, and P. J. Hertzog. A type 1 interferonsignahg factor, ISF2 1, encoded on chromosome 2 1 is distinct fiom receptor components and their down-regulation and is necessary for transcriptional activation of interferon- regulated genes. J. Biol. Chem. 272 (34), 2 1045-5 1, 1997. IO 1 .Horisberger, M. A. Interferons, Mx genes, and resistance to influenza virus. Am- J. Respir. Crit. Care Med., 152, S67-S7 1, 1995. 102.Huang, A. Y. C., P. Golumbek, M. Ahmadzadeh, E. Jaffee, D. Pardoll, and H- Levitsky. Role of bone marrow-derived cells in presenting MHC class 1-restricted tumor antigens. Science, 264,961-965. 103.Huber, A. R., S. L. Kunkel, R. F. Todd, and S. J. Weiss. Regulation of transendothelial neutrophil rn igntion by endogenous interleukin-8. Science, 254,99- 102, 1991. lO4.i-i~~~G., M. Silhol, and B. Lebleu. Microinjected interferon does not promote an antiviral response in HeLa cells. Biochem. Biophys. Res. Comrn. 1 10 (l), 155-160, 1983. IOj.Hunziker, W., and H. J. Geuze. lntracellular trafficking of lysosomal membrane proteins. BioEssays, 18 (5), 379-389. 106.Igarashi, K., T. Ogasawara, K. Ito, T. Yutsudo, and Y. Takeda. Inhibition of elongation factor 1-dependent am inoacyl-RNA binding to ri bosornes by Shiga-like toxin 1 (VT I ) fiom Escherichia coli 0 1 57:H7 and by Shiga toxin. FEMS Microbiology Letters, 4491-94, 1987. 107.Inokuchi, J.-L., and N.S. Radin, Preparation of the active isomer of 1-phenyl-2-decanoylamino-3- morpholino- 1 -propanol, inhibitor of murine glucocerebroside synthetase. J. Lipid Res., 28,565-57 1, 1987. 108.Inward, C. D., J. Williams, I. Chant, J. Crocker, D. V. Milford, P. E. Rose, and C. M. Taylor. Verocytotoxin- l induces apoptosis in ver0 cells. J. Infect., 30,2 13-2 18, 1995. 109.Isrnaili. A., D. J. Philpott, M. T. Dytoc, and P. M. Sherman. Signal transduction responses following adhesion of verocytotoxin-producing Escherichia coli. Infection and Immunity, 63 (9), 33 16-3326, 1995. I lO.Isrnaili, A.,. E. McWhirter, M. Y. Handelsman, J. L. Brunton, and P. M. Sherman. Divergent signal transduction responses to infection with attaching and effacing Escherichia coli. Infection and Immunity, 66 (3), 1688- 1696, 1998. 1 1 1 .Ito, H., M. Nishibuchi, and Y. Takeda. Analysis of the antigenic difference benveen ver0 toxin 2 (VT2) and VT2 variant (VT2vh) of verotoxin producing ficherichia coli by a site-directed mutagenesis. FEMS M icrobiology Leners, 79,27-30, 1991. 1 I 2.I U PAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN), Nomenclature of glycolipids. Carbohydrate Research, 3 12, 167- 175, 1998. 113.Jacewicz, M., D. W. K. Acheson, D.G. Binion, G. A. West, L. L. Lincicorne, C. Fiocchi, and G. T. Keusch. Responses of human intestinal microvascular endothelial cells to shiga toxins I and 2 and pathogenesis of hemorrhagic colitis. Infection and Immunity, 67 (3), 1439- 1444, 1999, 114.Jackson, M. P., E. A. Wadolkowski, D. L. Weinstein, R. K. Holmes, and A. D. O'Brien. Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis. J. Bacteriol. 172,653-658, 1990. 1 15. Jackson, M. P., and A. D. O'Brien. Structure-function relationships of shiga toxin and the shiga-like toxins to bacterial enterotoxins and ribosome-inactivating proteins. In Recent advances in verocytotoxin- producing Escherichia coli infections. Edited by M. A. Karmali, and A. G. Goglio. 1994. 116.fackson. M. R., T. Nilsson, and P.A. Peterson. Identification of the consensus motif for retension of transrnern brane proteins in the endoplasmic reticulum. EMBO J., 9,3153-3 162, 1990. 1 17.Johannes. L., D. Tenza, C. Antony, and B. Goud. Retrograde transport of KDEL-bearing B-fragment of Shiga toxin. J. Biol. Chem., 272 (3 l), 19554-61, 1997. 1 lS.Johnson, G. D., and E. J. Holborow. In " Immunochemistry". Weir, Henenberg, Blackwell and Herzenberg. 1986. 1 19.Johnson, K. F., and S. Kornfeld. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J. Biol. Chem., 267, 171 10-15, 1992. IîO.Kalderon, D., B. L. Roberts, W. D. Richardson, and A. E. Smith. A short amino acid sequence able to speciQ nuclear location. Cell, 39,499-509, 1984. 121.Kalisiak. A., J. G. Minniti, E. Oostenvijk, L. J. Old, and D. A. Scheinberg. Natural glycosphingolipid expression in B-cell neoplasms. uit. J. Cancer. 49 (6),837-845, 1991. 122.Kanfer, J. N., and S.I. Hakomori. In Handbook of Iipid research. Vol. 3, Sphingolipid Biochemisny. PIenum Presss, New York, 1983. 123.Kaplan, B. S., T. G. Cleary, and T. G. Obrig. Recent advances in understanding the pathogenesis of the hernolytic uremic syndromes. Pediatric Nephrology, 4,276-283, 1990. 124.Karpman, D., A. Andreasson, H. Thysell, B. S. Kaplan. and C. Svanborg. Cytokines in childhood hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Pediatric Nephrology, 9 (6), 694- 699, 1995- 125.Karmali, M. A. Infection by verocytotoxin-producuig Escherichia coli. Clin. Microbiol. Rev., 2, 15-38, 1989. 126.Katagiri, Y. U., T. Mori, T. Taguchi, T. Takeda, N. Kiyokawa, and J. Fujimoto. Activation of Src family kinase Yes occurs in the early phase of human renal tubular cell apoptosis induced by Shiga toxin binding to globotriaosyl ceramide (Gb,/CD77) in low density, detergent-insoluble microdomains- J. Biol. Chem. (Submitted), 1999. 127.KeuschTG, T., D. W. K. Acheson, L. Aaldering, J. Erban, and M. S. Jacewicz. Comparison of the effectsof Shiga-like toxin 1 on cytokine- and butyrate pretreated human umbilical and saphenous vein endothelial cells. J. Infect. Dis., 173, 1184-1 170, 1996, 128.Khine. A. A., and C. A. Lingwood. Capping and receptor-mediated endocytosis of cell-bound verotoxin (shiga-like toxin) 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization. J. CeIl. Physiology, 161,319-332, 1994. 1 29.KhineT A.A., M. Firtel, and C.A. LinWood. CD77(Globotriaosylceramide) determines the intracellular target of CD 19 and regulates CD 19 mediated endocytosis: Functional relationship between CD77 and CD 19 in germinal center B-cell apoptosis. f. Cellular Physiology, 176,28 1-292, 1998. 130.Kiarash, A., B. Boyd, and C. A. Lingwood. Glycosphingolipid receptor function is modified by fatty acid content. Verotoxin 1 and verotoxin 2c preferentially recognize different globotriaosyl ceramide fatty acid homologues. J. Biol. Chem. 269 (15), 1 1138-46, 1994. 13 l .Killion. J. J., R. Fishbeck, M. Bar-Eli, and Y. Chernajovsky. Delivery of interferon to intracellular pathways by encapsulation of interferon into multilamellar liposomes is independent of the status of interferon receptors. Cytokine, 6 (4), 443-449, 1994. 132.Kiyokawa, N., T. Taguchi, T. Mon, H. Uchida, N. Sato, T Takeda, and J. Fujimoto. Induction of apoptosis in normal human renal tubular epithelial cells by Escherichia coli shiga toxins 1 and 2. J. of Infectious Diseases, 178, 178- 184, 1998. 133.Knight, A. M.. J. M. Lucocq, A. R. Prescott, S. Ponnambatarn, and C. Watts. Antigen endocytosis and presentation mediated by human membrane IgG 1 in the absence of the IgdIgp dimer. EMBO Journal, 16 (1 3), 3842-3850, 1997. 134.Kojima, N, and S. Hakomori. Synergistic effect of two cell recognition systems: Glycosphingolipid- glycosphingolipid interaction and integrin receptor interaction with pericellular matrix protein. Glycobiology, 1 (6), 623-630, 199 1. I35.Kojima, N, M. Shiota, Y. Saciahira, K. Handa, and S. Hakomori. Ce11 adhesion in a dynamic flow system as compared to static system. Glycosphingolipid-glycosphingolipid interaction in the dynamic system predominates over lectin- or integin- based mechanisms in adhesion of BI6 melanoma cells to none activated endothelial cells. J. Biol. Chem. 267 (24), 17264-70, 1992- I36.Konowalchuk, J., J.I. Speirs, and S. Stavric. Vero response to a cytotoxin of Escherichia coli. Infection and Immunity, 18 (j),775-779, 1977. 137.Koopmar1, Ci., R. M. Keehnen, E. Lindhout, W. Newman, Y. Shimizu, G. A. van Seventer, C. de Groot, and S. T. Pals. Adhesion through the LFA- 1 (CD 1 laKD18)-ICAM 1 (CD53) and the VLA-4 (CD49d)- VCAM 1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152 (S), 3760- 3767, 1994. 138.Kozlov, Y. V., M. M. Chernaia, M. E. Fraser, and M. N, James. Purification and crystalization of Shiga toxin from Shigella dysenreriae J. Mol. Biol. 232 (2), 704-706, 1993. 1 39. Kurosaki, T. Molecular mechanisms in B ceIl antigen receptor sigaIling. Cunent Opinion in Immunology, 9. 309-3 18, 1997. 14O.LaCasse. E. C., M. T. Saleh, B. Patterson, M. D. Minden, and J. Gariepy. Shiga-like toxin purges human lymphorna from bone manow of severe combined irnmunodeficient mice. Blood, 88 (5), 156 1 - 1567, 1996. 141.LaCasse, E. C.,M. R. Bray, B. Patterson, W. Lim, S. Perampalarn, L.G. Radvanyi, A. Keating, A.K. Stewart, R. Buckstein, J. S. Sandhu, N. Miller, D. Banerjee, D. Singh, A.R. Belch, L.M. Pilarski and J. Gariepy. Shiga-like toxin-l receptor on human breast cancer, lymphorna, and myeloma and absence from CD34(+) hematopoeitic stem cells: implications for ex vivo tumor purging and autologous stem ceIl transplantation. Blood, 94 (8): 290 1- 10, 1999. 1 42. Ladisch, S., H, Becker, and L, Ulsh. Immunosuppression by human gangliosides: 1. Relationship of carbohydrate structure to the inhibition of T cell responses. Biochim Biophys Acta, 23, 1125 (2), 180-188, 1992. 143.Ladisch, S., A. Hasegawa, R Li, and M. Kiso, Immunosuppressive activity of chemically synthesized gangliosides. Biochemisûy, 34, 1 197- 1202, 1995. 144.Larnaze, C., and S. L. Schmid. The emergence of clathrin-independent pinocytic pathways. Current Opinion in Cell Biology. 7,573-580, 1995. 145.Lannet-t, H., K. Gorgas, 1. Meibner, F. T. Wieland, and D. Jeckel. Functional organization of the Golgi apparatus in glycosphingolipid biosynthesis. J. Biol. Chern., 27 (5), 2939-46, 1998. 146.Lavie, Y., H. Cao, S. L. Burstein, A. E. Giuliano, and M. C. Cabot. Accumulation of glucosylceramide in multidmg-resistant cancer cells. J. Biol. Chem. 271, 19530-36, 1997. 147.Le Borgne, R, and Hoflack, B. Protein transport from the secretory to the endocytic pathway in rnarnmalian cells. Biochemica et Biophysica Acta, 1404, 195-209, 1998. 148.Lee, C-K., H. A. R. Bluyssen, and D. E. Levy. Regulation of interferon-a responsiveness by the duration of Janus kinase activity. J. Biol. Chern, 272 (35),2 1872-77, 1997. 149.Lee, R-S.. E. Tartour, P. van der Bruggen, V. Vantomme, 1. Joyeux, B. Goud, W. H. FrÏdman, and L. Johannes. Major histocompatibility complex class 1 presentation of exogenous soluble tumor antigen fiised to the B-fragment of Shiga toxin. Eur. J. Immunol., 28,2726-2737, 1998. 15O.Lew, D, J., T. Decker, 1. Strehlow, and J. E. Damell. Overlapping elernents in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons. Mol. Cell. Biol. 11 91), 182-191, 1991. 15 1 .Li, J, and R. M. Roberts. Structure-hnction relationships in the interferon-t (IFN-r ). J. Biol. Chem. 269 (40), 24826-33, 1994. 15î.Lindberg A. A., J. E. Brown, N. Stromberg, M. Westling-Ryd, J. E. Schultz, and K. A. Karisson. Identification of the carbohydrate receptor for shiga toxin produced by Shigella dysenrriae type 1. J. Biol. Chem., 262, i 779- 1785, 1987. 153.Lindhout. E., and C. de Groot. Follicular dendritic cells and apoptosis: Iife and death in the germinal center. Histochem. J., 27, 167- 183, 1995. 154-Lindhout, E., A. Lakeman, and C. de Groot. Follicular dendritic cells inhibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of preexisting endonucfease. J. Exp. Med., 181, 1985- 1995, 1995. I55.Linggood, M. A., and J. M. Thompson. Verotoxin production among porcine strains of Escherichia coli and its association with edema disease. J. Med. Microbiol., 24,359-362, 1987. 156.Lingwood, C. A., H. Law, S. Richardson, M. Petric, S. L. Brunton, S. DeGrandis, and M. Karmali. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in virro. J. Biol. Chem., 262 ( 1 8), 8834-39, 1987. 157.Lingwood, C. A., and S. C. K. Yiu. Glycolipid modification of a-interferon binding: Sequence similarity between a-interferon receptor and the verotoxin (Shiga-like toxin) B-subunit. Biochem. J. 283 (l), 25-26, 1992. 158.Lingwood, C. A. Verotoxins and thei glycolipid receptors. Adv. In Lipid Research. 25, 189-21 1, 1993. 159.Lingwood, C. A. Verotoxin-binding in human renal sections. Nephron, 66.2 1-28, 1994. I6O.Lingwood, C. A. Aglycone modulation of glycolipid receptor function. Glycoconj. J., 13,495403, 1996. 16 1 .Lingwood, C. A. Role of verotoxin receptors in pathogenensis. Trends in Microbiol., 4, 147-153, 1996. 162.Lingwood, C.A.. A. A. Khine, and S. Arab. Globotriaosyl ceramide (Gb,) expression in human tumor cells: intracelluIar trafficking defines a new retrograde transpon pathway fiorn the cell surface to the nucleus, which correlates with sensitivity to verotoxin. Acta Biochem Polonica, 45 (2)?35 1-359, 1998. 163.Lingwood, C. A. Glycolipid and Bacterial pathogenesis. In "Saccharides in Chemistry and Biology, a Comprehension Handbook. (in press), 1999. 164.tippincott-Schwartz, J., L. Yuan, C. Tipper, M. Amherdt, L. Orci, and R. D. Klausner. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanisrn for regulating organelle structure and membrane trafic. Cell, 67.60 1-6 16, 199 1. 165.Liu, B., J. Liao, X. Rao, S. A. Kushner, C. D. Cheung, D. D. Chang, and K. Shuai. Inhibition of Statl- mediated gene activation by PiAs 1. Proc. Natl. Acad. Sci. USA., 95, 10626-3 1, 1998. 166.Liu, Y-J., D. E- Joshua, G.T. Williams, C. A. Smith, J. Gordon, and 1. C. M. MacLennan. Mechanism of antigen-driven selection in germinal centres. Nature, 342,929-93 1. 167.Lord, J. M.. and L. M. Roberts. Toxin entry: Retrograde transport through the secretory pathway. J. Cell. Biol, 140 (4). 733-736, 1998. 168.Louise, C. B., T. G. Obrig. Shiga toxin-associated hemolytic-uremic syndrome: combined cytotoxic effects of Shiga toxin, interleukin-l beta, and tumor necrosis factor alpha on human vascular endothelial cells in vitro. Infection and immunity, 59,4 173-41 79, 199 1. 169.Louise. C. B., S. A. Kaye, B Boyd, C-A. Lingwood, and T. G. Obrig. Shiga toxin-associated hemolytic uremic syndrome: Effect of sodium butyrate on sensitivity of human ubbilical vein endothelial cells to Shiga toxin. Infect. Immun. 63 (7),2765-2769, 1995. 170-MacLeod, D. L., and C. L. Gyles. Purification and characterization of an Escherichia cofi shiga-like toxin II variant. Infection and Immunity, 58, 1232- 1239, 1WO. 17 1 .MacLeod, D. L., and C. L. Gyles. Purification and characterization of a Escherichia col; shiga-like toxin 11 variant. Infection and Immunity, 59, 1300- 1306, 199 1. 173.Majoul, 1. V., P. 1. Bastiaens, and H. D. Soling. Transport of an extemal Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: Studies with cholera toxin in vero cells. J. Cell. Biol., 133. 777-789, 1996. 173.Mallard, F., D. Tenza, A. Claude, J. Salamero, B. Goud, and L. Johannes. Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of Shiga toxin B-fragment transport. J. Cell. Biol., 143 (4)- 973-990, 1998. I74.Maloney, M. D. and C. A. Lingwood. CD19 has a potential Cd77 (globotriaosy1 ceramide)-binding site with sequence similarity to verotoxin B-subunits: lmplications of molecular mimicry for B ce11 adhesion and enterohemorrhagic Escherichia coli pathogenensis. J. Exp. Med., 180, 19 1-20 1, 1994. 175.Mangeney, M., C. A. Lingwood, S. Taga, B. Caillou, T. Tursz, and J. Wiels. Apoptosis induced in Burkitt's lymphorna cells via Gb3/CD77, a glycolipid antigen. Cancer Research, 53,53 14-53 19, 1993. 176.Mangeney, M., G. Rousselet, S. Taga, T. Tursz, and J. Wiels. The fate of human CD77' germinal center B lymphocytes after rescue from apoptosis. Molecular Immunology, 32 (5), 333-339, 1995. 177.Marques, L. R. M., M. A. Moore, J. G. Wells, 1. Kaye Wachsmuth, and A. D. O'Brien. Production of Shiga-like toxin by Escherichia cofi.J. Infectious Diseases, 154 (2), 338-34 1, 1986. 178.Marques, L. R. M., J. S. M. Peiris, S. J. Cryz, and A. D. O'Brien. ficherichia col; strains isolated from pigs with edema disease produce a variant of shiga-like toxin 11. FEMS Microbiology Leaers, 44, 33-38, 1987. 179-Martinez, O, B. Goud. Rab proteins. Biochemica and Biophysica Acta, 1404, 10 1- 1 12,1998. 180.iMayor, S., K. G. Rothberg, and F. R Maxfield. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science, 264, 1948- 195 1, 1994. 18 1 .Meurs, E., K. Chong, J. Gatabru, N. S. B. Thomas, 1. M. Kerr, B. R G. Williams, and A. G. Hovanessian. Molecular cloning and charaterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell, 62,379-390, 1990. 182.Montecucc0, C., E. Papini, and G. Schiavo, Bacterial protein toxins peneuate cells via a four-step mechanism. FEBS Letters 346-92-98, 1994. 183.Montecucc0, C. Protein toxhs and membrane transport- Current Opinion in Ce11 Biology. 10, 530-536, 1998. 184.Mukhejee, S., R. N. Ghosh. and F. R. Maxfield. Endocytosis. Physiological review, 77 (9, 759-803, 1997. 185.Munr0, S., and H. R. Pelham. A C-terminal signal prevents secretion of luminal ER proteins. Cell, 48, 899- 907, 1987. 186.Munr0, S. An investigation of the role of trammembrane domains in Golgi protein retention. EMBO J., 14, 46954704, 1995. 187.Myers, D., X. Jun, K. Waddick, C. Forsyth, L. Chelstrom, R Gunther, N. Turner, J. Bolen, and F. Uckun. Membrane-associated CD19-LYN complex is anendogenous p53-independent and Bcl-2-independent regulator of apoptosis in human B-lineage lymphorna cells. Proc. Natl. Sci. Acad., 92, 9575-9579, 1995. 188-Nadler, L., K. Anderson, G. Marti, M. Bmes, E. Park, J. Daley, and S. Schlossman. B4, a human 8 lymphocyte associated antigen expressed on normal, mitogen activated and malignant B lymphocytes. J. Irnmunol., 13 1,244-250, 1983. 189.Naiki. M., and D. M. Marcus. An immunochernical study of the human blood group P, PI, and Pk glycosphingolipid antigens. Biochemistry, 14,4837484 1, 1975. I90.Nambiar, M. P., and H. C. Wu. Exp. Cell Res, Ilimaquinone inhibits the cytotoxicities of ricin, Diphhtheria toxin, and other protein toxins in vero cells. Exp. Cell Res., 2 19,671 -678,2 19,67 1 - 678, 1995. 19I.Nicke1, W., and F, T. Wieland. Biosynthetic protein transport throuph the early secretory pathway. Histochem. CelI. Bioi., 109,577-486, 1998. 193.Nilsson. T., P. Slusarewicz, P., M. H. Hoe, and G. Warren. Kin recognition, A model for the retension of Golgi enzymes. FEBS Lett, 330, 1-4, 1993. 193.Nilsson, T., and G. Warren. Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus.. Current Opinion in Cell Biology, 6,517-52 1, 1994. I94.Noda, M., T. Yutsudo, N. Nakabayashi, T. Hirayama, and Y. Takeda. Purification and some propenies of Shiga-like toxin ffom Escherichia coli 0157:H7 that is irnmunologically identical to Shiga toxin. Microb Pathog, 2 (5), 339-349, 1987. 195.Norvell, A., L. Mandik, and G. Monroe. Engagement of the antigen-receptor on immature murine B lymphocytes results in death by apoptosis. J. Immunol., 4404-44 13, 1995. 196.Novick, P.. and M. Zerial. The diversity of Rab proteins in vesicle transport. CurrentOpinion in Cell Biology, 9,496-504, 1997. 197.Nudelmar1, E., R. Kanagi, S. Hakomori, M. Parsons, M. Lipinski, J. Wiels, M. Fellows, and T. Tursz A glycolpind antigen associated with Burkitt's lyrnphoma defined by a monoclonal antibody. Science, 220, 509, 1983. 198.NyhoIm, P-G, G. Magnusson, 2. Zheng, R. Norel, B. Binnington-Boyd, and C. A. LinWood. Two distinct binding sites for globomaosyl ceramide on verotoxins: identification by molecular modelling and confirmation using deoxy analogues and a new glycolipid receptor for al1 verotoxins. Chemisay and BioIogy. 3,263-275, 1996. 199.0'Brien, A., T. A. Livety, M. E. Chen, S. W. Rothman, and S. B. Formal. Escherichia coli 0157:H7 strains associated with haemomhagic colitis in the united States produce a Shigella dysentriae I (Shiga) like cytotosin. Lancet, 702, l983a 2OO.O'Brien. A. D., and G. D. LaVeck. Purification and characterization of a Shigella àysenrriae I -1ike toxin produced by Escherichia coli. Infection and Immunity, 40 (3,675-683, 1983b. 201 .O'Brien, A. D., M. A. Karmali, and S. M. Scotland. A proposal for rationalizing the nomenclature of the Escherichia coli cytotoxins. In Recent advances in verocytotoxin-producing Escherichia coli infections. Edited by M. A. Karmali, and A. Ci. Goglio. 1994. 202.01Brien, a. D., and R. K. Holmes. Shiga and Shiga-like toxins. Microbiological Review, 51 (2), 206-220, 1987. 203.0brig, T. G., C. B. Louise, C. A. Lingwood, B. Boyd, L. Barley-Maloney, and T. O. Daniel. Endothelial hetrogeneity in Shiga toxin receptors and responses. J. Biol. Chem., 268, 15484- 15488, 1993. 204.0bri3, T. G., T. P. Moran, and J. E. Brown. The mode of action of Shiga toxin on peptide elongation of eukaryotic protein synthesis. Biochem. J., 244 (2), 387-294, 1987. 305.0hmi, K., N. Kiyokawa, T. Takeda and J. Fujimoto. Human microvascular endothelial cells are strongly sensitive to shiga toxins- Biochem. And Biophys. Research communications, 25 1, 137- 14 1, 1998. 206.0hyama, C., Y. Fukushi, M. Satoh, S. Saitoh, S. Orikasa, E. Nudelman, M. Straud, and S. Hakomori. Changes in glycolipid expression in human testicular tumor. Int, J. Cancer, 45 (6), 1040-1044, 1990. 207.01ie1 R. A., B. Fenderson, K. Daley, J. .W. Oosterhuis, J. Murphy, and L. H. Looijenga. Glycolipids of hurnan primary testicular germ celi tumors. British J. of Cancer, 74 (I), 133- 140, 1996. 2O8.OrIandiT P. A., P. K. Curran, and P. H. Fishman. Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for infracellular traficking in toxin action. 1. Biol. Chem., 268 (I6), 120 10-16, 1993. 209.0TRourke, L., R. Tooze, and D. T. Fearon., Co-receptors of B lymphocytes. Current Opinion in Irnmunology, 9,324-329, 1997- 2 1 O. Pagano, R. E. Lipid trafic in eukaryotic ce1 1s: mechanisms for intracellular transport and organelle-specific enrichment of Iipids. Cum. Opinion in Cell Biol., 2, 652663, 1990. 21 I.PaIlinari, A., H. Pang, and C. A. Lingwood. Binding of verocytotoxin 1 to its receptor is influenced by differences in receptor fatty acid content. Biochemistry, 3 1 (S), 1363-1370, 1992. 212.Pante, N., and U. Aebi. Toward the molecular dissection of protein import into nuclei. Current Opinion in Cell Biology. 8, 397-406, 1996. 2 1 3.Parton, R G. UItrasnictural localization of gangliosides; GM 1 is concentrated in caveolae. J. Histochem. Cytochem., 32, 155- 166, 1994. 214.Pavlovic, J., T. Zurcher, O. Haller, and P. Staeheli. Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein. J. Virol., 64 (7), 3370-3375, 1990. 215.Peaker, C. J. G. Transmembrane signalling by the B-cell antigen receptor. Current Opinion in Immunology, 6,359-363, 1994. 2 1 6.Pelham, H. R.B. Multiple targets for brefeldin A. Cell, 67,44945 1. 199 1. 217.Pelham, H. R. B., and S. Munro. Soning of membrane proteins in the secretory pathway. Cell, 75. 603- 605,1993. 21 S.Pelham, H. R. B., Sorting and retrieval between the endoplasmic reticulum and Golgi Apparatus. Current Opinion in Cell Biology. 7,530-535, 1995. 2 19.Pestka, S., J. A. Langer, K. C. Zoon, and C. E. Samuel. Interferons and their actions. Annu. Rev. Biochem., 56, 727-777, 1987. 220.Pestka, S. The interferon receptors. Seminars in Oncology, 24 (3), Suppl9, S9- l8-S9-40, 1997. 221.Petric, M., M. A. Karmali, S. Richardson, and R Cheung. Purification and biological properties of Escherichia coli verocytotoxin. FEMS Microbiology Letters, 4 1,63-68, 1987. 322.Peny. D. K. and Y. A. Hannun. The role of ceramide in ceIl signaling. Biochem. Biophys. Acta, 1436, 233-243, 1998. îZ.Pfeffer, L. M., C. Wang, S. N. Constantinescu, E.Croze, L-M- Blattt, A. P. Albino, and D. M. Nanus. Human renal cancers resistant to IFN's antiproliferative action exhibit sensitiveity to lm's gene-inducing and antiviral actions. 1. Urology, 156, 1867-1 87 1, 1996. 33J.Pfeffer. L. M. Biological activities of natural and synthetic type 1 interferons. Seminars in Oncology, 24 (3), S9-63-S9-69, 1997. 225.Pfeffer, L. M., C. A. DinarelIo, R. B. Herberman, B. R. G. Williams, E. C. Borden, R. Bordens, M. R. Walter, T. L. Nagabhushan, P. P. Trotta, and S. Pestka. Biological properties of recombinant a-interferons: 40h annivarsriry of the discovery of interferons. Cancer Research, 58,2489-2499, 1 998. 236.Powers, M. A., and D. I. Forbes. Cytosolic factors in nuclear transport: What's importin? Cell, 79, 93 1- 934, 1994. 227.Pryer, N. K., L. J. Wuestehube, and R Schekman. Vesicle-mediated protein sorting. Annu. Rev. Biochem. 61,471-516, 1992. 228.Pudymaitis, A., G Armstrong, and C. A. LinWood. Verotoxin-resistant cell clones are deficient in the glycolipid gIobotriosylceramide: Differential bais of phenotype.. Arch. Of Biochem. And Biophys., 286 (2). 448-452, 1991. 229.hdymaitis, A. and C. A. LinWood. Susceptibility to verotoxin as a Function of the ceIl cycle. J. of Cell. Ph ysiology, 150,632-639, 1992. 230.PuIczynski, S., A. M, Boesen, and O M Jensen. Antibody-induced modulation and intracellular transport of CD 1 O and CD 19 antipens in human-B ceIl lines: An immunofluorescence and irnmunoelectron microscopy study. Blood. 8 1 (6), 1549- 1557, 1993. 23 1 .Rabhovitch, M. Professional and non-professional phagocytes: an introduction. Trends Cell Biol., 5, 85- 88, 1995. 232. Ramotar, K., B. Boyd, G. Tyrrell, J. Garïepy, and C. A. Lingwood. Characterization of Shiga-like toxin 1 B subunit purified fiom overproducing clones of the SLT-1 B cistron. Biochem. J., 272,805-8 1 1, 1990. 233.Rawn, I. D. In Biochemistry. Neil Patterson Publishers, 1989. 234.Rernis, R. S.. K. L. McDonald, L. W. Riiey, N. D. Pub, J. G. Wells, B. R Davis, P. A. Blake, and M. L. Cohen. Sporadic cases of hemorrhagic colitis associated with Escherichia coli 0157:H7. Ann. Intern, Med., 10 1,624-626, 1985. 235.Richardso11, S. E., M. A. Kannali, L. E. Becker, and C. R Smith. nie histopathology of the hemolytic uremic syndrome associated with verocytotoxin producing Escherichia coli infections. Human Pathology, 19 (9), 1102-1 108, 1988. 236.Riley, L.W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Hebert, E. S. OIcott, L. M. Johnson, N. T. Hargett, P. A. Blake, and M. L. Cohen, Hemorrhagic colitis associated with a rare kcherichia coli serotype. N. Engl. I. Med., 308 (1 2), 681-685, 1987. 237.Roberts, R. M. Interferon-tau and pregnancy. J. Interferon & Cytokine Research. 16(4), 271-273, 1996. 238.Robinson, M-S. The role of clathrin, adaptors and dynamin in endocytosis. Current Opinion in Cell Biology, 6, 538-54, 1994. 239.Robinson, L. A., R. M. Hurley, C. A. LinWood, and D. Ci. Matsell- Escherichia coti verotoxin binding to human paediatric glomerular mesangial cells. Pediatric Nephrology, 9,6,700-704, 1995. 240.Robson, W. L. M.. A, K. C. Leung, and B. S. Kaplan. Hemolytic-uremic syndrome. Current problems in Pediatrics, 16-33, 1993. 24 1 .Rosen, R. Patching and capping of cell membrane receptors as examples of morphogenetic movement. I. Theor. Biol., 80, 149- 153, 1979. 242.Rosenshine. I., M. S. Donnenberg, J- B. Kaper, and B- B. Finlay. Signal transduction between enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPEC induces tyrosine phosphorylation of host ceIl proteins to initiate cytoskeletal rearrangment and bacterial uptake. EMBO J, 1 1,3551-3560, 1992. 243.RosenwaId, A. G., C. E. Machamer, and RE. Pagano. Effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway. Biochemistry, 3 1, 358 1 -3590, 1 992. 244.Rothberg, K. Ci., J. E. Heuser, W. C. Donzell, Y. Ying, J. R. Glenney, and R. G. W. Anderson. Caveolin, a protein component of caveolae membrane coats. Cell. 68,673-682, 1992. 245.Rothman, J. E., and G. Warren. Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Current Biology, 4 (3),220-233, 1994. 246.Rothrnan, J. E,, and F. T. Wieland. Protein soning by mspon vesicles. Science, 272, 227-234, 1996. 247.Rothstein, T. Signals and susceptibility to programmed death in B cells. Current Opinion in Immunology, 8, 362-37 1, 1996. 24S.Ruggenenti, P., J. Lutz, and G. Remuai. Pathogenesis and treatrnent of thrombotic microangiopathy. Kidney International, 5 1 (S-58), S-97-S- 10 1, 1997. 249.Rutherford, M.N., A. Kumar, B. Coulombe, D. Skup, D. H. Carver, and B. R Williams. Expression of intracellular interferon constituitively activates ISGF3 and confers resistance to EMC viral infection. J. Interferon and Cytokine Research, 16 (7)- 507-5 10, 1996. 250.Salarna, N. R., and R. W. Schekman. The role of coat proteins in the biosynthsis of secretory proteins. Current Opinion in Cell Biology, 7, 536-543, 1995. 25 1 .Samuel, C. E. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology, 183, 1- 1 1, 199 1. 252.Samue1, J. E., L. P. Perera, and S. Ward. Cornparison of the glycolipid rsceptor specificities of shiga-like toxin type II and shiga-like tosin type II variants. Infection and Immunity, 58.61 1-618, 1990. 253.Sandhoff, K., and A. Klein. Intracellular traffkking of glycosphingolipids: role of sphingolipid activator proteins in the topology of endocytosis and lysosomal digestion. FEBS Letters, 346, 103- 107, 1994. 254.Sandhoff K., and T. Kolter, Topology of glycosphingolipid degradation. Trends in Cell Biology, Vol. 6, page 98-1 03, 1996. 255.Sandvig, K., S. Olsnes, J. E. Brown, O. W. Petersen, and B. van Deurs. Endocytosis fiom coated pits of shiga toxin: A glycolipid-binding protein fiom Shigelfa ùysentriae 1. J. Cell. Biol., 108,133 1- 1343, 1989. 256.Sandvig, K., K. Prydz, M. Ryd, and B. van Deurs. Endocytosis and intracellular transport of the glycolipid- binding ligand Shiga toxin in polarized MDCK cells. J. Cell. BioI. 1 13 (3,553-562, 199 1. 357.Sandvig7 K., K. Prydz, S. H. Hansen, and B. van Deurs. Ricin transport in brefeldin A-treated cells: Correlation beween Golgi structure and toxic effect. J. Cell. Biol. 1 15 (4), 971-98 1, 1991. 258.Sandvig, K., K. Prydz, and B. van Deurs. Endocytic uptake of ricin and shiga toxin. In "Endocytosis", NATO Asi series, Vol. H 62, 1992. 259.Sandvig, K., M. Ryd, O. Garred, E. Schweda, P. K-Holm, and B. van Deurs. Retrograde transpon from the Golgi compIex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and CAMP.J. Cell Biol., 126 (l), 53-64, 1994. 260.Sandvig, K, and B. van Deurs. Endocytosis and intracellular sorting of ricin and Shiga toxin. FEBS Letters, 346,99- 102. 1994. 26 1 .Sandvig. K, and B. van Deurs. Endocytosis, Intracellular transpoe and cytotoxic action of Shiga toxin and Ricin. Physiological Review. 76 (4), 949-966,1996. 262.Schapir0, F. B., C. A. Lingwood, W. Furuya, and S. Grinstein. pH-independent retrograde targeting of glycolipids to the Golgi complex. Am. J. Physiol. CeII Physiol., 43, C319-(2332, 1998. 263.Schee1, J., R. Pepperkok, M. Lowe, G. Grifflths, and T. E. Kreis. Dissociation of coatomers is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum. 1. Cell. Biol., 137 (2), 3 19- 333, 1997. 264.Schekman, R., and L. Orci. Coat proteins and vesicle budding. Science, 271, 1526-1 533, 1996. 265.Schindler, C., K. Shuai, V. R. Prezioso, and S. E. Dantell, Jr. Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science, 257,809-8 13,1992. 266.Schintzer, J. E., J. Liu, and P. Oh. Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J. Biol. Chern., 270, 14399- 14404, 1995. 267.Schwarzmann, G., and K- Sancihoff' Metabolism and intracellular transport of glycosphingolipids. Biochemistry, 29 (91), 10865-71, 1990. 268.Schwar~A. L. Receptor cell biology: receptor-mediated endocytosis. Pediaûic research, 38 (6),835-843, 1995. 269.Scotland, S. M., H. R. Smith, and B. Rowe. Two distinct toxins active on vero ceils fiorn Escherichia coli 0 157. Lancet, 885, 1985. 270.Sen, G. C., and P. Lengyel. The interferon system. A bird's eye view of its biochernistry. J. Biol. Chern. 267 (8), 50 17-5020, 1992. 271 .Simons, K., and E. Ikonen. Functional rafts in cell membranes. Nature, 387 (9,569-572, 1997. 272.Srna1-t~E. J., Y-S Ying, P. A. Conard, and R. G. W. Anderson. Caveolin rnoves fiom caveolae to the Golgi Apparatus in response to cholesterol oxidation. J. Cell. Biol. 127 (S), 1 185- 1 197, 1994. 273.Smythe, E., and G. Warren. The mechanism of receptor mediated endocytosis. Eur. J. Biochem., 202,689- 699, 199 1. 274.SoIter, D.. and B. D. Knowles, Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA- 1 ). Proc. Natl. Acad. Sci. USA, 74, 5565-70, 1978. 275.Song W., H Cho, P. Cheng, and S. K. Pierce. Entry of B ceIl antigen receptor and antigen into class II peptide-loading cornpartment is independent of receptor cross-linking. S. Immunol., 42553263, 1995. 276.Stein. P. E., A. Boodhoo, G. J. Tyrrell, J. L. Brunton, and R. J. Read. Crystal structure of the cell-binding B oligomer of verotoxin-1 from E-coli. Nature, 355, 748-750. 277.Stirling, J. W., and P. S. Graff. Antigen unmasking for immunoelectron microscopy: Labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J. Histochern. Cytochem., 43, 1 15-123, 1995. 278.Stites, D. P., and A. 1. Terr. ln Basic Human Immunology. Appleton & Lange Publishing, 1991. 279.Stow, J. L. Regulation of vesicular transport by GTP-binding proteins. Current Opinion in Nephrology and hypertension, 4,42 1-425, 1995. 280.Sugita, M., J. T. Dulaney, and H. W. Moser. Ceramidase deficiency in Faber's disease (Lipogranulomatosis). Science, 178, 1 100-02, 1972. 28 1 .Taga, S., M. Mangeney, T. Tursz, and J. Wiels. Differential repulation of glycosphingolipid biosynthesis in phenotypically distinct Burkitt's lymphoma ce11 tines. Int. J. Cancer. 6 1-261-267, 1995. 282.Taga, S., K. Carlier, Z. Mishal, C. Capoulade, M. Mangeney, Y. Lecluse, D. Coulauâ, C. Tetaud, L. L. Pritchard, T. Turs~and J. Wiels. Intracellular signaling events in CD77-mediated apoptosis of Burkitt's lymphoma cells. Blood, 90 (7). 2757-2767, 1997. 383.Tashir0, H, S. Miun, 1. Kurose, D. Fukumura, H. Suzuki, M. Suematsu, M. Yoshioka, M. Tsuchiya, A. Kai, and Y. Kudoh. Verotoxin induces hemorrhagic lesions in rat srnall intestine. Temporal alteration of vasoactive substances. Digestive Diseases and Sciences. 39 (6), 1230- 1238, 1994. 284.TayIor, F. B., B. S. Coller, A. C. K. Chang, G. Peer, R. Jordan, W. Engellener, and C. T. Esmon. 7E3 F(abl),, a monoclonal antibody to the platelet GPIIb/IIIa receptor, protects against microangiopathic hemolytic anemia and microvascular thrombotic renal failure in baboons treated with C4b binding protein and a sublethal infusion of Escherichia coli. Blood, 89, 1 1,4078-4084, 1997. 285.Tedder, T. F., L.-J., Zhou, and P. Engel. The CD19KD21 signal transduction complex of B lymphocytes. Irnmunol. Today. 15,437342,1994. 286.Tesh, V. L., J. E. Samuel, L. P. Perera, J. B. Sharefkin, and A. D. O'Brien. Evaluation of the role of shiga and shiga-like toxin in mediating direct damage to human vascular endothelial cells. J. of Infectious Diseases, 164, 344-352, 199 1. 287.TeshTV. L.. and A. D. O'Brien. nie pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol Microbiol., 5 (8), 18 17-1 822, 1991. 288.Tesh, V. L., J. A. Burris, J. W. Owens, V. M. Gordon, E- A. Wadolkowski, A. D. O'Brien, and J. E. Samuel. Comparison of the relative toxicities of shiga-like toxins type 1 and type II for mice. Infection and Irnmunity, 6 1 (8), 3392-3402, 1993. 289.Tesh, V. L., B. Ramegowda, and J. E. Samuel. PurifÏed shiga-like toxins induce expression of proinflarnmatory cytokines from murine peritoneal macrophages. Infection and Immunity, 62, 5085-5094, 1994. 290.Tilg H. New insights into the mechanisms of interferon alfa: An immunoregulatos, and anti-inflammatory cytokine. Gastroenterology, 1 12, 101 7-102 1, 1997. 291 .Townsley, F. M., D. W. Wilson, and H. R. B. Pelham. Mutational analysis of the human KDEL receptor: distinct structural requirements for Golgi retention, ligand binding and retrograde transport. EMBO J., 12 (7), 282 1-2829, 1993. 292.Tran, D., J-L. Carpentier, F. Sawano, P. Gorden, and L. Orci. Ligands intemalized through coated or noncoated invaginations follow a common intracellular pathway. Proc. Natl. Acad. Sci. USA., 84, 7957- 796 1, 1987. 293.Tsubatq T., M. Murakami, and T. Honjo. Antigen-receptor cross-linking induces peritoneal B-ceIl apoptosis in normal but not autoimmunity-prone mice. Current Biology, 4 (1 ), 8-17. 294.Tyrre11, G. J., K. Rarnotar, B. Toye, B. Boyd, C. A. Lingwood, and J. L. Brunton. Alteration of the carbohydrate binding specificity of verotoxins fkom Gala 1-4Gal to GalNAcp 1-3Gala 1-4GaI and vice versa by site-directed mutagenesis of the binding subunit. Proc. Natl. Acad. Sci. USA. 89, 524-528, 1992. 295.Uckun, F. M., A. L. Burkhardt, L. Jarvis, X. Jun, B. Stealey, 1. Dibirdik, D. E. Myers, L. Tuel-Ahlgren, and J. B. Bolen. Signal transduction through the CD1 9 receptor during discrete developmental stages of human B-ceIl ontogeny. J. Biol. Chem. 268 (28), 21 172-84, 1993. 296.Upadhyaya1 K., K. Banvick, and M. Fishaut- The importance of nomenal involvement in hemolytic-uremic syndrome. Pediatrks, 65, 1 15- 120, 1980. 297.Uze, G., G. Lutfalla, and K. E. Morgensen. A and b interferons and their receptor and their friends and relations (Review). J. Interferon Cytokine Res., 15, 3-26, 1995. 398.Van de Kar, N. C. A. J., L. A. H. Monnens, M. A. Karmali, and V. W. M. van Hinsbergh. Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome, Blood, 80, 2755- 2764,1993. 299.Van de Kar, N. C., R.W. Sauenvein, P. N. Demacker, G. E. Grou, V. W. van Hinsbergh, and L. A. Monnens. Plasma cytokine levels in hemolytic uremic syndrome. Nephron, 7 1 (3), 309-3 13, 1995. 5OO.Van Deurs, B., O,W. Petersen, S. Olsnes, and K, Sandvig. The Way of Endocytosis. International Review of Cytology, 1 17, 13 1- 177, 1989. 30 I .Van Helvoort, A., and G. van Meer- Intracellular lipid heterogeneity caused by topology of synthesis and specificity in transport. Exarnple: sphingolopids. FEBS Letters, 369, 18-21, 1995. 302.Van heivoort, A., A. J. Smith, H. Sprong, 1- Fritzsche, A. H. Schinkel, P. Boest, and G van Meer. MDRI P- glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell, 87, 507-5 17, 1996. 303.Van Senen, P. A., L. A. H. Monnens, R. G. G. Versartten, L.P. W. J. van den Heuvel, and V. W. M. van Hinsbergh. Effects of verocytotoxin-1 on nonadherent human monocytes: Binding characteristics, protein synthesis, and induction of cytokine release. Blood, 88, 174- 1 83, 1996, 304.Van Senen, P. A., V. W. van Hinsbersh, T. J. van der Velden, N. C. van de Kar, M. Vermeer, J, D. Mahan, K. J. Assmann, L. P. van den Heuvel, and L. A. Monnens. Effects of TNF alpha on verocytotoxin cytotoxicity in purified human glomemlar microvascular endothelial cells. Kidney International, 5 1 (4), 1245- 1256, 1997a. 3OS.Van Senen, P. A., V. W. van Hinsbergh, L. P. van den Heuvel, T. J. var- der Velden, N. C. van de Kar, R. J. Krebbers, M. A. Karmali, and L. A. Monnens.Verocytotoxin inhibits mitogenesis and protein synthesis in purified human glomerular mesangial cells without affecting cell viability: evidence for nvo distinct mechanisms. J. Am. Soc. Nephrol., 8 (12), 1877-1888, 1997. 306.Vasconcelos, A. C., and K. M-Lam. Apoptosis induced by Ulfectious bursal disease virus. J. Gen. Vùol., 75, 1803-1 806, 1994. 307.Veldrnan R.J., K. Klappe, D. Hoeksm and J. W. Kok. Interferon-gamma-induced differentiation and apoptosis of HT29 cells: dissociation of (glucosyl) cerarnide signaling. Biochem. Biophys. Res Commun. 247 (3): 802-8, 1998. 308.Voorhees, P., E. Deignan, E. van Donselaar, J. Humphrey, M. S. Marks, P. J. Peters, and J. S. Bomifacino. An accidic sequence within the cytoplasmic domain of hrin hnctions as a determinant of rm-Golgi network localization and intemalization from the cell surface. EM80 J., 14,496 1-4975, 1995. 309.Waddel1, T., S. Head, M. Pemc, A. Cohen, and C. A. Lingwood. Globotriosyl ceramide is specifically recognized by the Escherichia col; verocytotoxin2. Biochem. Biophys. Res. Commun., i 52 (2), 674-679, 1988. 3 IO-Waddell, T., A. Cohen, and C. A. LinWood. Induction of verotoxin sensitivity in receptor-deficient celI lines using the receptor glycolipid globotriosyIceramide. Proc. Natl. Aca. USA, 87, 7898-7901, 1990. 31 I.Wadolkowski, E. A., L. M- Sung, J. A. Burris, J. E. Samuel, and A. D. O'Brien. Acute renal tubular necrosis and death of mice orally infected with Escherichia coli stranis that produce Shiga-like toxin type II. Infect Immun., 58 (1 2), 3959-3965, 1990. 3 12. Wakita, H., K. Nishimura, Y. Tokura, F. Furukawa, and M. Takigawa. Inhibitors of sphingolipid synthesis modulate interferon (1FN)-gamma-induced intercellular adhesion molecule (1CAM)-1 and human leukocyte antigen (HLA)-DR expression on cultured normal human keratinocytes: possible involvment of ceramide in biotogic action of IFN-gamma. J. lnvest Dermatol, IO7 (3): 336-342, 1996- 313.Weinstein, D. L., M. P. Jackson, L.P. Perera, R. K. Holmes, and A. D. O'Brien. In vivo formation of hybrid toxins comprising shiga toxin and the shiga-like toxins and role of the B subunit in localization and cytotoxic activity. Infection and immunity. 57 (12), 3743-3750, 1989. 3 14.Wenk, J., P. W. Andrews, J. Casper, J. Hata, M. F. Pera, A. van Keitz, 1. Damjanov, and B. A. Fenderson. GIycolipids of germ cell tumors: extended globo- series glycolipids are a hallmark of human embryoma carcinoma cells. Int. J. of Cancer, 58 (1 ), 108- 1 15, 1994. 3 1 S. Wiels, J., E. H. Holrnes, N. Cochran, T. Tursz, and S. Hakomori. Enzymatic and organizational difference in expression of Burkitt's lymphoma and lymphoblastoid cell lines. J. Biot. Chem., 259 (23), 14783-87, 1 984. 3 16.WielsTJ., M. Mangeney, C. Tetaud, and T-Tursz. Sequential shifts in the three major glycosphingolipid series are associated with B cell differentiation. Int. Immunol., 3, 1289-1300, 1991. 3 17. Williams, B. R. G. Signal transduction and transcriptional regulation of interferon-a stimulated genes. J. Interferon Res., 1 1,207-2 13, 199 1. 3 18. Williams, B. R- G. Role of the double-stranded RNA-activated protein kinase (PKR)in cell regulation. Biochemical Society Transactions. 25, 509-5 13, 1997. 3 19. Williams, B. R G., and S. J. Haque. Interacting pathways of intefieron signating. Seminars in Oncology, 24 (3), Suppl9, S9-7049-77, 1997. 320.Wood, S. A., J. E. Park, and W. J. Brown. Brefeldin A causes a micronibule-mediated fusion of the trans- Golgi network and early endosomes. Cell, 59 1-600, 1991, 321.Yamakawa, T., and Y. Nagai. Glycolipids at the cell surface and their biological fiinctions. Trends in Biochem. Sci., 3, 128-13 1, 1978. 322.YamasakiTC., Y. Natori, X-T. Zeng, M. Ohmura, S. Yarnasaki, Y. Takeda, and Y. Natori. Induction of cytokines in a hurnan colon epithelial cell line by shiga toxin 1 ((Stxl) and StxZ but not by non-toxic mutant Sm 1 which lacks N-glycosidase activity. FEBS Letters, 442,23 1-234, 1999. 323.Yamashita, T., R. Wada, T. Sasaki, C. Deng, U. Bierfreund, K. Sandhoff, and R L. Proia. A vital role for glycosphingolipid synthesis during development and differentiation. Proc. Natl. Acad. Sci. USA., 96, 9 142-9147, 1999. 324.Yang C-H.,A. Murti, and L. M. Pfeffer. STAT3 complements defects in an interferon-resistant cell line: Evidence for an essential role for STAT3 in interferon signaling and biological activities. Proc. Natl. Acad. Sci. USA., 95,5568-5572, 1998. 335.Yang C-H., W. Shi, L. Basu, A. Murti, S. N. Constantinescu, L. Blatt, E. Croze, J. E. Mullersman, and L. M. Pfeffer. Direct association of STAT3 with the IFNAR-1 chain of the human type 1 interferon receptor. J. Biol. Chem. 271 (14). 8057-8061, 1996. 326.Yonehara, S., A. Ishii, and M. Yonehara-Takahashi. Cell surface receptor-mediated internalization of interferon: Relation to the antiviral activity of interferon. J. Gen. Virology, 64,2409-2418, 1983. 327.Yoshida, T., C.Chen, M. Zhang, and H. C. Wu. Dismption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp. Cell Res., 192,389-395, 1991. 328.Yutsud0, T., T. Honda, T. Miwatani, and Y. Takeda. Physicochemical characterization of A and B subunits of shiga toxin and reconstitution of holotoxin from isolated subunits. Microbiol. Immunol. 3 1 (3), 189- 197, 1987. 329.Zhong Z., Z. Wen, and J. E. Damell, Jr. Stat3 and Stat4: Members of the family of signal transducers and activators of transcription. Proc. Natl. Acad. Sci. USA., 9 1.4806-48 10, 1994. 330.Zhou, L-J., D. C. Ord, A. L. Hughes, and T. F. Tedder. Structure and domain organization of the CD19 antigen of hurnan, mouse, and guinea pig B lymphocytes. 147 (4), 1424- 1432,199 1. 33 1 .Zoja, C., D. Coma, C. Farina, G. Sacchi, C. Lingwood, M. P. Doyle, V. V. Padhye, M. Abbate, and G. Remuzzi. Verotoxin glycolipid teceptors determine the localiztion of microangiopathic process in rabbits given verotoxin-1 . J. Lab. Clin. Med., 120 (2), 229-238. 33î,.zoon, K-C., H. Arnheiter, D. 2. Nedden, D. I. P. Fitzgerald, and M. C. Willingham. Human interferon alpha enters cells by receptor-rnediated endocytosis. Virology. 130, 195-203, 1983.