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