GPI-ANCHORED PROTEINS IN RECONSTITUTED LIPID BILAYERS:
STRUCTURE, FUNCTION, AND CLEAVAGE BY PI-SPECFIC PHOSPHOLIPASE C
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by MARTY T. LEHTO
In partial fulfilrnent of requirements
for the degree of
Doctor of Philosophy
August 2001
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Marty T. Lehto Advisor: University of Guelph, 200 1 Professor F.J. Sharom
Many eukaryotic proteins are anchored to the ce11 surface by a
glycosylphosphatidylinositol (GPI) moiety. One of these proteins, the enzyme ecto-
5'-nucleotidase (5'-NTase), was purified fiom porcine lymphocytes and reconstituted into
defined lipid bilayers. The GPI anchor was removed fiom al1 5'-NTase molecules
following cleavage by PI-specific phospholipase C from Bncillz
(Bt-PI-PLC). Anchor cleavage was modulated by the composition of the membrane
bilayer, suggesting that lipid molecular properties and bilayer packing may affect the
ability of PI-PLC to gain access to the GPI anchor. Catalytic activation of 5'-NTase was
observed following Bi-PI-PLC cleavage fiorn lipid bilayers. The degree of activation
depended on the lipid composition of the reconstituted vesicles. Insertion of the GPI
anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-NTase, possibly
via conformational changes in the enzyme, and activity is restored upon release fiom the membrane.
A kinetic study of the reiease of 5'-NTase fiom membrane bilayers by Br-PI-PLC
indicated that lipid fluidity and bilayer packing were the most important factors influencing cleavage activity. Very high rates of cleavage were observed in fluid lipids with a low phase transition temperature (Td,lymphocyte plasma membrane, and in lipid mixtures that form rafts. Arrhenius plots of the rate of anchor cleavage in various lipids showed a characteristic break at the Tm of the bilayer. The introduction of charged species into the membrane bilayer had little effect on anchor cleavage, indicating that membrane surface charge is less important in the regulation of Bt-PI-PLC activity.
The GPI-anchored protein placental alkaline phosphatase (PLAP) was labelled with 7-dimethylarnino-comarin-4-acetic acid @MACA) or Oregon Green 488 (06488)
(fluorescent donors) and reconstituted into lipid bilayer vesicles containing increasing mole fractions of (7-nitrobenz-2-oxa- 1,3-diazol-4-y1)- l,2-dihexadecanoyl-sn-glycero-3- phosphoethanolarnine (NBD-PE) or octadecyl rhodarnine B (C,,RhoB) (acceptors), respectively. Resonance energy transfer between the two donor/acceptor pairs was analysed to estimate the distance between the fluorescent label on PLAP and the membrane surface. The results indicated that the protein portion of PLAP is Iocated at a distance of 8-
12 A fi-om the bilayer surface, suggesting that the protein lies close to the membrane, possibly resting on the surface. ACKNOWLEDGEMENTS
1 would like to sincerely thank my supervisor, Dr. Frances I. Sharom, for her tremendous amount of guidance and support throughout my research. 1 would also like to thank the rnembers of my advisory and examination cornmittee: Dr. P.D. Josephy, Dr. R.
Keates, Dr. E. London and Dr. E. Meiering.
1 would like to thank the members of my research lab, especially Joseph Chu for his technical support over the years, for helping me with the DSC scans, and for preparing most of the Thy-l used in this research.
1 would like to thank rny parents, Martin and Muriel Lehto, for their love and support. I would also like to thank the rest of my family and oiends.
Finally, 1would like to thank my beautifid wife Natalie for her incredible arnount of love, support and friendship. 1could not have done it without you Natalie. TABLE OF CONTENTS ABSTRACT
ACKNOWrdEDGEMENTS i
TABLE OF CONTENTS ii
LIST OF TABLES vii
LIST OF FIGURES viii
GLOSSARY OF ABBREVIATIONS
CHAPTER 1: INTRODUCTION
1.1 General background
1.2 Structure of the GPI anchor
1.2.1 Use of phospholipases to elucidate GPI anchor structure
1.2.2 Detailed structure of the GPI anchor
1.3 Biosynthesis of GPI anchors
1.4 Biological significance of GPI-anchored proteins
1.4.1 Release of GPI-anchored proteins through cleavage by endogenous phospholipases
1.4.2 Lateral mobility of GPI-anchored proteins
1.4.3 Distribution and 1ocaIization of GPI-anchored proteins
1.4.3.1 The lipid raft hypothesis
1.4.3.2 In vivo evidence for the existence of membrane rafts
1.4.3-3 Localization to caveolae
1.4.4 Importance of the GPI-anchor in signal transduction
1.5 5'-Nucleotidase (5'-NTase) (EC 3.1.3 -5)
1.5.1 General properties
1.5.2 Irnmunological role
1.6 Other GPI-anchored proteins .. II 1-6.2 Human placenta1 alkaline phosphatase (EC 3.1.3.1 ; PLAP) 25
1.7 Fluorescence sîudies of proteins 28
1.7.1 General fluorescence theory 28
1.7.2 Fluorescence resonance energy transfer (FRET) 30
RATIONALE AND RESEARCH OBJECTIVES 33
CHAPTER 2: RELEASE OF 5'-NUCLEOTIDASE BY PI-SPECIFIC PHOSPHOLIPASE C: EFFECT OF GPI ANCHOR CLEAVAGE ON THE CATALYTIC PROPERTIES OF THE ENZYME
2.1 Abstract 37
2.2 Introduction 38
2.3 Materials and methods 40
2.3.1 Materials 40
2.3.2 General methods 41
2.3 -3 Purification of porcine-lymphocyte 5'-NTase 41
2.3.4 Reconstitution of porcine-lymphocyte 5'-NTase 42
2.3.5 Cleavage of detergent-solubilized 5'-NTase by Bt-PI-PLC 42
2.3.6 Cleavage of membrane-bound 5'-NTase by Bt-PI-PLC 43
2.3.7 Kinetic analysis of 5'-NTase enzyrnatic activity 44
2.4.1 Purification of porcine-lymphocyte 5'-NTase 44
2.4.2 Reconstitution of purified 5'-NTase 46
2.4.3 Kinetics of 5'-AMP hydrolysis by detergent-solubilized and membrane-bound 5'-NTase 50
2.4.4 Cleavage of detergent-solubilized and membrane-bound 5'-NTase by Bt-PI-FLC 52 2.4.5 Activation of 5'-NTase following cleavage fiom various membrane systems
2.5 Discussion
CHAPTER 3: BACTERIAL PI-SPECIFIC PHOSPHOLIPASE C: MODULATION OF ANCHOR CLEAVAGE ACTIVITY BY THE PROPERTIES OF THE LIPID BILAYER
3.1 Abstract 68
3.2 Introduction 69
3.3 Materials and methods 71
3 -3.1 Materials 7 1
3 -3.2 Purification of porcine lymphocyte 5'-NTase 7 1
3 -3.3 Reconstitution of porcine lymphocyte 5'-NTase 71
3 -3.4 Cleavage of 5'-NTase by Bt-PI-PLC 73
3.3 -5 Preparation of detergent-resistant membranes (DRM's) 74
3.3-6 Di fferential scanning calorimetry 75
3.4 Results 75
3.4.1 Kinetics of cleavage of 5'-NTase by Bt-PI-PLC 75
3.4.2 Effect of acyl chain length and unsaturation on Bi-PI-PLC cleavage of 5'-NTase 77
3.4.3 Effect of lipid bilayer surface charge on the cIeavage of 5'-NTase by PI-PLC 8 1
3.4.4 Effect of lipid phase state on the cleavage of 5'-NTase by Bt-PT-PLC 83
3.4.5 Effect of lipid raft components on the cleavage of 5'-NTase by Bt-PI-PLC 90
3.4.5.1 Effect of gangliosides on the cIeavage of 5'-NTase by Bt-PI-PLC 90
3.4.5.2 Effect of Thy- 1 on the cleavage of 5'-NTase by Bt-PI-PLC 96 3.4.5.3 CIeavage of 5'-NTase in SCRL vesicles by Bt-PI-PLC
3.5 Discussion
CHAPTER 4: PROXIM.ITY OF THE PRQTEIN MOIETY OF THE GBI- ANCHORED PROTEIN PLAP TO THE MElMlBRANE SURFACE: A FLUORESCENCE RESONANCE ENERGY TRANSFER STUDY
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
Materials
Purification of placenta1 alkaline phosphatase (PLAP)
Assay for PLAP activity
Absorption spectra and fluorescence excitation.emission spectra
Fluorescent labeling of PLAP
Preparation of reconstituted vesicles containing PLAP
Dynarnic light scattering
Resonance energy transfer measurements
Determination of parameters for FRET analysis
Analysis of the distance between donor and acceptor
4.4 Results
4.4.1 Purification of PLAP
4.4.2 Fluorescent labelling and reconstitution of PLAP
4.4.3 Resonance energy transfer
4.5 Discussion CHAPTER 5: SUMRlARY AND CONCLUSIONS
5.1 Surnmary and conclusions
5.2 Suggestions for fùture work
REFERENCES LIST OF TABLES
Table Title Page
Purification of 5'-NTase fiom porcine lymphocytes 45
Release of detergent-solubilized and membrane-bound 5'-NTase by PI-PLC 56
Sumrnary of kinetic parameters for 5'-NTase before and after cleavage by PI-PLC 59
Catalytic-centre activities for 5'-NTase before and afier cleavage by PI-PLC 60
Bt-f 1-PLC cleavage of 5'-NTase in different lipid environments at 37 OC 78
Effect of various components on the cleavage of 5'-NTase by Bt-PI-PLC 85
Activation energies for Bt-PI-PLC cleavage of 5'-NTase in different lipid environments 88
Activation energies for the cleavage of 5'-NTase by Bt-PI-PLC in proteoliposomes containing various components 92
Purification of PLAP kom human placenta 117
Spectral parameters for donor and acceptor pairs 123
vii LIST OF FIGURES
Figure Title Page
Structure of the GPI anchor
The pathway of GPI precusor formation
Cleavage of phosphatidylinositol by PI-PLC, and structure of PI- PLC from B. cereus in cornplex with glucosaminyl(a 1+6)-D- myo-inositol
Overall reaction scheme for the hydrolysis of 5'-AMP by 5'-NTase
Overall structure of the PLAP dimer
Fluorescence resonance energy transfer between donor and acceptor fluorophores
Fluorescence energy transfer between two fluorophores
SDSPAGE analysis of purified porcine-lymphocyte 5'-NTase
Determination of the bilayer to hexagonal phase transition of egg PE
Syrnrnetry of reconstitution of 5'-NTase into various lipids
Kinetics of catalysis by detergent-solubilized and membrane- bound 5'-NTase
Cleavage of detergent-solubilized and membrane-bound 5'-NTase by PI-PLC
Kinetics of 5'-NTase before and after cleavage of the GPI anchor by PI-PLC
Initial rate of Bt-PI-PLC cleavage of 5'-NTase at 37 "C
Cleavage of 5'-NTase by Bt-PI-PLC in porcine lymphocyte plasma membrane vesicles
Effect of acyl chain length and unsaturation on the initial rate of Bt-PI-PLC cleavage of 5'-NTase in phosphatidyIcholine proteoliposomes DSC scans of various phospholipid bilayers
Effect of lipid bilayer surface charge on the initial rate of Bt-PI-PLC cleavage of 5'-NTase in DMPC proteoliposomes
Arrhenius plots of the initial rate of Bt-PI-PLC cleavage of 5'-NTase in different lipid systems
Effect of GM, on 5'-NTase cleavage by Bt-PI-PLC
Detergent solubility of 5'-NTase in porcine lymphocyte plasma membrane and proteoliposornes of various lipids
Effect of lipid raft components on the initial rate of Bt-PI-PLC cleavage of 5'-NTase
Effect of lipid bilayer fluidity on the activity of Bt-PI-PLC
Purification of PLAP fiom human pIacenta
Size distribution of reconstituted vesicles containing PLAP
Overlap of the fluorescence emission spectra of the donors with the UV-visible absorption spectra of the acceptors
Fluorescence resonance energy transfer between donors and acceptors
Curve fitting of the FRET data from the different donors and acceptors
Curve fitting of the FRET data fiom the different donors and acceptors
Models of dimeric PLAP in relation to the bilayer surface GLOSSARY OF ABBREVIATIONS
wavelength of fluorescence emission maximum
Aes wavelength of fluorescence excitation maximum
5'-NTase ecto-5'-nucleotidase
AChE acetylcholinesterase
AFM atomic force microscopy
1,8-ANS 1-aniIinonaphthalene-8-sulfonic acid
APase alkaline phosphatase
Bc-PI-PLC PI-PLC from Bacillus cerezrs
BS A bovine semalbumin
Bt-PI-PLC PI-PLC from Bacillus thziringiensis
C,,RhoB octadecyl rhodarnine B chloride
CHAPS 3 [(3-cholamidopropyl)dimethyI~onio]-I -propanesuifonate cIP myo-inositol 1,2-(cyc1ic)-phosphate
Con A concanavalin A
DAF decay accelerating factor
DAG diacyIglycero1
DiCP dicetyl phosphate
DLS dynamic light scattering
DMACA 7-dimethylaminocoum~n-4-aceticacid
DMPC dimyristoylphosphatidylcholine
DMSO dirnethylsuIfoxide
DOPC dioleoylphosphatidyicholine D-PDMP
DRM detergent resistant membrane
DSC differential scanning calorimetry
Ex, activation energy
EC50 concentration of Bt-PI-PLC required to release 50% of 5'-NTase
in soluble form
ER endoplasmic reticulum
FPLC fast protein liquid chromatography
FRAP fluorescence recovery after photo bleaching
FRET fluorescence resonance energy transfer
GPI glycosylphosphatidylinositol
GPI-PLD glycosylphosphatidylinositol-specific phospholipase D
GSL glycosp hingolipid
IBS interfacial binding surface
LFA-3 lymphocyte-function-associated molecule 3
MB-PE Marina Blue 1,2-dihexadecanoyl-sn-glycero-3-phospho-
ethanolamine
MDCK ~Madin-Darbycanine kidney cells
NBD-PE (7-nitrobenz-2-oxa- 1,3-diazol-4-YI)- l,2-dihexadecanoyl-sn-
glycero-3-phosphoethanolarnine
NMR nuclear magnetic resonance
Oregon Green 488
procyclic acidic repetitive protein
phosphatidylcholine PE phosphatidylethanolamine phosphonate L-histidyldiazobenzylphosphonic acid
PT phosphatidylinositol
PI-PLC phosphatidylinositol-specific phospholipase C
PLAP placenta1 alkaline phosphatase
PM plasma membrane
PMPC palmitoylm~stoylphosphatidylcholine
PNP p-nitrophenylphosphate
SA stearylamine
S.D. standard deviation
S.E.M. standard error of the arithmetic mean
SCRL sphingolipid/cholesterol-rich liposomes
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SM sphingomyelin
TBS Tris buffered saline
TBSBSA TBS containing 1% BSA
TCR T-ceIl receptor
Tm gel to liquid-crystalline phase transition temperature
TX-1 00 Triton X-100
TX-I 14 Triton X- 1 14
xii CHAPTER 1 : INTRODUCTION
1.1 General background
As originally proposed in the Singer-Nicolson mode1 [279], al1 integral membrane proteins were thought to be anchored to the ce11 surface via one or more transmembrane domains interacting hydrophobically with the membrane bilayer. In the mid-198O1s, studies of variant surface glycoprotein (VSG) kom Tvpanosorna bnrcei [73] led to the first description of a new type of membrane anchor, involving the covalent attachment of the C-terminal amino acid of the protein to a glycosylphosphatidylinositol (GPI) moiety, which is inserted into the lipid bilayer. Since that time, over 100 stmcturally and functionally diverse proteins from a variety of eukaryotic sources (marnrnalian, plant, yeast and protozoan) have been identified as GPI-anchored. These proteins include lymphocpe and trypanosome surface antigens (e.g. Thy-1, VSG), adhesion molecules
(e.g. lymphocyte-function-associated rnolecule [LFA-3]), exofacial ecto-enzymes (e-g.
5'-nucleotidase [S-NTase], alkaline phosphatase [APase] and acetylcholinesterase
[AChE]) and receptors (e.g. folate receptor) (reviewed in [72,73,126]). The prion protein, which has received much attention in recent years as the causative agent of spongiform encephalopathies (Creutzfeldt-Jakob disease, 'mad cow disease' and scrapie), has long been known to be a GPI-aqchored protein (reviewed in [259]). The functional diversity of the many GPI-linked proteins is very broad. However, there does not appear to be any distinct or cornmon functional relevance for the use of the GPI anchor. 1.2 Structure of the GPI anchor
1.2.1 Use of phospholipases to elucidate GPI anchor structure
The initial observation that APase (thought to be an integral protein) could be released from the membrane bilayer by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) [135,172,282] was the first clue that there may be another form of membrane attachrnent for proteins. This work suggested that the protein was linked, probably covalently, to a phosphatidylinositol phospholipid. Since then, PI-PLC's have been used extensively to identie and elucidate the basic structure of the GPI anchor.
Bacterial PI-PLC has been purified from Bacfis cerezu, BaciZZzu thringiensis,
Clostridium novyi and Staphylococclts aureus. These enzymes have a high affinity for the GPI anchor and cm cleave the anchor in vitro to release a soluble protein and diacylglycerol @AG), which remains embedded in the membrane. Triton X- 1 14
(TX-114) partitionhg revealed that the protein enters the aqueous phase afier anchor cleavage, while the DAG remains in the detergent phase [l27,128].
1.2.2 Detailed structure of the GPI anchor
The first complete structure of the GPI anchor was elucidated for VSG, a GPI- anchored protein from i? brucei [74] (see Figure 1.1). The core tetrasaccharide Mana
1+2Mana 1-+6Mana 1+4GIcN is glycosidically linked to the 6-hydroxyl group of phosphatidyIinosito1. A notable feature of the glucosamine is that it is non-acety lated, and thus differs from glucosamine found in rnost biological systems. The terminal mannose in the tetrasaccharide is linked via a phosphodiester bond to ethanolamine
(EtN), which is amide-linked to the a-carboxyl group of the C-terminal peptide of the protein. Variations in the structure include the attachrnent of galactosyl side chains (Rq in te' 1 rn Figure 1.1), and additional phosphoethanolamine or rnannosyl residues, to the first mannose residue (R3in Figure 1.1). The DAG (diacylglycerol or 1 -alkyl-2-acyl glycerol) portion usually contains alkyl or acyl chains, with an unsaturated chain at the snl position and a saturated chain at the sr22 position [63]. The type of fatty acyl or alkyl groups linked to the glycerol may Vary. In some anchors, such as AChE [251] and the procyclic acidic repetitive protein (PARP) [79], the inositol ring residue is acylated, usually with palmitate. The above mentioned differences in structure are species- and tissue-specific and may be responsible for the resistance to phospholipase cleavage observed for certain subsets of GPI-anchored proteins.
The structure of the GPI anchor of other proteins has been partially or cornpletely elucidated. These include human AChE [56,251,2521, rat brain Thy-1 [124], Leishmania major surface protease (gp63) [262], and Trypanosorna cmzi 1G7 antigen 1991.
1.3 Biosynthesis of GPI anchors
The GPI biosynthetic pathway was first elucidated for VSG of T. brucei, by means of a combination of chemical and enzyrnatic analysis, nuclear magnetic resonance
(NMR) spectroscopy, mass spectrometry, and pulse-chase radiolabelling of cell-f?ee extracts [187,195]. The GPI biosynthetic pathway consists of the sequential transfer of sugar residues to PI which is bound to the membrane of the rough endoplasmic reticulum
(ER). The complete anchor is then attached to a newly translated protein. Most of the proteins involved in GPI anchor biosynthesis have been identified in both yeast and mammals. Only the mamrnalian biosynthetic rnachinery will be discussed here (Figure
1.2).
A GlcNAc unit is first transferred f?om UDP-GlcNAc to PI to form GlcNAc-PI
[62]. The mamrnalian al-6Glc-NAc transferase is a complex of at least four protein
4 subunits: PIG-A [204], PIG-C [138], PIG-H [147] and Gpilp [319,337]. The PIG-A subunit is probably the site of catalysis [18,148] and Gpilp may be involved in the assembly and stability of the GIcNAc-PI synthetic cornplex. Both PIG-C and PIG-H have unhown roles 13 181.
The N-de-acetylation of GlcNAc-PI is the next step in the biosynthesis of GPI
[62,116]. A catalytic subunit of de-N-acetylase, PIG-L, has been identified in rats
[214,291] and may be in complex with an as-yet-unidentified regulatory subunit [3 183.
The next step in the process is the acylation of the inositol ring at the 2-position
[45,326] using acyl-CoA as the irnmediate acyl donor [64]. Inositol acylation casses the
GPI anchored protein to become resistant to PI-PLC cleavage [251]. However, most marnrnalian GPI-anchored proteins are sensitive to PI-PLC cleavage because they Iack an acylated inositol. This suggests that a deacylase activity may exist in mammals [280] and, indeed, it was discovered that deacylation takes place in the ER within 5 min of the transfer of the protein to the GPI anchor [42].
The GlcN(acylinositol)-PI is then sequentially mannosylated [187,195], using as the donor dolichol-P-mannose [194], which had previously been mannosylated by GDP- mannose. Dolichal-P-rnannose is thought to be the mannosyl donor because mammaliw, ceIl lines that cannot synthesize dolichol-P-rnannose fail to express Thy-1 and other GPI- anchored proteins [57]. Transfection of the defective ce11 lines with the yeast gene for dolichol-P-mannose synthase resulted in normal expression of the GPI-anchored proteins.
Of the three mannosyltransferases (GPI-MT-1, -II and -III), information has only been obtained for GPI-MT-1 and GPI-MT-III. The PIG-B pene is defective in a ce11 line lacking GPI-MT-III activity [238,301]. The PIG-B gene product is an ER membrane protein with a large intralurnenal C-terminus and a small cytoplasmic N-terminus. The
PIG-M gene is responsible for GPI-MT-1 activity [179].
The next step in the biosynthetic pathway is the transfer of EtN-P to the
6-position of the terminal mannose residue to form EtN-P-Man,-GlcN(acy1inositol)-PI, which is also hown as glycolipid A' [73]. The donor for the EtN-P transferase is probably phosphatidyiethanolamine [ 193,1961. Finally, the complex undergoes a series of fatty acid remodelling steps to form glycolipid A [188].
The addition of the pre-formed GPI anchor to the polypeptide has been exarnined for VSG and several other proteins. The GPI-anchored protein contains a cleavable hydrophobic N-terminal signal sequence and is first translated on the rough ER and translocated through the ER membrane. Within 1 to 2 minutes after protein translation and insertion, the preformed GPI anchor is added. A domain of 15-20 amino acids at the
C-terminal is displaced by glycolipid A, probably by nucleophilic attack catalyzed by a transamidase enzyme [63,189]. Two proteins have been identified that make up the mammalian transamidase enzyme: Gpi8p [15] and Gaalp [115]. The Gpi8p protein is probably the site of catalysis in the transarnidase enzyme [3 181, and Gaalp is most likely responsible for the recognition of the GPI cleavage/attachment site and regulation of the specificity of the transarnidase [221]. The C-terminal anchor attachent sequence consists of 15-20 hydrophobic arnino acid residues at the extreme C-terminus. It is the hydrophobicity of this C-terminal segment, rather than the specific arnino acid sequence itself that directs GPI-anchor attachent. Just upstrem of the hydrophobic sequence is a spacer of 5-10 hydrophilic residues. Both the length of this region and the presence of 1 to 3 small arnino acid residues appear to be important for anchor addition
[14,90,200,205,334]. The anchor is always added to one of the small arnino acid
7 residues, either Gly, Ser, Ala, Cys, Asn or Asp [325]. Following the addition of
glycolipid A, the mature GPI-anchored protein travels to the Golgi for modification of the
GPI-anchor glycan, and glycosylation at other sites on the protein [63].
1.4 Biological significance of GPI-anchored proteins
1.4.1 Release of GPI-anchored proteins through cieavage by endogenous phospholipases Glycosylphosphatidylinosito1-specific phospholipase C (GPI-PLC) has been
discovered in T. brucei [75] and in several rnarnrnalian cells and tissues, inchding human
neutrophils [133], bovine brain [302], and rat liver [292]. Since these tissues are also
known to express GPI-anchored proteins, endogenous phospholipases may serve as
important biological factors. These enzymes may allow specific down-regulation of the
expression of GPI-anchored proteins on the ce11 surfâce while simultaneously increasing
the soluble protein in circulation. This theory has been supported by evidence that
soluble foms of GPI-anchored proteins, such as 5'-NTase [285], Thy-1 [4], and APase
[305], can be found in circulation.
Other evidence supports the possible role of phospholipases in the regulation of
GPI-anchored proteins. The protective glycoprotein coat of lr: bnlcei is made up of millions of GPI-anchored VSG proteins. To evade the immune response of the host organism, the surface coat undergoes antigenic variation, and removal of the old surface coat rnay be achieved by the activity of trypanosomal GPI-PLC [186]. Activated neutrophils release CD16 receptors for the constant region of IgG through the specific
action of endogenous GPI-PLC's and these receptors are believed to mediate immune
responses once in circulation [133]. Endogenous phospholipases cm cleave APase from
rat intestine [71] and a human carcinoma ce11 line [248]. Insulin cm cause the release of several GPI-anchored proteins firom myocytes [258] and skeletal muscle [152] by
activating an endogenous GPI-PLC. However, not al1 of the GPI-anchored proteins on
the insulin-sensitive cells are released, suggesting that some anchors are modified and
thus resistant to GPI-PLC cleavage. Recently, an endogenous GPI-PLC was found to
release renal dipeptidase fkom porcine proximal tubules in vitro [225].
Since bacterial PI-PLC's are able to cleave GPI anchors in eukaryotes, structural
studies of PI-PLC found in bacteria may yield important information about the structure
of GPI-PLC found in eukaryotes. The crystal structure of PI-PLC fiom B. cerezts has
been determined both in fkee form (at 2.5 A resolution; PDB 1PTD), and in complex with
myo-inositol (at 2.6 A resolution; PDB 1PTG) [Ill]. The protein has eight p-strands
forrning an a/P barre1 and there are no hydrogen bonds between the P-5/P-6 strands. The
carboxyl- and amino-terminus are in proximity, and the myo-inositol is in a deep active
site pocket. To understand how PI-PLC interacts with the GPI anchor, the crystal
structure of PI-PLC from B. cereus was determined at 2.2 A resolution in cornplex with
glucosaminyl(al+6)-D-myo-inositol (PDB 1GYM) (see Figure 1.3B), which is part of
the core of al1 GPI anchors [112]. The myo-inositol moiety of GlcN(ct1+6)Ins occupies
the same position as fiee myo-inositol, whereas the glucosamine moiety lies exposed to
solvent at the entrance to the active site. This shows that the catalytic mechanism for
cleavage of PI is similar to that of GPI (Figure 1.3A). The glycan moiety has very little contact with the enzyme, which may explain why it cm cleave GPI anchors with varying glycan structures, The crystal structure of PI-PLC fkom Listeria monocytogenes (PDB
IAOD) has also been solved at 2.0 A resolution and proved to be very similar to the structure of PI-PLC fiorn B. cereus [206]. PI-PLC PI-PLC P --p"k
Figure 1.3 Cleavage of phosphatidylinositol by PI-PLC, and structure of PI-PLC from B. cereus in complex with glucosaminyl(al-+6)-D-myo-inositol (A) The proposed twa-step mechanism for the cleavage of PI by PI-PLC. The position of the glycan portion of the GPI anchor is indicated by an X (see Figure 1.1). (B) The overaIl structure of PI-PLC is shown in nbbon representation. The proposed interfacial binding surface (IBS; helk 42-48 and ioop 237-243) is indicated in red. This figure was made with Swiss PDB viewer Ver. 3.6b (PDB IGYM; [112]). Another phospholipase, glycosyIphosphatidylinositol-specific phospholipase D
(GPI-PLD), has been discovered in a nurnber of mamrnalian tissues including human senim and placenta [54] as well as rat sem[55], rabbit sem[175], and bovine serum, brain and Iiver [113,117,118,130]. Synthesized in the pancreas and then released into circulation [198], the endogenous GPI-PLD rnay interact with other marnmalian tissues, resulting in down-regulation of GPI-anchored protein expression on tissue surfaces, and an increased concentration of these proteins in the plasma. Detergent-solubilized GPI- anchored proteins are susceptibIe to GPI-PLD cleavage, and al1 assays for GPI-PLD cleavage are carried out in detergent [55,130,292]. In the absence of detergents, GPI-
PLD's show a greatly reduced or undetectable ability to release GPI-anchored proteins from the ce11 membrane [130,174,294]. The GPI-PLD enzyme may have to be activated in some way before it is able to cleave GPI-anchored proteins from cells [174]. Trypsin can cleave GPI-PLD into distinct degradation products, which remain associated and show increased activity towards GPI-anchored pro teins. In rat liver, GPI-PLD is enriched in a Iysosomal fiaction [107]. Neuroblastoma cells take up GPI-PLD fiom the medium and proteolytically process it in acidic intracellular compartments, resulting in fkagrnents of molecular masses between 101 and 39 kDa that are still active towards GPI- anchored proteins [106]. These results suggest that the GPI-PLD enzyme acts on its substrates after uptake fiom senun and proteolytic processing in an intraceMar compartment.
1.4.2 Lateral mobility of GPI-anchored proteins
The technique of fluorescence recovery after photobleaching (FRAP) was used to measure the lateral mobility of several GPI-anchored proteins. In FRAP, a laser is used to bleach the membrane proteins (or lipids) containing a fluorescent label, and the
11 specific area that was bleached is monitored to observe the recoveiy of fluorescence.
Most GPI-anchored proteins have Iateral mobilities (measued as the lateral diffusion coefficient, D) 10-fold higher than those of polypeptide-anchored proteins, typically in the range of 1-5 x IO-' cm2/sec. For exarnple, both Thy-1 in lymphoid cells and fibroblasts, and APase in cells that express APase naturally or following DNA transfection, showed D values approaching those of phospholipids (D z IO-' cm2/sec)
[218]. However, some GPI-anchored proteins are immobile, and have much lower D values. For example, VSG has a difhsion coefficient of -1 x 10-Iocm2/sec in both intact cells, and in BHK ce11 membranes following transfection [32]. The adhesion protein PH-
20 from guinea pig sperm has a diffbsion coefficient dependent on the maturity of the sperm (-2 x IO-"- -5 x cm2/sec ) [234]. The GPI-anchor thus appears to play an important role in the lateral mobility of GPI-anchored proteins.
1.4.3 Distribution and localization of GPI-anchored proteins
Most GPI-anchored proteins are located at the cell surface and face the extracellular space. Some exceptions are elongation factor EF-la on the ER of Chinese hamster fibroblasts [log], a fiaction of PH-20 in the acrosome of guinea pig sperm [234] and a fraction of decay accelerating factor (DM) in neutrophils [16]. The latter two proteins face the lumen of intracellular compartrnents and may be reIeased to the ceIl surface under regulatory control by the cell. This suggests a possible role for the GPI anchor in regulated secretion of proteins [165].
The attachrnent of a GPI anchor appears to direct a protein to the apical surface of polarized cells. In Madin-Darby canine kidney (MDCK) cells, at least six endogenous
GPI-linked proteins are localized to the apical surface [166]. The GPI-anchored Thy-1 protein is delivered exclusively to axonal membranes of hippocampal neurons [66], and
12 5'-NTase and APase are delivered to the apical membrane of hepatocytes [2]. The PH-20 adhesion protein fkom guinea pig sperm is localized in the anterior region of the mature sperm. The presence of the GPI-anchor itself, and not the identity of the protein it anchors, seems to be responsible for polarized expression. In fact, the GPI anchor signal sequence can be attached (through molecular biological techniques) to proteins known to be distributed in the basolateral membrane, such as the viral envelope glycoproteins HSV gD-1 or VSV G-protein [26,164], or to a regulated secretory protein such as hurnan growth hormone [164], and the new protein becomes GPI-anchored and is targeted to the apical surface.
1.4.3.1 The lipid raft hypothesis
Glycosphingolipids (GSL) appear to play an important role in the sorting of GPI- anchored proteins to the apical surface. One proposa1 suggests that apical membrane proteins may be sorted with GSL and delivered preferentially to the apical surface of the ce11 [275,276]. Glycosphingolipids are found in the outer leaflet of the plasma membrane, where they can self-aggregate and form patches when mixed with phospholipids (reviewed in [3 171). Most GPI-anchored proteins in the plasma membrane are insoluble in cold nonionic detergents such as TX-100 (reviewed in [128,171]).
Cellular GSL and sphingomyelin are also insoluble in cold TX-IO0
[52,lOl, 297,297,3441. A class of apically targeted GPI-anchored proteins in MDCK cells associates with GSLs and other GPI-anchored proteins in the Golgi, suggesting that protein-sphingolipid microdomains fom in the Golgi and are transported to the plasma membrane [30]. Al1 of these observations led to the development of the lipid raft hypothesis [274], which has gained increasing support in recent years. The plasma membrane is no longer envisioned as a unifonn surface, as the fluid-mosaic mode1
13 suggests, but is likely made up of specialized regions containing specific lipids and proteins .
Indirect support for the lipid raft hypothesis has corne fiom a variety of studies which most commonly used cold nonionic detergents (such as TX-100) to generate insoluble membrane fiagrnents of mammalian cells. These membrane fragments have been called detergent-resistant membranes (DRM's) 1301, detergent-insoluble glycolipid- enriched membranes PIG's) [23 11, glycolipid-enriched membranes (GEM's) [256],
Triton-insoluble membranes (TIM's) 1871, low-density Triton-insolub le fractions
(LDTI1s) [230] and Triton-insoluble floating fiactions (TIFF) [210]. DRMs are rich in sphingolipids, glycosphingolipids, cholesterol, GPI-anchored proteins and certain non- receptor tyrosine kinases, but are relatively poor in phospholipids and trammembrane proteins [30,43,76,103,274,288,290]. Cholesterol-binding agents, such as saponin and
P-cyclodextrin, cm disrupt the detergent-resistant clustering of GPI-anchored proteins
[39,85,137,163]. Also, it appears that interactions between saturated acyl chains, but not necessarily cholesterol, are important for the detergent resistance of GPI-anchored proteins [263]. A recent study used atornic force microscopy (AFM) to visualize the membrane domains remaining after detergent extraction, and found that detergent- resistant plasma membrane fiagrnents form domains of 15-20 pm', which May be associated with the cytoskeleton [91]. Another AFM study using mode1 membranes of the ganglioside GM,, cholesterol and DPPC supported on mica was able to mesure submicron-sized domains analogous to those found in DRM's [345].
Lipid rafts have been implicated in a nwnber of important processes, including cellular signalling (discussed later), budding of enveloped viruses, such as influenza [26 1,3471 and HN-1 [216,3351 fiom the plasma membrane, bacteria-host ce11 interaction
[271,272] and the processing of arnyloid precursor protein, which is implicated in the development of Alzheimer's disease [229].
Several lines of evidence support the idea that lipid rails are in the liquid-ordered phase l,, a phase that is intermediate between the solid gel phase and the fluid liquid crystalline phase. The 1, phase is characterized by tight acyl chain packing and relatively extended acyl chains (similar to the gel phase), but the acyl chains have rapid lateral mobiIity in the bilayer (similar to the liquid crystalline phase) [3]. Artificial 1, phase liposomes are insoluble in TX-100 [263]. A detergent-kee in vitro approach using a fluorescence-quenching technique demonstrated that separation of fluid and 1, phases can be achieved in mixtures of phospholipids, sphingolipids and cholesterol at physiological conditions of temperature and lipid composition [l].
Detergent insolubility is a valuable tool for studying the protein and lipid requirements for raft formation. However, DRM1s probably do not accurately reflect the size and morphology of membrane microdomains in vivo. This is partly due to the fact that cells must be chilled to 4 OC before detergent extraction occurs. Chilling probably stabilizes the 1, phase and enhances its detergent resistance, which may explain why a surpnsingly high fiaction of membrane lipids are present in DRM's [27,28] md why the composition of DRM's cari resemble that of the plasma membrane [83]. However, an underestimation of raft components cm occur if raft lipids and proteins are solubilized by detergent even after chilling [7,77,223]. These temperature effects have raised some doubts as to whether lipid rafts exist at al1 at physiological temperatures, or whether raft formation is actually an artifact of the detergent extraction procedure. A detergent-fiee method for their isolation is necessary to better understand the morphology and
composition of rafts in the plasma membrane of living cells.
1.4.3.2 In vivo evidence for the existence of membrane rafts
If rafts actually exist in living cells, it should be possible to visualize them by
light microscopy. However, several raft markers such as GPI proteins and gangliosides
appear to be uniformly distnbuted on the ce11 surface when visualized microscopically
unless they are clustered by antibodies or other agents [28,141]. The clustering of one
marker can sometimes cause the redistribution of another rnarker without the existence of
a direct interaction between the two, which strongly suggests that the two markers are
colocalized to membrane rafts [27,lOS, 143,264,3291. Clustering may cause the
coalescence of rafts that are too small to visualize microscopically, or may increase the
affiniiy of certain molecules for rafts, and thus increase their concentration in rafts to a
detectable level [29].
A single particle tracking study of Thy-1 (a GPI-anchored protein) and the
ganglioside GM, (a GSL) on fibroblasts was the first to estimate the size of membrane
microdomains in vivo [269]. Both raft markers were transiently confined to domains of
260-320 nrn in diarneter whereas a non-raft marker showed littie confinement. The
confinement was sensitive to a GSL synthesis inhibitor, which provided evidence for the
importance of GSL in membrane microdomains. Another single particle tracking study
looked at the motion of a bead attached to a GPI-anchored protein confined to a srnaII area using a laser trap [236]. This group suggested that lipid rafts are considerably smaller, cholesterol-stabilized complexes of 26 + 13 nrn in size, diffusing as a single entity. The lateral motion of single fluorescently labelled lipid molecules was imaged in the plasma membrane of human coronary artery srnooth muscle cells [264]. Lipid
16 domains of 0.7 pm in size covered -13% of the total membrane area and were descnbed
as the in vivo equivalent of DRM's. Another group used fluorescence microscopy to
show that the GPI-anchored folate receptor clustered into domains of <70 nrn in diameter
in a cholesterol-dependent rnanner [328]. However, fluorescence microscopy studies of
three GPI-anchored proteins and a GSL found no evidence for clustering in the membrane
[149,150]. These apparent discrepancies could be expIained if only a small fraction of
molecules were closely clustered [328] or if the molecules were uniformly distributed
within the rafts 1291. Biochemical evidence for in vivo clustenng was obtained by a study
of the chemical crosslinking of GPI-anchored proteins in MDCK cells [84]. Crosslinking
efficiencies between GPI-anchored proteins were independent of their concentration on
the ce11 surface suggesting that they were sequestered into raft structures at a constant
density. Crosslinking could be disrupted by the depletion of cholesterol, whereas
detergent treatment substantially increased chemical crosslinking. A follow-up study
using the same crosslinking approach showed thzt exogenous application of gangliosides
could displace GPI-anchored proteins fiorn membrane microdomains [277].
1.4.3.3 Localization to caveolae
Some GPI-anchored proteins may be concentrated in specialized non-clathrin
coated membrane invaginations called caveolae (reviewed in [5]). Caveolae, or
plasmalemmal vesicles, require cholesterol for their integrity and are highly enriched in
GSL and the transmembrane protein caveolin. Caveolae are resistant to solubilization by non-ionic detergents, and their high cholesterol and sphingolipid composition leads to the
formation of the 1, phase. In fact, DRM's were initially thought to be caveolae [40].
However, the observation that DRM's could be isolated £iom cells that lack caveolae and caveolin has led to a clarification of this view [82,95]. In some reports, DRM's that
17 contain caveolins are referred to as caveolae and DRM's lacking caveolins are called caveola-related domains (CRD's) [283].
Caveolae are thought to hction in important cellular processes such as potocytosis (the uptake of srnaIl molecules or ions), non-clathrin mediated endocytosis, signal transduction, and Ca" and cholesterol homeostasis. Clustenng of GPI-anchored proteins in these biologically important membrane invaginations suggests that GPI- anchored proteins are important for the proposed cellular fùnctions of caveolae. The GPI anchor appears to be a! important component in the distribution and localization of GPI- proteins to these membrane domains.
1.4.4 Importance of the GPI-anchor in signal transduction
GPI-anchored proteins appear to be involved in signal transduction in several ce11 types, most notably in lymphocytes (rrviewed in [38,47,6 1,120,136,2531)- Antibody crosslinking of a number of GPI-anchored proteins in lymphocytes and other haematopoietic cells leads to protein tyrosine and sennehhreonine phosphory!ation, intracellular calcium mobilization and activation of transcription factors. Some examples of GPI-anchored proteins that elicit T-ce11 activation include 5'-NTase [185], Thy-1
[176], Ly-6/TAP [18 11 and DAF [53]. T-cells expressing a GPI-anchored fom of DAF were able to be activated, whereas cells expressing transmembrane DAF did not show a response [270]. This study and other evidence has suggested that, for this set of proteins, the existence of the GPI anchor is necessary to induce T-ce11 activation [255,300].
A question has &sen as to how the signal elicited by GPI-anchored proteins can be transmitted across the ce11 membrane since GPI-anchored proteins exist exclusively on the outer leaflet of the plasma membrane. Protein tyrosine kinases (PTKs) appear to co- isolate with most GPI-anchored proteins in detergent-resistant complexes, suggesting that
18 signalling via GPI-anchored proteins could proceed through Src farnily PTKs
[43,67,270]. These PTKs include p56*~k, and p53/56h, which are bound to the imer leaflet of the plasma membrane by at least two fatty acyl chains [245]. This dual acylation of the kinase appears to be the dnving force for the CO-isolationof PTKs with
GPI-anchored proteins in DM'S [ 146,256,3271. However, the exact nature of the influence of the outer Ieaflet on the inner leaflet of the plasma membrane during DRM fonnation is not hlly understood [136]. Thus, the involvement of the GPI anchor in signalling arises fYom its localization to the lipid rafts, which appear to act as "signalling platforms", where participating molecules are concentrated close together. The current understanding of the GPI-anchored protein signalling process is that crosslinking leads to coalescence of microdomains, which generates threshold levels of activation signals through clustered microdomain-associated Src fmily PTKs [105,274]. Administration of compounds that disrupt rafts, such as the cholesterol depleting reagent saponin and
D-threo- 1-phenyl-2-decanoylarnino-3-morpholino- I -propanol (D-PDMP, which disrupts
GSL synthesis), cm abolish signalling though rafts [143].[145]
There may still exist a transmembrane protein to link the exofacial GPI-anchored protein signal with the cytosolic protein tyrosine kinase. The GPI-anchored proteins may associate with the transmembrane T-ce11 receptor (TCR) signal transducer [253]. For exarnple, TCWCD3-deficient T-cells fail to proliferate following crosslinking of the GPI- anchored proteins Thy-l [98], Ly-6 [303] or hurnan CD55 [323]. Also, T-ce11 clones deficient in GPI anchor biosynthesis show reduced TCR signalling [343]. There may also be another, as-yet-unidentified, trammembrane protein that links GPI-anchored proteins with the inner leaflet of the plasma membrane [119,287]. Clearly, there is a large body of evidence that suggests an important role for GPI-anchored proteins in signalling.
19 1.5 5'-Nucleotidase (5'-NTase) (EC 3.1.3.5)
1.5.1 General properties
5'-NTase is a ubiquitous enzyme that catalyzes the breakdown of nucleotides to nucleosides (reviewed in [351]). A 5'-NTase activity has been found both in soluble form and in membrane-bound fom, the membrane form being a GPI-anchored ectoenzyme.
The arnino acid sequence of 5'-NTase has been detemined in three mammalian species
(rat liver [202]; human placenta [203]; bovine liver [304]) and an elasmobranch fish
(brain from electric ray, [331]), as well as in bacteria and plants. The mature protein consists of 548 amino acid residues with a molar mass of 61-63 ma. A C-terminal region of uncharged hydrophobie amino acid residues is replaced with the GPI-anchor on
Ser-523. There are four potential N-linked glycosylation sites for the enzymes from human placenta, electnc ray and bovine brain, and five glycosylation sites for the enzyme fiom rat liver. The three mamrnalian forms dispIay 85-90% arnino acid identity, whereas the electric ray displays -61% amino acid identity with the mammalian forms. There are essentially four known forms of the enzyme, one membrane-bound GPI-anchored form and three soluble forms, although one of the soluble forms appears to be derived from the cleaved membrane-bound forrn. Only the membrane-bound form of 5'-NTase will be discussed further.
The GPI-anchored form of 5'-NTase hydrolyzes exclusively 5'-nucleoside monophosphates, with S'-AMI' being the preferred substrate (KM values in the low micromolar range; see Figure 1.4). Competitive inhibitors of the enzyme include ADP,
ATP and adenosine 5'-[cc$-methyleneldiphosphate, whereas the lectin concanavalin A
(Con A; which binds to the N-linked oligosaccharide chains) is a non-competitive inhibitor. Zinc appears to be required for 5'-NTase activity, since the purified enzyme
20 Figure 1.4 Overall reaction scheme for the hydrolysis of 5'-AMP by 5'-NTase Reaction scheme for the assay described in Section 2.3.7, showing the hydrclysis of [2-3H]-5'-~~~to yield adenosine and inorganic phosphate. fi-om chicken gizzard is tightly associated with hvo Zn2' ions [8 11 and decreased 5'-NTase
activity is noted in the lymphocytes of patients with ~n"deficiency [191].
GPI-anchored 5'-NTase exists as a homodimer with an apparent molar mass of 132 kDa
on sucrose density gradient sedimentation [169]. The 5'-NTase homodimer was
originally thought to be crosslinked by interchain disulfide bridges
[9,33,59,80,97,104,2 131 and intact disulfide bridges were proposed to be essential for
enzyme activity [go]. However, a recent report claims to demonstrate that the subunits in
5'-NTase dimers are linked not by disulfide bonds, but by non-covalent bonds [183].
The apparent molar mass of the monomeric subunit ranges from 60 to 80 kDa depending
on the source of the enzyme and the electrophoretic gel system used.
As mentioned previously, the ecto-5'-NTase fom is a GPI-anchored protein, and
several lines of evidence support this fact. The enzyme can be released from a variety of
tissues and cellular systems by PI-PLC [97,f 28,l5 l,l64,166,173,202,224,273,293,294,
307,3 1O,3 13-3 15,3221, and fiom skeletal muscle membranes by an insulin-activated
endogenous phospholipase C [152]. Isolated, detergent-solubilized enzyme contains
equimolar amounts of myo-inositol [8,l5 11. The C-terminal kagrnent of rat liver and
human placenta1 5'-NTase ends at Ser-523, the predicted C-terminal extension having
been cleaved off and repIaced with the GPI anchor [203,219]. Analysis of bovine liver
5'-NTase has yielded similar results [304]. The GPI anchors of rat liver and human placental 5'-NTase contain ethanolamine, mannose (3 mol), glucosamine (1 mol) and
inositol (1 mol), which would indicate a common GPI-core structure. The fatty acids
include stearic acid and rnyristic acid [203,219]. Analysis of the five different rnolecular
mass species of bovine brain GPI anchor revealed the existence of the comrnon core
structure, as weI1 as variations including additional mannose, N-acetylhexosarnine, and
22 ethanolamine phosphate residues [306]. An additional ethanolamine phosphate residue
attached to the mannose next to glucosamine was cornrnon to al1 five forms of bovine
liver 5'-NTase studied.
Ecto-5'-NTase is a well-studied enzyme with a great deal of biological
significance. The main function of the enzyme appears to be to supply the ce11 with
nucleosides, which cm enter the ce11 via passive transporters, where nucleotides cannot.
However, production of extracelIular adenosine may also be an important fünction for
5'-NTase, since adenosine causes a vâriety of physiological responses, including
vasodilation, a decrease in glomerular filtration rate, inhibition of renin release, inhibition
of neurotransmitter release, inhibition of the immune and inflamrnatory response, and
lipolysis [51,94,260,296]. 5'-NTase has also been implicated in transrnembrane
signalling. Poly- and monoclonal anti-5'-NTase antibodies can induce human T
lymphocytes to proliferate, and to express interleukin-2 receptors and secrete interleukin-
2, which suggests the existence of transmembrane signalling mediated by GPI-anchored
5'-NTase [3 l3,3 141. As rnentioned earlier, 5'-NTase has been found clustered in specialized DRM fractions along with other GPI-anchored proteins and protein tyrosine kinases that are components of the lymphocyte signalling pathway. There may also be a role for 5'-NTase in cell-ce11 and cell-matrix interactions. Vogel et al. observed that
5'-NTase fi-om electric rxy electric organ and cat cerebral cortex expresses the HNK-I sugar epitope, which is associated with a number of other ce11 surface proteins involved in cell-ce11 and cell-matrix interactions [330]. Numerous reports also suggest that
5'-NTase interacts with, and is modulated by, the extracellular matrix proteins fibronectin and laminin [44,60,293,295]. The role of 5'-NTase in cells of the immune system has also been widely studied and wilI be discussed in the next section.
23 1-52 Immunological role
5'-NTase has long been an important marker of lymphocyte differentiation and maturation [3 15,3 161. Enzyme activity in peripheral T-cells is about ten-fold higher than that in thyrnocytes [70]. Other studies have revealed that the activity of B-cells fiom adult penpheral blood is several-fold higher than in the blood of fetal spleen and cord tissue [13,316]. Activated lymphocytes appear to rely heavily on ecto-5WTase for their purine nucleotide requirements. Mitogen-stimulated human T-cells and rapidly dividing
B lyrnphoblastoid cells are able to use the catalytic activity of 5'-NTase to supply their total purine requirements [3 121, and anti-5'-NTase antibodies completely suppressed ce11 proliferation [6]. This shows that 5'-NTase is the only enzyme on the lymphocyte surface that is capable of degrading extracellular purine nucleotides to nucleosides. 5'-NTase deficiency appears to play an important role in a variety of disease States and is observed in the lymphocytes of patients with immunodeficiency diseases [2 11 ,î4 1,3 15,3 161
Omem's syndrome [89], Duchenne muscular dystrophy [278], and during chernotherapy treatment [28 1] and aging [23].
1.6 Other GPI-anchored proteins
Two other proteins were used as mode1 GPI-anchored proteins in this study:
Thy-1 fiom rat brain and human placental alkaline phosphatase (PLAP).
1.6.1 Thy-1
Thy-1 is a well characterized GPI-anchored surface glycoprotein expressed on neurons, lymphocytes, fibroblasts and a small number of other ce11 types [12,339]. It is the major glycoprotein found in rodent thymocytes and adult neuronal cells [Il,35,184].
Thy-1 has an apparent rnolecuIar weight of 25-29 kDa on SDS-PAGE, with three N- linked carbohydrate chains accounting for -30% of the mass [36,338]. The secondary
24 structure of Thy-1 is high in P-sheet content and very low in a-helical content, in accordance with the fact that Thy-1 belongs to the immunoglobulin superfamily [36].
The function of Thy-1 has not been fully elucidated. It has been implicated as a receptor molecule or adhesion protein because of its location on the ceIl surface and the presence of an immunoglobulin fold in its structure [46]. In fact, Thy-1 appears to be a receptor for the toxic channel-foming protein, aerolysin [2151, although this observation does not explain the endogenous function of Thy-1. In lymphocytes, Thy-1 appears to be involved in the adhesion of thymocytes to thymic epithelial cells [ 11 O. 139,1781. Thy- 1 associates with protein tyrosine kinases [loi] and CO-irnmunoprecipitates with heterotrimeric G protein a-subunits in lymphocytes [284], suggesting a possible role for
Thy-1 in ce11 signalling. There is also evidence that Thy-1 may play a crucial role in the maturation of thyrnocytes by controlling apoptosis of the cells during developrnent [ 1321.
In fact, thyrnocytes in Thy-1 knockout mice fail to mature properly [131]. In neurons,
Thy-1 associates with protein tyrosine kinases and heterotrimenc G protein a-subunits, suggesting that, as in lymphocytes, Thy-1 may play an important role in ceIl signaIIhg
[114]. Thy-1 is also involved in the prevention of neurite outgrowth and the GPI anchor plays a crucial role in this process [320,321].
1.6.2 Hurnan placental alkaline phosphatase (EC 3.1 Al; PLAP)
Alkaline phosphatase is a general tem that describes non-specific phospho- monoesterases with optimal activity at alkaline pH. Alkaline phosphatases in human tissues and serurn exhibit considerable heterogeneity with respect to net molecular charge, size and antigenic distinction [65]. Human placental alkaline phosphatase (EC
3.1.3.1; PLAP) is synthesized in the placental syncytiotrophoblast, becorning detectable in materna1 circulation afier the twelfth week of pregnancy [207]. PLAP is thought to be
a dimer and the rnolecular weight on SDS gels is 67/130 kDa depending on running
conditions [68]. PLAP is readily distinguished from other human alkaline phosphatase
isozymes by its heat stability, differential sensitivity to inhibition by certain L-amino
acids, and reactivity with specific antisera or monocional antibodies [65,207].
Alkaline phosphatase was the first protein shown to be released fiom the plasma
membrane by bacterial PI-PLC [135,172]. PLAP was shown to be anchored to the
plasma membrane by a GPI anchor through metabolic labelling experiments in cultured placenta and carcinoma ce11 Lines [129,144,308] and chernical analysis of purified PLAP
[199,220]. The exact structure of the GPI anchor of PLAP has been determined, and
consists of the GPI core structure described in Section 1.2.1 ; Thr-Asp-ethanolamine-PO,-
Mana 1- 2Mana 1- 6Mana 1- 4GlcN- (sn- 1-O-alkyl -2-0- acylglycerol -3-PO,- 1-rnyo- D-
inositol) with an additional ethanolamine phosphate group that is most likely attached to the 2-position of the reducing terminal a-mannose residue. Recently, the crystal structure of PLAP was solved at 1.8 A resolution (Figure 1.5; PDB 1EW2) [157]. The structure confirms that PLAP is an allosteric dimer with a long N-terminal a-helix that extends fkom one monomer and embraces the other (coloured red in Figure 1.3, and the exchange of a residue from one monomer into the active site of the other. Both structural elements probably contribute to the allostery of PLAP. Missing fkom the structure are the
C-terminal residues, 480-513 and consequently, the GPI anchor, which prevents the visualization of the localization of the protein in relation to the GPI anchor (and the membrane bilayer.) Figure 1.5 Overall structure of the PLAP dimer The overall structure of PLAP is shown in ribbon representation. Monomer 1 is shown in light green and monomer II in violet. The N-terminal cc-helix and loop (residues 1-24) are displayed in red, and both the N- and C-terminus me labelled in yellow for monomer 1 and white for monomer II. Residues 480-5 13 were not resolved in the crystal structure. This figure was made using Swiss PDB viewer Ver. 3.6b (PDB 1EW2; [157]). 1.7 Fluorescence studies of proteins
1.7.1 General fluorescence theory
Fluorescence spectroscopy is an invaluable tool for detennining whether an interaction occurs between two biological molecules, using the technique of flucrescence resonance energy transfer (FRET). To better understand this method, knowledge of the simple theory of fluorescence is necessary and will be discussed briefly. The electronic energies of molecules are restricted to discrete energy levels that can be described by an energy level diagram. Figure 1.6 shows two electronic levels, the lower level or ground state (G), and one upper or first excited state S, 11561. Some vibrational levels are also shown for each state, which indicate the energy of various modes of vibration of the molecule (e-g. the bending and stretching of various covalent bonds). The absorption of light energy causes the molecule to change fiom a lower to a higher energy level
(indicated by solid vertical arrows in Figure 1.6). Light energy can be absorbed only if the wavelength (A) of the exciting light corresponds to the difference between the ground state and excited state energy levels. The molecuIe in its lowest unexcited ground state
(G) may absorb more than enough energy to reach the lowest electronic excited state
[156], and the excess energy cm be absorbed as vibrational energy, exciting the rnolecule to one of the higher energy vibrational levels. This vibrational energy is rapidly dissipated as heat and the molecule relaxes to the lowest vibrational level of S,. In returning fiom S, to G, energy can be released through the emission of light
(fluorescence) or by a non-radiative transition (which will not be discussed here). The emitted fluorescent light will have lower energy (Le. a longer wavelength) than the absorbed light because energy is lost in dropping to the lowest level of S,; therefore, emitted fluorescent light always has a longer wavelength than the exciting light. Also,
28 Donor excited state Acceptor excited state
I Donor ground state Accepter gromd state
Figure 1.6 Fluorescence resonance energy transfer between donor and acceptor fluorophores when returning frorn S, to G, the molecule may amive at one of the upper vibrational
levels of G instead of the absolute ground state, and this vibrational energy will be
dissipated as heat. If there are many absorbing molecules, the fluorescent light ernitted
will cover an envelope of wavelengths, resulting in a fluorescence peak of finite width at
a characteristic wavelength.
1 .%2 Fluorescence resonance energy transfer (FRET)
Extnnsic fluorophores exist that can be bound to macromolecules such as proteins
(e.g. fluorescein, rhodarnine). These fluorophores absorb at a specific wavelength and
emit energy with a characteristic fluorescence spectnim. Figure 1.7 illustrates the
concept of FRET. If a protein contains a fluorophore that absorbs energy at hl and emits
fluorescent light centred at h, (Figure 1.7A), and & is a wavelength that cmbe absorbed by a fluorophore on a second molecule (Figure 1.7B), resonance energy transfer can occur if the two fluorophores are within a certain distance of each other. Fluorescence is thus 'transferred' from the donor to the acceptor and is emitted at A,, the emission wavelength of the acceptor fluorophore (Figure 1.7D). We see a reduction in emission of doilor at &, and an increase in fluorescence of emission of acceptor, at A,.
The emission spectnim of the donor must overlap the absorption spectnun of the acceptor for FRET to take place (Figure 1.7C).
The efficiency of energy transfer, E, is a function of the separation distance of the donor and acceptor fluorophores and is described by the following equation [Z 561:
E = R~I(R~+ R:) which can be rewritten as:
R = R,[(l - E)IEJ1'6 Figure 1.7 Fluorescence energy transfer between two fluorophores (A) Fluore ence spectra of fluorophore 1 showing excitation at A, and ernission at &. (B) Fluorescence spectra of fluorophore 2 showing excitation at &' and emission at h,. (C) Overlap of the emission of fluorophore 1 (&) and the excitation of fluorophore 2 (XJ (D) FRET behveen fluorophore 1 and 2. Following excitation at h, there is a reduction in emission of fluorophore 1 (L,) and a sensitized emission of fluorophore 2 (A,). where R is the distance between donor and acceptor (range of O to 60 A), and R, is a constant, related to each donor-acceptor pair, that can be calculated fiom certain parameters of the absorption and emission spectra of each molecule.
To determine if two fluorescently-labelled molecules are close together (in the range R above), three wavelengths are considered: h,, which can excite the donor but not the acceptor; h,, which is a waveiength in the emission spectnun of the donor (but not the
acceptor); and A,, which is a wavelength emitted only by the acceptor. By monitoring a decrease in the fluorescence emission of the donor at X,, or an increase in the fluorescence of emission of the acceptor at &, one cm determine whether the two molecules associate. RATIONALE AND RESEARCH OBJECTIVES
Cleavage of GPI-anchored proteins by phospholipases may influence their overall biological role in vivo. One of the goals of this research was to study the cleavage of a
GPI-anchored protein by PI-PLC. Two recent studies have shown that cleavage of the
GPI-anchor somehow modulates the confonnation of the attached protein. Barboni et al. were able to show that cleavage of the GPI-anchor of purified rat and mouse brain Thy-1 by PI-PLC induced a conformational change in the protein [IO]. Another study showed that cleavage of dipeptidase from pig kidney microvillar membranes using PZ-PLC caused an activation of the enzyme, possibly due to the removal of conformational constraints caused by the anchor [25]. The latter study also reported that there was no detectable activation of 5'-NTase activity in this system. However, an earlier study in our laboratory suggested that there is an increase in the activity of lymphocyte 5'-NTase upon cleavage of the GPI anchor [268]. "High KM"and "low KM1'5'-NTase enzymes have been found in several mamrndian tissues [121], and in rat liver, the "low KM1'enzyme was identified as the cleaved form of the membrane-bound ("high KM")5'-NTase [235], suggesting a change in the kinetic properties of the enzyme following removal of the GPI anchor. Cleavage of plasma membrane 5'-NTase from chicken gizzard by PI-PLC also yielded an enzyme with a lower KM and higher V,, [294]. Activation of 5'-NTase following PI-PLC cleavage may be specific to certain tissue or ce11 types. A more precise study of the kinetic parameters of 5'-NTase before and after cleavage of the GPI anchor is necessary to fûIIy resolve the biochemistry of this apparent activation and address the discrepancies in the literature. Earlier experiments in our laboratory have also revealed that membrane surface charge and fluidity may affect the action of PI-PLC [268]. This observation suggests that membrane composition may modulate the activity of PI-PLC towards the anchor of GPI- anchored proteins, but this hypothesis has not been investigated in detail. Therefore, fùrther systematic study is needed to determine if alterations in membrane characteristics and biophysical properties, such as surface charge and fluidity, have any effects on PI-
PLC cleavage of GPI-anchored proteins. Many GPI-anchored proteins tend to aggregate in the ce11 membrane, into GSL-cholesterol-rich clusters, which form insoluble complexes (lipid rafts) after extraction with the nonionic detergent TX- 100
[76,103,17 1,263,2741. Alterations in membrane composition (GSL, sphingolipids, cholesterol) may also affect PI-PLC cleavage and more study is required to elucidate these effects.
Our laboratory previously reported that reconstituted 5'-NTase demonstrated a decrease in activation energy when the bilayer was converted fiom the solid gel phase to the fluid liquid crystalline phase [268]. 5'-NTase fiom rat enterocytes aIso displayed a break point on Arrhenius plots, which coincided with a lipid thermotropic transition 1241.
These results suggest that the protein portion of 5'-NTase may be in direct contact with the lipid bilayer. Evidence fkom modelling studies of the GPI-anchored lymphocyte antigen Thy-1 suggest that this may indeed be the case. The glycan portion of the GPI anchor of Thy-1 is predicted to lie either between the lipid surface and the protein in a tightly folded conformation [IO], or in a carbohydrate-binding pocket within the protein itself [240], and it rnay thus impose a particular conformation on the protein. In both models, the protein domain of Thy-1 is visualized as being very close to, or in contact with, the bilayer. A direct measure of the distance between a GPI-anchored protein and
34 the bilayer using a FRET approach is important to demonstrate whether or not the protein portion is in contact with the membrane.
A more thorough investigation of the behaviour and membrane interactions of
GPI-anc hored proteins is necessary in order to better understand the molecular behaviour that arises fkom this novel form of anchoring in the plasma membrane. The objectives of this research were to study the biochemical and biophysical properties of biologically important GPI-anchored proteins, with respect to the following.
modulation of their catalytic properties by phospholipase C
effect of the membrane environment on anchor cleavage by bacterial PI-PLC, using a
kinetic approach
measurement of their proximity to the membrane surface, irsing a FRET approach. CHAPTER 2: RELEASE OF 5'-NUCLEOTIDASE BY PI-SPECIFIC PHOSPHOLIPASE C: EFFECT OF GPI -4NCHOR CLEAVAGE ON THE CATALYTIC PROPERTIES OF THE ENZYME
This work has been published: Marty T. Lehto and Frances J. Sharom (1 998) Biochemical J., 332, 10 1- 109 2.1 Abstract
Many hydrolytic enzymes are atîached to the extracellular face of the plasma membrane of eukaiyotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme fimction of anchor cleavage by PI- specific phospholipase C (PI-PLC). We have exarnined this question for the GPI- anchored protein 5'-NTase (5'-nbonucleotide phosphohydroiase; EC 3.1.3.5) both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid biIayer vesicles, using BaciZZus thuringiensis PI-PLC. Membrane-bound, detergent-solubilized, and cleaved 5'-NTase al1 obeyed Michaelis-Menten kinetics, with a
KMfor 5'-AMP in the range 1 1-1 6 PM. The GPI anchor was removed fkom essentially al1 5'-NTase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-NTase was present in detergent solution, dirnyristoylphosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared to the native plasma membrane, egg phosphatidylcholine, and a sphingolipid/cholesterol-rkh mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterizeci by an increase in V,,, was observed following PI-PLC cleavage of reconstituted 5'-NTase fiom vesicles of several different lipids. The highest degree of activation was noted for 5'-NTase in egg phosphatidylethanolamine. An increase in Vmx was also noted for a sphingolipid cholesterol-rich mixture, the native lymphocyte plasma membrane, and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl- phosphatidylcholine showed litîle activation. KM generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-nch mixture.
37 Insertion of the GPI anchor into a Iipid bilayer appears to reduce the catalytic efficiency of 5'-NTase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.
2.2 Introduction
Covalent modification of proteins with a GPI-anchor is cornmonly employed as a mode of membrane attachment in a wide range of eukaryotes, including parasitic protozoa, the yeast Saccharomyces cerevisirre, higher plants, and mamrnals [72,19O].
GPI-anchored proteins are characterized by the presence of a hydrophobie peptide sequence at the C-terminus, which is removed by a putative transamidase enzyme in the
(ER) which also attaches the preformed anchor [289,324]. Well over 100 proteins are known to be GPI-anchored; they are a fûnctionally diverse group, encornpassing extracellular coat proteins, hydrolytic enzymes, adhesion proteins, surface antigens, and receptors.
The exact function of the GPI anchor has been the subject of speculation
[78,126,165]. The anchor confers rapid lateral rnobility on some plasma membrane proteins, and it has been proposed that the anchor enables the proteins themselves to pack tightly, which may be especially important for protozoan surface coat glycoproteins. The
GPI anchor appears to act as an intracellular targetting signal in poIarized epithelial cells.
GPI-anchoreà proteins are sorted into glycolipid-enriched membrane subdomains pnor to transport to the apical membrane surface 1301. Because of their insolubility in TX-100 at
4"C, these domains can be isolated as detergent-resistant membranes (DRMs). DRMs also exist in reconstituted liposomal systems [263], and it was suggested that acyl chain interactions are important in their formation. It has also been suggested that certain GPI- anchored proteins, e.g. the folate receptor, are involved in the high affinity cellular uptake
38 of small molecules by the process of potocytosis, which involves specialized membrane invaginations known as caveolae [126]. GPI anchors are also involved in transmembrane sigrialhg. The products of phospholipase cleavage of the anchor, inositol phospho- glycans, are proposed to be mediators in the action of insulin and several other agents, and crosslinking of GPI-anchored proteins by antibody can stimulate T-lyniphocyte activation [78,246,254,257]. Simons and Ikonen [274] have proposed that GPI-anchored proteins exist at the membrane surface within sphingolipid-cholesterol "rafts", which serve as relay stations in transmembrane signalling. Finally, hydrolysis of the anchor by specific phospholipases C and D results in release of the protein in soluble fom, and this process may play a role in modulating the expression and function of GPI-wxhored proteins at the ce11 surface.
The GPI-anchored ectoenzyme 5'-NTase is found on the plasma membrane of many ce11 types, and is widely distibuted from plants to mamrnals (reviewed in [351]).
The enzyme converts extracellular 5'-AMP to adenosine, which acts via adenosine receptors in a varie@ of physiological signalling processes. 5'-NTase plays an important role in purine salvage in lymphocytes, where it is known as CD73, and has also been implicated in transmembrane signalling in vitro in T-lymphocytes [246]. The enzyme is
N-glycosylated, and binding of a variety of lectins to N-linked carbohydrates on 5'-NTase is known to inhibit activity [266,267], suggesting that some communication exists between the glycan chain(s) and the catalytic site.
Previous work in our laboratory led to the isolation and reconstitution of porcine lymphocyte 5'-NTase into lipid bilayers, where it retains enzyrnatic activity that is inhibited by lectin binding [169,267]. Reconstitution of purified GPI-anchored proteins into bilayers of defined phospholipids provides a powerful tool to delineate the effects of
39 membrane properties on the behaviour and interactions of this class of proteins. More recently, we explored the cleavage of 5'-NTase and two other GPI-anchored enzymes,
AChE and Nase, by bactenal PI-PLCs [268]. 5'-NTase showed an increase in activity of
20-25% following release corn the lyrnph~cyte membrane surface by Bacillrcs thuringiensis PI-PLC (Bt-PI-PLC). In the present study, we have characterized the catalytic activation of 5'-NTase kinetically, in both the native lymphocyte plasma membrane, and also following reconstitution of purified protein into bilayers of several different Iipids. Results indicate that enzymatic activation results fiom lowered catalytic turnover when the GPI anchor is inserted into membrane lipids, suggesting that the GPI anchor may affect the protein confornation of 5'-NTase. In addition, we show that the physicochemical properties of the membrane cm modulate both the susceptibility of the
GPI anchor to Bi-PI-PLC cleavage, and the extent of catalytic activation observed on anchor removal.
2.3 Materials and metbods
2.3.1 Materials
Egg phosphatidylethanolmine (Egg PE) and egg phosphatidylcholine (Egg PC) were supplied by Avanti Polar Lipids (Alabaster, AL,). 3-[(3-Cholamidopropy1)- dimethylammonio]- 1-propane-sulphonate (CHUS), dirnyristoylp hosphatidylcho line
(DMPC), sphingomyelin (SM) (fkom bovine erythrocytes and bovine brain), cholesterol, galactocerebrosides (Type II £tom bovine brain), and TX-114 were purchased fkom Sigma
Chemical Co. (St. Louis, MO). Recombinant PI-PLC f?om Bacillirs thuringiensis
(Bi-PI-PLC) (300 Ulml; expressed in B. subtilis) was obtained frorn Oxford
GlycoSciences Inc. (Bedford, MA). One unit (U) of enzyme activity released 1 pmol Pi fiom phosphatidylinositol per min at 37 OC, pH 7.5. N~'"I was purchased fkom ICN
Biomedicals (Costa Mesa, CA).
2.3.2 General methods
The protein content of plasma membrane and partially purified 5'-NTase was determined by the method of Peterson [233] using BSA (Sigma, crystallized and lyophilized) as a standard. SDS-PAGE was carried out in polyacrylamide gels according to Laernmli [155j, followed by silver staining or autoradiography. Purified 5'-NTase was labelled on tyrosine residues with "'1 using Iodobeads (Pierce Chemical Co.. Rockford,
IL), according to the manufacturer's instructions.
2.3.3 Purification of porcine-lymphocyte 5'-NTase
Porcine mesenteric lymph nodes were supplied by the Meat Laboratory at the
University of Guelph within a few minutes of slaughter, and plasma membrane vesicles were prepared according to the method of Maeda et al. [177]. Briefly, homogenized porcine mesenteric Iyrnph nodes (30 g) were layered over a sucrose cushion (41% wh), followed by ultracentrifugation at 95 000 g. Lymphocyte 5'-NTase was purified as previously described [169,267], bjr solubilizing plasma membrane in 50 rnM CHAPS and performing two sequential affinity chromatography steps, the first using lentil lectin-
Sepharose 4B (Pharrnacia Canada, Baie D'Urfé, QC) the second using 5 '-AMP-Sepharose
(Sigma Chemical Co., St Louis, MO), One ml of puified 5'-NTase solution in CHAPS
(S'-AMI' colurnn eluate) was concentrated to -5-10 pl using a Microcon 30 microconcentrator (30 kDa cutoff; Amicon Inc., Beverly, MA) which was pre-treated wiih 1% @dv) powdered milk to prevent nonspecific binding of protein to the membrane.
The concentrated 5'-NTase sarnple was diluted with 0.5 ml of distilled water and concentrated once more to reduce the CHUS concentration. The final volume was 41 70 pl, representing a 14-fold concentration. The protein content of the concentrated
5'-NTase sarnple was determined by the method of Peterson [233], using BSA as a standard.
2.3.4 Reconstitution of porcine-lymphocyte 5'-NTase
Purified 5'-NTase was reconstituted into lipid bilayer vesicles using a modification of the detergent dialysis technique described previously [169,267,268]. A mixture of the desired lipids (1 -3 mg) in MeOH-CHCI, was evaporated to dryness in a small glas tube using N, gas, and pumped under vacuum for 1 h to remove al1 traces of organic solvent. The dned lipid was dissolved in 12.5 rnM CHAPS in 50 rnM Tris-HCI,
0.1 M NaCl, 0.2 rnM dithiothreitol, 0.02% sodium azide, 0.7 mM CaCI,, 0.7 rnM MgCl,,
0.7 mM MnCI, (pH 7.4), and mixed with purified 5'-NTase in the same buffer. The detergent was removed by dialysis in Spectrapor 4 tubing (12- 14 kDa cutoff) against 3 changes (a total of 3 L) of 20 mM Tris-HC1 buffer (pH 7.4). The resulting lipid bilayer vesicles had a final 1ipid:protein ratio of 150-200:l (wlw). Purified 5'-NTase was reconstituted into the following lipid systems: DMPC, egg PC, SM, egg PE, and sphingolipid/cholesterol-rich liposomes (SCRL; consisting of egg PC: egg PE: SM: cerebrosides: cholesterol, 1: 1 : 1 : 1 :2 mole ratio) [263].
2.3.5 Cleavage of detergent-solubilized 5'-NTase by Bt-PI-PLC
B-1-PLC (expressed in B. subtilis) was used to cleave the GPI anchor of
5'-NTase. Experiments were canied out to deterrnine the concentration of Bt-PI-PLC which effected maximal cleavage of purified detergent-solubilized 5'-NTase, and the concentration of Bt-PI-PLC required to release 50% of the 5'-NTase in soluble form
(defined as the EC,,). Increasing concentrations of Bt-PI-PLC in 50 rnM Tris-HCI @H 7.4) were incubated for 90 min at 37 OC with purified 5'-NTase in 12.5 rnM CHAPS. The
GPI-anchored form of 5'-NTase in CWSwas separated from the soluble forrn by two-
phase separation in TX-114, based on a modification of the method of Bordier [22].
Eriefly, a 20 pl aliquot of 5'-NTase in CHAPS was made up to 50 pL with 50 rnM Tris-
HC1 (pH 7.4) containing increasing concentrations of Bt-PI-PLC. Following incubation
for 90 min at 37 OC, 250 pl of 50 mM Tris-HC1 (pH 7.4) and 20 pl of pre-condensed
TX-114 were added. The mixture was cooled on ice for 3 min, warrned to 37 OC for 5
min, and then centrifuged at 14 700 g for 1 min at 37 OC in a microcentrifuge to separate
the phases. The upper aqueous phase and the lower detergent phase were removed into
separate microcentrifuge tubes. TX- 114 (20 pl) was added to the upper aqueous phase,
200 pl of 50 rnM Tris-HC1 (pH 7.4) was added to the lower detergent phase, and a second
phase separation was performed on each of the two original phases. Following this, the
two upper phases were combined (400 pl in total), as were the two Iower phases (40 pl,
mixed with 360 pl of 50 mM Tris-HC1, for a total of 400 pl), and the 5'-NTase activity
was deterrnined using 20 pl aliquots of each.
2.3.6 Cleavage of membrane-bound 5'-NTase by Bt-PI-PLC
Previous work in Our laboratory showed that Bt-PI-PLC was inactivated in a tirne-dependent fashion following adsorption to DMPC bilayers; however, loss of activity could be completely prevented by addition of 1% (wlv) BSA to cleavage reaction mixtures containing lipid vesicles [268]. To determine the phospholipase concentration at which maximal cleavage of 5'-NTase occurred, and the EC,,, increasing concentrations of Bt-PI-PLC in 50 rnM Tris-HCI/l% (w/v) BSA (pH 7.4) were incubated for 90 min at
37 OC with either lymphocyte plasma membrane vesicles, or purified 5'-NTase
43 reconstituted into various lipids. Followïng incubation, the lymphocyte plasma membrane or lipid vesicles were collected by centrifugation (41 000 g for 10 min), and the pellet and supernatant were assayed for enzyrnatic activity of the GPI-anchored fom and the cleaved soluble fom of 5'-NTase, respectively (see below).
2.3.7 Kinetic analysis of 5'-NTase enzymatic activity
Lymphocyte plasma membrane vesicles, purified 5'-NTase in CHAPS, or purified
5'-NTase reconstituted into various lipids were incubated for 90 min at 37 OC, with the appropriate concentration of BI-PI-PLC that yielded maximal cleavage. Aliquots were removed for kinetic analysis of 5'-NTase activity. The enzymatic activity of 5'-NTase was detemined by measuring the release of 2-[)~]adenosinefiom 5'-[2-'HIAMP as described by Sharom et al. [267] and optimized by Loe et al. [169] (see Figure 1.4). The initial rate of substrate hydrolysis was detemined for increasing concentrations of the substrate 5'-Am, and the kinetic data were fined to the Michaelis-Menten equation using the Curve-Fit fûnction of the SigmaPlot programme (SPSS Inc., Chicago, IL).
2.4 Results
2.4.1 Purification of porcine-lymphocyte 5'-NTase
Porcine lymphocyte 5'-NTase was successfully purified using a two step affinity chromatography procedure, first on lentil lectin (which isolates a-D-mannose-containing glycoproteins), then on Sepharose containing the covalently bound substrate, 5'-AMP
(Table 2.1). The zwitterionic detergent CHAPS proved highly suitable for the purification procedure, resulting in a high degree of solubilization of the lymphocyte plasma membrane (-80% of the total protein). Retention of 5'-NTase activity during the procedure was also high; 26% of the catalytic activity of the CHMS extract was recovered in the final purified, concentrated enzyme preparation. SDS-PAGE analysis of 44 Table 2.1 Purification of 5'-NTase from porcine lymphocytes
Sarnples kom various stages of the purification procedure were assayed for 5'-NTase activity as descnbed in Section 2.3.7. Activity is expressed as 5'-AMP hyclrolyzed and is presented as the mean h S.E.M. (n=3).
Stage of Protein Total 5'-NTase 5'-NTase specific Fold purification activity activity Purification
(mg) (pmoVmin) (pmoVmin per mg protein)
Plasma membrane 63.8 * 0.8 1.89 k 0.01 0.030 + 0.001 1
CWSextract 49.8 st 2.6 2.20 =t0.02 0.044 + 0.002 1.5
Lentil lectin 7.0 * 0.2 2.33 * 0.02 0.33 + 0.01 11.2 column eluate
5'-AMP column 0.046 k 0.002 0.57 + 0.02 12.36 * 0.73 416 eluate highly purified 5'-NTase following '"1-labelling resulted in a major band with an apparent M, of 81 kDa (Figure 2.1, lanes 4 and 5). It is important to note bat 5'-NTase is a low abundance protein in the lymphocyte membrane (see Figure 2.1), and a typical purification protocol yie1ded less than 50 pg of enzyme. However, 5'-NTase is highly catalytically active, and nanogram mounts could be readily measured using the assay systern described in Materials and Methods. The apparent level of purification of
5'-NTase wûs >400-fold from the plasma membrane, -280-fold fiom the CHAPS extract
(Table 2.1).
2.4.2 Reconstitution of purified 5'-NTase
Purified 5'-NTase was reconstituted into lipid bilayers of various natural and synthetic phospholipids, including DMPC, egg PC, SM, egg PE, and a sphingohpid cholesterol-rich mixture (SCRL), using a detergent dialysis technique previously developed in our laboratory. Over 95% of the 5'-NTase enzymatic activity was removed from the reconstitution mixtures following harvesting of the vesicies by centnfkgation.
Reconstitution resulted in large unilarnellar vesicles, as assessed by fluorescence microscopy in the presence of the lipid-soluble fluorescent marker l-anilinonaphthalene-
8-sulfonic acid (1 &ANS). PE species have a tendency to shift from the bilayer phase to the hexagonal (H,,) phase as the temperature is increased. These two phases can readily be distinguished by their charactenstic "P-NMR spectra [122]. The temperature at which the two phases interconvert depends on the acyl chain composition of the particular PE.
For egg PE, the temperature at which the lipid starts to convert kom the bilayer phase to the hexagonal phase is just below 40 OC. "P-NMR experiments showed that the egg PE used in this study was largely in the bilayer phase at 37 OC (Figure 2.2A). The transition Figure 2.1 SDS/PAGE analysis of purified porcine-lymphocyte 5'-NTase Lymphocyte plasma membrane (lane 1, 15 pg of protein), CHAPS-solubilized plasma membrane (lane 2, 10 pg of protein) and the glycoprotein fraction (lane 3, 10 pg of protein) were subjected to SDSRAGE analysis in a 12% polyacrylamide gel, followed by staining with silver. 1251-~abelled,purified 5'-NTase in CHAPS solution (lane 4), and DMPC vesicles containing reconstituted 125~-5'-~~ase(lane 5) were separated in a 12% gel, and detected by autoradiography. The position of the molecular-mass markers is indicated on the lefi, and an arrow indicates the 5' -NTase band. 4- 4- Bilayer phase peak
Figure 2.2 Determination of the bilayer to hexagonal phase transition of egg PE (A) "P-NMR spectroscopy of egg PE sarnples at different temperatures showing the bilayer phase pedc at - -1 1 ppm and the hexagonal phase (H,,) peak at - +8 ppm. (F3). For determination of the bilayer to HI, phase transition, the H,, to bilayer ratio (from A) was plotted versus the temperature. 37 39 41 43
Temperature (OC) fiom the bilayer to the hexagonal phase started to occur at 38-39 "C, and was largely complete by 45 OC (Figure 2.2B).
The syrnrneûy of 5'-NTase reconstitution was assessed by addition of increasing concentrations of CHAPS to the lipid structures. The assay for 5'-NTase measures the activity of outward-facins enzyme, and the 5'-NTase facing the vesicle lumen remains cryptic unless the bilayer is permeabilized to 5'-AMP by the addition of detergent.
5'-NTase was inserted into the vesicles approximately symmeû-ically for DMPC, SCRL, and egg PE, since 56%, 48%, and 46%, respectively, of the total enzyrne activity was measurable in the absence of detergent, indicating that it is present at the outer surface of the vesicle. The balance of the enzyme activity (44- 54% of the total) remained cryptic until after the addition of permeabilizing arnounts of CHAPS (Figure 2.3). Reconstituted
5'-NTase retained hl1 catalytic activity for over 5 weeks when the reconstituted systems were stored at 4 OC in 20 mM Tris-HC1 buffer (pH 7.4), emphasizing the stability of the enzyme under these conditions.
2.4.3 Kinetics of 5'-AMP hydrolysis by detergent-solubilized and membrane- bound 5'-NTase A major objective of this study was to determine whether 5'-NTase undenvent a change in kinetic parameters following cleavage ficorn a membrane surface by
Bt-PI-PLC. The first step was, therefore, to characterize the kinetics of purified
5'-NTase in both detergent solution and a membrane environrnent. 5'-NTase is a homodimer with interchain disulphide bridges, which are essential for enzymatic activity
(reviewed in [351]). We have previously show that 5'-NTase remains in the dimeric form (as indicated by gel filtration fast protein liquid chromatography (FPLC)) following solubilization of lymphocyte plasma membrane by CHAPS [169]. Lnitial rate 220 -
O 20 40 60 80 1O0 CHAPS concentration (mM)
Figure 2.3 Symmetry of reconstitution of 5'-NTase into various lipids 5'-NTase activity following reconstitution into various lipids (DMPC, ; SCRL, A; egg PE, *)was detemined in the presence of increasing concentrations of CHAPS, which permeabilizes the vesicles and reveals cryptic inward-facing enzyme. Data points are presented as the mean & range for duplicate determinations of 5'-NTase activity, as a percentage of control sarnples with no added detergent. rneasurernents were carried out with purified 5'-NTase in CHAPS solution, in the absence of added lipids, using a 5'-AMP concentration range fiom 1 pM to over 300 FM. The data were fitted to the Michaelis-Menten equation by non-linear regression analysis
(Figure 2.4A), and the kinetic parameters KMand V,, were extracted (Table 2.3). The same expenment was carried out for 5'-NTase in native lymphocyte plasma membrane
(Figure 2.4B) and reconstituted DMPC vesicles (Fipure 2.4C). In each case, the kinetic data fitted well to the Michaelis-Menten equation, so it appears that 5'-NTase follows classical Michaelis-Menten kinetics despite existing as a homodimer.
2.4.4 Cleavage of detergent-solubilized and membrane-bound 5'-NTase by Bt-PI-PLC Purified 5'-NTase, both in CHAPS solution and reconstituted into various lipids. was incubated with increasing concentrations of recombinant Bt-PI-PLC. We previously showed that the form of 5'-NTase which exists following Bt-PI-PLC treatrnent is hydrophilic, as assessed by TX-114 partitioning, indicating that the GPI anchor had been removed fi-om the enzyme [268]. The hydrophilic soluble form of
5'-NTase was separated From the rernaining enzyme with an intact anchor by either TX-
1 14 partitioning (in the case of CHAPS-solubilized 5'-NTase), or by centrifugation (in the case of membrane-bound 5'-NTase). For 5'-NTase solubilized in CHAPS, cleavage by
Bt-PI-PLC resulted in Ioss of enzymatic activity fkom the hydrophobic detergent layer, and its appearance in the aqueous phase (Figure 2.5A). In the case of plasma membrane or reconstituted 5'-NTase, the disappearance of zctivity fiorn the pellet corresponded to its appearance in the supernatant (Figures 2.5B and C). The GPI anchor of essentially al1
5'-NTase molecules was cleaved by Bt-PI-PLC, whether the enzyme was solubilized in detergent solution (Figure 2.5A), reconstituted into lipid bilayer vesicles of DMPC O 50 100 150 200 250 300 350 Concentration AMP (PM) Figure 2.4 Kinetics of catalysis by detergent-solubilized and membrane-bound 5'-NTase The initial rates of AMP hydrolysis were determined for purified 5'-NTase in CHAPS solution (A), porcine lymphocyte plasma membrane vesides (B) and purified 5'-NTase reconstituted into DMPC vesicles (C). Data points are presented as the means I S.E.M. (n=3). The solid lines represent the best fit of the data points to the Michaelis-Menten equation, as deterrnined by non-linear regression analysis. - PI-PLC concentration (UImL)
0.001 0.0i 0.1 1 PI-PLC concentration (UImL) 140 1 V C T
0.01 o. 1 1 10 PI-PLC concentration (UImL)
Figure 2.5 Cleavage of detergent-solubilized and membrane-bound 5'-NTase by PI-PLC (A) Purified 5'-NTase in CHAPS solution was incubated with increasing concentrations of Bt-PI-PLC for 90 min. at 37 OC, and enzyme retaining an intact GPI anchor was separated from the cleaved enzyme using TX-114 extraction. Enzyme activities in the lower detergent phase (0)and the upper aqueous phase (e)were then determined as described in Materials and Methods. Plasma membrane vesicles (B) and DMPC vesicles containing reconstituted 5'-NTase (C) were incubated with increasing concentrations of PI-PLC for 90 min. at 37 OC, and the membrane-bound enzyme was separated fiom the cleaved soluble enzyme by centrifugation. Enzyme activities in the membrane pellet (O) and the soluble supernatant (a)were then deterrnined as described in Materials and Methods. Data points represent the mean * range for duplicate determinations. (Figure ZSB), or present in native lymphocyte plasma membrane vesicles (Figure 2 SC).
This indicates that there is no pool of lymphocyte 5'-NTase that is intrinsically resistant to
Bt4I-PLC cleavage.
Phospholipase cleavage of 5'-NTase was Mercharacterized by detemination of the quantitative parameter EC,,, defined as the concentration of Bt-PI-PLC required for removal of the GPI anchor fiom 50% of the 5'-NTase pool. For 5'-NTase solubilized in
CHAPS,EC,, was estimated fiom the curve representing enzyme activity in the TX-114 layer (Figure 2.5A). For 5'-NTase in native plasma membrane, or reconstituted into various lipid systems, EC,, was deterinined Eom the curves for enzyme activity of the pellet, as shown in Figures 2.5B and C. As indicated in Table 2.2, the EC,, values for cleavage of reconstituted 5'-NTase spanned a very broad range, fiom 0.008 U/ml for egg
PC, to 1.5 U/ml for SM. Lipids fell into two distinct groups: those with low EC, values, and those with hi& EC,, values. Native plasma membrane, egg PC and SCRL al1 showed high efficiency of 5'-NTase cleavage, with EC,, values in the range 0.008-0.03
U/ml. On the other hand, Bt-PI-PLC worked much less efficiently on 5'-NTase when it was present in CHAPS micelles, DMPC, egg PE, and SM, where EC,, values fell in the range 0.53- 1.5 U/ml. This wide difference in susceptibi1it-y to cleavage suggests that the properties of the host lipid, such as bilayer packing, fluidity, and headgroup molecular properties, may affect the ability of Br-PI-PLC to gain access to the GPI anchor of
5'-NTase.
2.4.5 Activation of 5'-NTase following cleavage from various membrane systerns
Substantial catalytic activation (-2 1%) was observed for lymphocyte plasma membrane 5'-NTase following cleavage of the GPI anchor by Bt-PI-PLC, and release in Table 2.2 Release of detergent-solubilized and membrane-bound 5'-NTase by PI-PLC
The EC,, was defined as the concentration of PI-PLC required to release 50% of the 5'-NTase in soluble form. For CHAPS-solubilized 5'-NTase, 5'-NTase with an intact anchor was separated f?om soluble 5'-NTase fkom which the anchor had been cleaved using TX-114. For membrane-bound 5'-NTase, the value of EC,, was detemined from a plot of enzyme activity in the pellet versus PI-PLC concentration, and was similar to the value determined f?om a plot of enzyme activity remaining in the supernatant versus PI-PLC concentration.
Plasma membrane CHAPS solution DMPC Egg PC Egg PE SCRL SM soluble forrn (Figure 2.6A). Activation following Bt-PI-PLC treatrnent was also noted
for 5'-NTase in CHAPS micelles (Figure 2.6B) and after reconstitution into certain lipids
and lipid mixtures (Figure 2.6C-F). In al1 cases, the V, (Table 2.3) and turnover
nurnber (Table 2.4) of the ectoenzyme were increased following release fkom the
membrane. The V,, of soluble 5'-NTase after release was more or less independent of
the Iipid system initially used to anchor the protein. The KM of 5'-NTase for AMP
hydrolysis remained essentially unchanged following release from the membrane for al1
lipids except SCRL, where a 28% decrease in KM was noted (Table 2.3). The largest
degrees of activation were observed for 5'-NTase reconstituted into egg PE (74%) and
SCRL (21%). Only a small activation of the enzyme was noted following cleavage of
5'-NTase fiom vesicles of DMPC and SM. The observation of activation in detergent
solution indicates that insertion of the GPI anchor into a lipid bilayerper se is not strictly necessary for catalytic activation following anchor cleavage.
The turnover nurnbers of soluble 5'-NTase lacking the GPI anchor fell in a narrow range (27-38 s-'; mean 3 1.9 s'l, Table 2.4), no matter which lipid system the enzyme had originally been reconstituted into. The turnover nurnber for cleaved 5'-NTase in the presence of CHAPS micelles was significantIy higher (Table 2.4), which may reflect the effect of detergent on the enzyme. The turnover number of 5'-NTase was decreased on reconstitution into certain lipids, especially egg PE and SCRL, and release fiom the membrane surface following Bt-PI-PLC cleavage restored the enzymatic activity (Table
2.4). These results suggest that the kinetic characteristics of 5'-NTase are affected by the presence of the GPI anchor. The intact anchor, when inserted into a detergent micelle, lipid bilayer or membrane, may change the conformation of the protein to reduce its O 50 100 150 200 250 300 Concentration 5'-AMP (FM) Concentration 5'-AMP (PM)
Figure 2.6 Kinetics of 5'-NTase before and after cleavage of the GPI anchor by PI-PLC Plasma membrane vesicles, 5'-NTase solubilized in CHAPS, and 5'-NTase reconstituted into various lipids were treated for 90 min at 37°C with the appropriate concentration of Bt-PI-PLC necessary for complete cleavage of the GPI anchor (see Figure 2.5). The initial rate of AMP hydrolysis was determined on identical samples before (0)and after (O) anchor cleavage, for native lymphocyte plasma membrane vesicles (A), CHAPS-solubilized 5'-NTase (B), and 5'-NTase reconstituted into egg PE (C), SCRL (D), egg PC (E), and DMPC (F). Data points are presented as the means * S.E.M. (n=3). The solid lines represent the best fit of the data points to the Michaelis-Menten equation, as determined by non-linear regression analysis. Table 2.3 Summary of kinetic parameters for 5'-NTase before and after cleavage by Pf-PLC
k (PM) V,, (pmoVmin per mg of protein)
Bound Cleaved () Bound Cleaved AV,, (%)
Plasma 13.4 f 1.0 13.4 t 1.0 0 O. 124 + 0.003 O. 150 + 0.003 2 1* membrane CHAPS DMPC
SCRL 15.7k1.2 11.2f0.5 -28* 15.3 f 0.3 18.6 + 0.2 21*
* Significantly different as detemined by the paired t-test. Table 2.4 Catalytic-centre activities for 5'-NTase before and after cleavage by PI-PLC
Bound CIeaved -- - - -. .- .- CHAPS 40.4 * 0.7 49.7 + 0.7 DMPC 29.6 =t 0.3 32.1 * 0.3 Egg PC 32.3 + 0.3 37.5 h 0.5 Egg PE 19.2 0.4 33.3 * 0.6 SCRL 22.0 k 0.4 26.6 * 0.3 SM 28.9 * 0.5 30.0 * 0.3 catalytic efficiency. This reduction in efficiency appears to depend on the nature of the bilayer or membrane into which the anchor is inserted.
2.5 Discussion
Release of GPI-anchored proteins by endogenous phospholipases has been proposed to play an important role in regulation of their surface activity, and may also generate second messengers that initiate transmembrane signalling processes
[78,162,254,257]. It is, therefore, important to have a detailed understanding of the potential consequences of such release. Our approach in the present work was to use the purified GPI-anchorec! ectoenzyrne 5'-NTaçe, reconstituted into defined phospholipids.
The advantage of using such lipid systems is that many biochemical and biophysical parameters, such as 1ipid:protein ratio, acyl chain length and fluidity, and headgroup charge, may be strictly controlled.
Porcine lymphocyte 5'-NTase is a disulfide-linked homodimer in its native state
[35 11, and remains in this form during purification and reconstitution [169]. Results of the present study indicated that 5'-NTase obeyed classical Michaelis-Menten kinetics whether it was present in native plasma membrane, solubilized in detergent, or reconstituted with phospholipids, indicating that there is no allosteric communication between the two monomers. Experiments also showed that 5'-NTase, whether it was membrane-bound, in detergent solution, or reconstituted, was completely cleaved by the action of Bt-PI-PLC. Therefore, no phospholipase-resistant pool of enzyme exists for
5'-NTase fiom this source. The resistance of some GPI-anchored proteins (e.g. human erythrocyte acetylcholinesterase, alkaline phosphatase) to release by PI-PLC has been demonstrated to arise frorn a change in the anchor structure via covalent modification of the inositol ring by esterification of an additional fatty acid moiety [251]. Such rnodified
61 anchors may remain susceptible to cleavage by GPI-PLD [341]. Highly PI-PLC-resistant populations have been reported for 5'-NTase in hepatocytes [273], and in the plasma membrane of liver fkom different species [346]. Since in the case of porcine lymphocyte
5'-Nase the entire population was cleaved by Bt-PI-PLC, covalent modification does not appear to be involved.
Koelsch et al. [153] reported that aminopeptidase P of rat small intestine bnish- border membrane vesicles, which had a normal unrnodified anchor structure, was resistant to cleavage by PI-PLC, but could be released by GPI-PLD. The anchor could be made accessible to PI-PLC by treatment of the membrane vesicles with papain, sonication, or solubilization with detergent. It was proposed that the GPI anchor of arninopeptidase P is sterîcally hindered, and only accessible to PI-PLC after disturbance of the membrane structure. Similar arguments were used to explain the PI-PLC resistance of alkaline phosphatase in mouse brush-border membrane vesicles [140].
We used the EC,, (the concentration of phospholipase required to release 50% of the 5'-NTase in soluble form) as a quantitative measure of the ease of cleavage of the
5'-NTase anchor in several different lipid environments by Bt-PI-PLC. Results showed that EC,, values varied over a range of almost 200-fold (see Table 2.2), depending on the lipid chosen for reconstitution. The 1ipid:protein ratio of the reconstituted preparations was controlled to be in the range 150-200:l in al1 cases, and gven the low protein content, steric crowding effects on the membrane surface should not be a factor. It appears, therefore, that the properties of the lipid alone can modulate cleavage by
Bt-PI-PLC. Thee systems (native membrane, egg PC, SCRL) allowed Bt-PI-PLC to cleave 5'-NTase very easily, yielding low EC,, values, whereas three lipids (DMPC, egg
PE, SM) and CHAPS micelles had high EC,, values, indicating that phospholipase 62 cleavage was considerably more difficult in these environrnents. We have shown that the susceptibility of GPI-anchored proteins to PI-PLC cleavage may depend on bilayer surface charge [268], however in the present case, the lipids and detergent used were al1 zwitterionic, so differences in headgroup charge cannot account for the observed variations. Headgroup structure also does not seem to play a role, since large differences in EC,, values were observed among the choline-containing lipids (PC and SM). This suggests that properties of the lipid acyl chains backing, fluidity) may be important in determining susceptibility to phospholipase cleavage. In this regard, our laboratory has previously shown that the ability of PI-PLC to cleave 5'-NTase is greatly lowered when the host lipid is in the rigid, tightly-packed gel phase, compared to the fluid, loosely- packed liquid crystalline phase [268]. It is possible that a tight packing density and low surface deformability may restrict the ability of the phospholipase to gain access to the
GPI anchor, which is located within the polar interfacial region of the bilayer.
There have been other reports that the susceptibility of GPI-anchored proteins to attack by phospholipase C is dependent on membrane Iipid composition. In Chinese hamster ovary cells deficient in sphingolipids, the GPI-anchored antigen CD 14 became hypersensitive to PI-PLC cleavage, relative to the same cells when supplemented with sphingolipids in the medium [102]. These results suggested that interaction of GPI- anchored proteins with sphingolipids, likely by formation of DIGs, can reduce the availability of the anchor to phospholipase action.
The results of the present study indicate that 5'-NTase is catalytically activated following release from a nurnber of different membrane systems. Catalytic activation of
GPI-anchored proteins following release fi-om the membrane surface by specific phospholipases has been reported for several enzymes. The hyaluronidase activity of the
63 PH-20 protein present in the plasma membrane of guinea pig sperm is released into the medium by PI-PLC with a large increase in enzyrnatic activity [92]. Early work by Low and Finean [173] reported recovery of substantially increased 5'-NTase activity in the soluble supernatant following treatment of intact pig lymphocytes with S~aphylococcus aureus PI-PLC. Dipeptidase kom porcine kidney rnicrovillar membrane is also activated following removal of the GPI anchor [25]. In this case, release of the enzyme fYom the membrane surface resulted in a 10-fold decrease in the KM of the enzyme, whereas Vmx remained essentially unchanged. The authors suggested that insertion of the GPI anchor into a lipid bilayer may result in conformational restraints on the active site, which are relzxed when the protein is released by PI-PLC cleavage, resulting in an increase in the affinity of the enzyrne for its substrate. Interestingly, 5'-NTase present in the sarne membrane did not show any apparent activation, which suggests that different constraints on the activity of 5'-NTase exist in the plasma membrane of lymphocytes relative to kidney cells in the pig.
In the case of porcine lymphocyte 5'-NTase, kinetic analysis indicated that catalytic activation is pnmarily the result of an increase in V, (or k,,J of the enzyme, rather than a change in KM(Tables 2.3 and 2.4). V,, appears to be lowered when the GPI anchor of the enzyrne is inserted into various lipids, especially egg PE. As might be expected, the VmKof soluble 5'-NTase, after release from the membrane, is more or less independent of the lipid system initially used to anchor the protein. Overall, the data indicate that the catalytic efficiency of 5'-NTase is reduced when its GPI anchor is inserted into a membrane environment or a detergent micelle. This restriction is relieved following release of 5'-NTase in soluble form by the action of Bt-PI-PLC. The kinetic properties of chicken gizzard 5'-NTase were also reported to be different depending on
64 whether the enzyme was detergent-solubilized or cleaved by B. thuringiensis PI-PLC
[294]. In this case, 5'-NTase with the anchor removed showed both an increase in V,, and a 2-fold decrease in KM compared to intact 5'-NTase in deoxycholate solution.
Clearly, 5'-NTase behaves differently depending on the species and tissue from which it is isolated.
The affinity of membrane-bound 5'-NTase for the substrate 5'-AM7 is quite high, wiîh KMin the range 1 1-16 pM depending on the lipid system employed (see Table 2.3).
This affinity remains essentially unchanged following Bt-PI-PLC release for the native plasma membrane and al1 the lipid bilayer systems used in this study, with the exception of SC=. The K, of 5'-NTase in SCRL bilayers was higher than for other lipids, and a significant decrease was observed following cleavage (Table 2.3), which suggests that a conformational constraint on substrate binding might exist in this case. The lipid composition of SCRL has been shown to encourage the formation of DRMs into which
GPI-anchored proteins preferentially locate [263], and this might lead to some conformational restriction which would affect KM. 5'-NTase has been reported to be present in DRMs in porcine lung membranes [228], intestinal epithelial cells [298] and mouse cerebellum [227].
It is possibIe that insertion of both GPI anchors of the 5'-NTase homodimer into
the bilayer imposes some conformational constraints on the protein structure, which are relieved following cleavage of one anchor. This explanation was suggested for
dipeptidase [25], where maximal catalytic activation was observed at a PI-PLC concentration 10-fold less than that required for complete release kom the membrane. In
contrast, in the present study, increased activity of 5'-NTase coincided with maximal release of the enzyme into the supernatant, making it unlikely that this mechanisrn is responsible for activation.
Alterations in protein structure and fùnction following anchor cleavage have also been observed for GPI-anchored proteins with no emqmatic activity. Two yeast CAMP receptor proteins displayed a -IO-fold decrease in the KMfor CAMP following treatment with PI-PLC or GPI-PLD [208]. Wang et al. [336] reported that covalent fatty acyi modification of the inositol ring of a folate receptor variant was associated with high binding affinity for reduced folates. PI-PLC cleavage of the modified anchor afier mild base treatment led to a large decrease in substrate binding affinity, whereas anchor removal f?om an unmodified folate receptor had no effect, suggesting that the modified anchor can influence protein conformation or topology with respect to the membrane.
Removal of the GPI anchor from Thy-1 by phospholipase C or D triggered a major change in structure of the protein, which greatly reduced binding of both polyclonal and monoclonal antibodies to the protein [IO]. Experimental evidence and molecular dynamics simulations indicated that removal of the phospholipid portion of the
GPI anchor causes a conformational change in the rernaining glycan, which in tum leads to a change in conformation on the opposite face of the Thy-1 protein. Changes in antibody binding following cleavage of the GPI anchor have also been noted for carcinoembryonic antigen [69]. Barboni et al. [IO] suggested that the GPI anchor might generally alter the conformation of proteins to which it is attached. It is possible that, as in the case of Thy-1, cleavage of the GPI anchor of 5'-NTase alters the protein conformation, which in tum increases the catalytic activity of the enzyme. CWAPTER 3: BACTERIAL PI-SPECIFIC PHOSPHOLIPASE C: MODULATION OF ANCHOR CLEAVAGE ACTIVITY BY THE PROPERTIES OF THE LIPID BILAYER 3.1 Abstract
Release of GPI-anchored ectoenzyrnes fkom the membrane by phosphatidylinositol-specific phospholipases may play an important role in modulating the surface expression and fûnction of this group of proteins. To investigate how the properties of the host membrane affect anchor cleavage, porcine lymphocyte ecto-5'-nucleotidase (5'-NTase; EC 3.1.3.5) was purified, reconstituted into lipid biIayer vesicles of various phospholipids, and cleaved using PI-PLC from BaciIIzts thrtringiensis
(Bt-PI-PLC). Bt-PI-PLC cleavage activity on the GPI anchor of 5'-NTase in lipid bilayers was highly dependent on the chah length and unsaturation of the constituent phospholipids. Very high rates of cleavage were observed in fluid lipids with a low phase transition temperature (Td, in lymphocyte plasma membrane, and in a lipid mixture that fomed rafts. Arrhenius plots of the rate of anchor cleavage in various lipids showed a characteristic break at the Tmof the bilayer, together with a discontinuity in the region around the Tm. The activation energy for GPI anchor cleavage was substantially higher in dimyristoylphosphatidylcholine (DMPC) bilayers in the gel phase compared to those in the liquid crystalline phase. The addition of cholesterol simultaneously abolished the phase transition and the large difference in cleavage rates observed around the Tm.
Inclusion of gangliosides GM, and GT,, (components of Iipid rafts) in the bilayer reduced the overall activity of Bt-PI-PLC, but the pattern of the Arrhenius plots remained unchanged. Both had similar effects, suggesting that bilayer surface charge has little influence on PI-PLC activity. 5'-NTase anchor cleavage was increased when dicetylphosphate was included in the bilayer, and decreased by stearylamine, although these changes probably arise fiom alterations in packing rather than surface charge. Incorporation into the bilayer of another raft component, the GPI-anchored protein
Thy-1, resulted in only srnall effects on PI-PLC-mediated cleavage of the 5'-NTase anchor. Taken together, these results suggest that Iipid fluidity and packing are the most important modulators of PI-PLC activity on GPI anchors.
3.2 Introduction
Intracellular PI-PLC (EC 3.1.4.1 1) catalyzes the cleavage of phosphatidyIinosito1
4,s-bisphosphate to produce diacylglycerol @AG) and myo-inositol phosphates in rnamrnalian cells [125,247,309]. The production of DAG (responsible for the activation of protein kinase C) and inositol 1,4,5-triphosphate (responsible for intracellular calcium rnobilization) by PI-PLC is an important factor in PI-mediated signalling pathways
[17,2 17,2321. Extracellular PI-PLC is secreted by several rnicroorganisms [74,134,170] although the exact physiological function of the enzyme is unclear. Bacterial PI-PLC cataIyzes the hydrolysis of membrane-bound PI to yield DAG and myo-inositol
1,2-(cyc1ic)-phosphate (cP) (see Figure 1.3A). The sarne enzyme slowly hydrolyzes the water-soluble cIP to D-myo-inositol-1-phosphate (1-1-P). The bacterial PI-PLC's appear to play an essential role as virulence factors in several pathogenic strains, including Staphylococnis aureus [182] and Listeria momcytogenes [34,192] possibly due to their ability to cleave the GPI anchor of rnarnmalian membrane proteins.
Many proteins are anchored to the extemal surface of eukaryotic cells by a GPI anchor, including extracellular coat proteins, hydrolytic enzymes, adhesion proteins, surface antigens, and receptors [72,190]. The GPI anchor appears to play a role in the increased lateral mobility of some plasma membrane proteins [2 181 and the intracellular targetting of proteins in polarized epithelial cells [30]. In addition, the GPI anchor is responsible for the clustering of this class of proteins into glycolipid-enriched lipid rafts that are resistant to extraction by nonionic detergents and play a central role in transmembrane signalling [5,27,222,274]. Hydrolysis of the anchor by specific phospholipases C and D results in the release of the protein in soluble form, and this process rnay play a roIe in modulaiing the display and fùnction of GPI-anchored proteins at the ce11 surface.
Water-soluble phospholipase enzymes tend to be more active towards an aggregated substrate. This phenomenon, termed 'interfacial activation', depends on the physicochemical nature as weIl as the organization and dynarnics of the interface, and has been studied quite extensively for phospholipase A2 (reviewed in [88,142]) and other phospholipases [249]. Bacterial PI-PLC displays interfacial activation towards both the membrane-bound substrate PI [ l6O,3 321 and the water-soluble substrate cP [349,3501; however, Iittle is known about the interfacial kinetics of bacterial PI-PLC cleavage of
GPI-anchored proteins in lipid bilayer vesicles. Recent work in Our laboratory explored the catalytic activation of a purified GPI-anchored protein, 5'-nucleotidase (5'-NTase), following cleavage of the GPI anchor by PI-PLC ffom Bacillus thltringiensis
(Bt-PI-PLC) (see Chapter 2) 11591. Results showed that the physicochemical properties of the membrane cm modulate both the extent of catafytic activation observed on anchor removal and the susceptibility of the GPI anchor to Bt-PI-PLC cleavage. In this study, the kinetics of cleavage of 5'-NTase by Bt-PI-PLC were exarnined in defined lipid bilayer vesicles to determine the effects on anchor cleavage activity of membrane surface charge, lipid fluidity and phase state, and the presence of lipid raft components. The results showed that membrane fluidity and packing were the most important factors
affecting GPI anchor cleavage by Bt-PI-PLC.
3.3 Materials and rnethods
3.3.1 Materials
Egg PE, egg PC, GMl, GTib7 dioleoylphosphatidylcholine (DOPC),
palmitoylmyristoyl-phosphatidyIcholine (PMPC), and galactocerebrosides (Type II fiom bovine brain) were supplied by Avanti Polar Lipids (Alabaster, AL). CHAPS, DMPC,
SM (fiom bovine erythrocytes and bovine brain), cholesterol, dicetyl phosphate (DiCP), stearylamine (SA), and TX-114 were purchased f?om Sigma Chernical Co. (St. Louis,
MO). Recombinant Bacillus thuringiensis PI-PLC (250-300 Uhl; expressed in B. subtilis) was obtained fiom Oxford GlycoSciences Inc. (Bedford, MA). One unit (U) of enzyme activity released 1 pmol Pi fi-om phosphatidylinositol per min at 37 OC, pH 7.5.
3.3.2 Purification of porcine lymphocyte 5'-NTase
5WTase was purified as described in Section 2.3.3 with the following modifications. Fractions containing 5'-NTase activity From the 5'-AMP-Sepharose colurnn (Sigma) were cornbined and dialyzed extensively against 20 mM ammonium bicarbonate buffer, pH 7.4 followed by lyophilization to dryness. The lyophilized enzyme was resuspended in 12.5 mM CHAPS in 50 rnM Tris/HCI/lSOmM NaCl (pH
7.4). The protein content of the concentrated 5'-NTase sarnple was determined by the method of Peterson [233].
3.3.3 Reconstitution of porcine lymphocyte 5'-NTase
Punfied 5'-NTase was reconstituted into lipid bilayer vesicles using a modification of the detergent dialysis technique described previously [169,267,268]. A rnixîure of the desired lipids (1-3 mg) in MeOH-CHC1, (brain cerebrosides were 71 dissolved in py-ridine to avoid CHCl, according to the manufacturer's instructions; Avanti
Polar Lipids [Alabaster, AL, U.S.A.]) was evaporated to dryness in a small glass tube using Nz gas, and purnped under vacuum for 1 h to remove al1 traces of organic solvent.
The dried lipid was dissolved in 12.5 rnM CHAPS in 50 mM Tris-HCI, 0.15 M NaCl,
0.02% sodium azide @H 7.4), and mixed with purified, concentrated, 5'-NTase in the sarne buffer. The detergent was removed by dialysis in Spectrapor 4 tubing (12- 14 kDa cutoff) against 3 changes (a total of 3 L) of 10 mM Tris-HCVO.1 M NaCl buffer (pH 7.4).
Lipid bilayer vesicles containing Thy-1 were prepared by the sarne method except dialysis hibing with a smaller pore size (3-6 kDa cutoff) was used for dialysis. Thy-1 was purified as previously descnbed [243] and resuspended in the sarne buffer as
5'-NTase before addition to CHAPS-solubilized lipids.
Following dialysis, lipid vesicles were harvested by centrifugation (41 000 g for
10 min) and then resuspended in 50 rnM Tris-HCI, 0.15 M NaCl, 0.02% sodium azide
(pH 7.4) (TBS buffer). The resulting lipid bilayer vesicles had a final 1ipid:protein ratio of 150- 200: 1 (w/w) for vesicles containing 5'-NTase. Vesicles containing Thy- 1 and
5'-NTase had a final 1ipid:protein ratio of 9.5: 1 (wlw). Purified 5'-NTase was reconstituted into the following lipid systems: DMPC, egg PC, DOPC. PMPC, sphingolipid/cholesterol-rich liposomes (SCRL; consisting of egg PC: egg PE: SM: cerebrosides: choiesterol, 1: 1 : 1 : 1 :2 mole ratio) [263], DMPC containing 5% (w/w) of the ganglioside GM, or GT,,, DMPC containing 5% GMl/22% cholesterol (w/w/w), DMPC containing 5% (w/w) dicetylphosphate (DiCP) and DMPC containing 5% (w/w) stearylamine (SA). 3.3.4 Cleavage of 5'-NTase by Bt-PI-PLC
Recombinant B. thuringiensis PI-PLC expressed in B. szibtilis was used to cleave the GPI anchor of 5'-NTase as in Sections 2.3.5 and 2.3.6, with the following modifications. An aliquot (20 pl) of membrane-bound 5'-NTase in TBS supplemented with 1% (w/v) BSA (pH 7.4) (TBS/BSA buffer) was made up to 50 pl with Bt-PI-PLC in the sarne buffer at the indicated temperature for the appropriate arnount of time. Two negative controis and one positive control were also assayed for each Bi-PI-PLC concentration used. One negative control and the positive control had TBS/BSA buffer added in place of Bt-PI-PLC at Hl, and the second negative control had Bt-PI-PLC added at the end of the experiment, just before the reaction was stopped. After the indicated tirne period, the cleavage reaction was stopped by the addition of 50 pI ice-cold
20% (v/v) TX-114 in TBS buffer (50 pl TBS was added to the positive control). The mixture was cooled on ice for 3 min, warmed to 37 OC for 3 min, and then centrifuged at
14 700 g for 3 min at room temperature (the positive control was not centrifùged). The upper aqueous phaie (2-3 x 20 pl aliquots) was assayed for the cleaved anchorless form of 5'-NTase using the radioactive assay descnbed in Section 2.3.7.
For detergent-solubilized 5'-NTase, cleavage experiments were performed as described above, except that special precautions (described below) had to be taken for the
TX-114 extraction step because the CHAPS content in the 5'-NTase sample interferes with the cloud point of TX-114. At the end of the cleavage reaction, 100 pl (instead of
50 pl) ice-cold 20% (v/v) TX-114 was added (100 pl TBS buffer was added to the positive control) and the sarnples were cooled on ice for 3 min, warmed to 37 OC for
3 min, and then centrifuged at 14 700 g for 3 min at 37 OC (instead of room temperature)
73 in a rnicrocentrifùge, to separate the phases. The upper aqueous phase was assayed for
the cleaved soluble form of 5'-NTase as described in Section 2.3.7.
Arrhenius plots were used to analyze the effects of lipid phase state on the
cleavage of 5'-NTase by Bt-PI-PLC. The Arrhenius equation postulates that the
temperature dependence of any reaction rate can be described by the following equation:
in k = In A - (EJRT)
where E,,, is the activation energy and corresponds to the standard enthalpy of activation
and ln A is a constant. A plot of log k vs. l/T (Arrhenius plot) is a straight line with slope
-Ea,J2.303R. According to classical kinetic theory, the constant A = PZ, where Z is the
fkequency of molecular collisions for a bimolecular reaction, and P is the probability that
the two molecules are correctly oriented for a reaction to take place. According to
transition state theory, the constant A is related to the activation entropy, AS', as follows:
AS' = R ln(ANh/RT) - R
3.3.5 Preparation of detergent-resistant membranes (DRM's)
To an aliquot (135 pl) of 5'-NTase in lipid vesicles was added 2% (vlv) TX- 100
(135 pl) to give a final TX-100 content of 1% (v/v). Following incubation on ice for 30
min, the sarnple volume was made up to 800 pl with 60% (w/v) sucrose to give a final
sucrose content of 40% (w/v). The sarnple was then placed at the bottom of a 5.2 ml ultracentrifuge tube and overlayed with 30% (wlv) sucrose (2.2 ml) followed by 5% (wlv) sucrose (2.2 ml). Following centrifirgation at 64 000 g for 3 h, 400 pl fiactions (total of
13) were collected kom the top of the tube and assayed for 5'-NTase activity as described in Section 2.3.7. Low density insoluble lipid matenal floated at the interface between the
5% and 30% sucrose layers and was collected in fractions 2-8, whereas, completely soluble and high density insoluble material remained in the 40% sucrose layer and was collected in fiactions 10-13. Fractions 2-8 were referred to as detergent resistant membranes (DRM's).
3.3.6 Differential scanning calorimetry
A Microcal MC-2 high-sensitivity differential scanning calorimeter (Microcal
Inc., Northampton, MA) was used to obtain calorirnetnc data for the different Iipid systems tested. Lipid vesicles (3 mg of lipid) in 3 ml of TBS were pre-warmed above the gel to liquid-crystalline phase transition temperature pnor to calonmetric analysis and then cooled to 4 OC. The lipid samples were analyzed at a scanning rate of 1.5 "C/min and each sample was scanned at least twice up to 40 OC, with highly reproducible results.
The calorimebic data were analyzed using Microcal Origin Scientific software (Microcal
Software Inc.). The phase transition (melting) temperature of the bilayer, Tm,was defined as the temperature at the peak maximum.
3.4 Results
3.4.1 Kinetics of cleavage of 5'-NTase by Bt-PI-PLC
In Chapter 2, the EC,, (the concentration of Bt-PI-PLC required to release 50% of the 5'-NTase in soluble form) was used to give an estimate of the ability of Br-PI-PLC to cleave the GPI anchor of 5'-NTase. Results showed that EC,, values varied over a range of almost 200-fold (see Table 2.2), depending on the lipid chosen for reconstitution.
One of the objectives of the current work was to hrrther investigate the cleavage ability of
Br-PI-PLC towards 5'-NTase by looking at the initial rates of cleavage in different lipid systems. Figure 3.1 shows that the initial rate of cleavage of 5'-NTase was linear over the chosen time range for porcine lymphocyte plasma membrane vesicles and purified PM i SCRL - - A egg PC DMPC
-
O 10 20 30 40 50 60 70 Time (min)
Figure 3.1 Initial rate of Bt-PI-PLC cleavage of 5'-NTase at 37 OC Porcine lymphocyte plasma membrane vesicles ( ) and purified 5'-NTase reconstituted into SCRL ( i), egg PC (A) and DMPC (*)proteoIiposomes were incubated with Bt-PI- PLC at 3 7 OC. The cleaved enzyme was separated fkom the bound enzyme by TX- 1 14 phase partitiming and the 5'-NTase activity of the cleaved enzyme was determined. Data points represent the mean S.E.M. (II-3). 5'-NTase reconstituted into proteoliposomes composed of various lipids. The initial rate
of cleavage foIlowed a similar pattern to the EC,, data, in that a high EC,, was associaied
with a low initial rate of cleavage (Table 3.1). The initial rates of cleavage were highest
for 5'-NTase in porcine lymphocyte plasma membrane vesicles, SCRL and egg PC
(which aIso had a low EC,,) and substantially lower in DMPC (which had a high EC,,).
In any kinetic study, it is aiso important to test whether the initial rates of activity
of an enzyme are linear with respect to the concentration of the enzyme. Figure 3.2
shows the initial rate of cleavage of 5'-NTase by increasing concentrations of Bt-PI-PLC
in porcine lymphocyte plasma membrane vesicles. The results showed that the cleavage of 5'-NTase was linear with respect to Bt-PI-PLC concentration. This experiment was repeated for the other 5'-NTase samples used in this study and al1 showed linearity with
Bt4I-PLC concentration (data not shown).
3.4.2 Effect of acyl chain length and unsaturation on Br-PI-PLC cleavage of 5'-NTase The initial rates of Bt-PI-PLC cleavage of 5'-NTase were examined in proteoliposornes made up of several different phosphatidylcholines to observe the effect of acyl chain length and unsaturation on anchor cleavage. Figure 3.3 shows that there is a substantial difference in the rate of cleavage, depending on acyl chain length and unsaturation. The initial rates of cleavage varied over a range of -280-fold for the different PC species tested (Table 3.1). The two lipid systems with the lowest initial rate of cleavage were those with saturated acyl chahs; DMPC and PMPC at 0.96% and 0.13% cleaved/mU Bt-PI-PLClmin, respectively. Palmitoylmy-ristoylphosphatidylcholine
(PMPC) differs fiorn DMPC in that one acyl chain is longer by only two carbon atoms, Table 3.1 Bt-PI-PLC cleavage of 5'-NTase in different lipid environments at
37 OC
The initial rates were calculated from the slopes of the lines in Figure 3.1. The error indicates the goodness of fit to a straight line by linear regression analysis. The EC,, values, defined as the concentration of Br-PI-PLC required to release 50% of the 5'-NTase in soluble form, are taken hmTable 2.3.
5'-NTase sarnple ECm Initial rate (units/ml) (% cIeaved/mU Bt-PI-PLClmin)
Porcine lymphocyte plasma 0.03 membrane SCRL 0.0 1
0.008
0.7 *
DOPC *
* not deterrnined 0.00 0.05 0.10 0.15 0.20 0.25 0.30 [Bt-PI-PLC] (m Ulm L)
Figure 3.2 Cleavage of 5'-NTase by Bt-PI-PLC in porcine lymphocyte plasma membrane vesicles Porcine lymphocyte plasma membrane vesicles were incubated with increasing concentrations of Bt-PI-PLC at 37 OC for 30 min. The cleaved form of 5'-NTase was separated from the membrane-bound form by TX- 114 phase partitioning. The enzymatic activity of the cleaved form was assayed, and the data are presented as the percent cleaved per min over the 30 min period. Data points represent the mean + S.E.M. (n=3). + DOPC
PMPC i DMPC
O 20 30 40 50 Time (min.)
Figure 3.3 Effect of acyl chain length and unsaturation on the initial rate of Bt-PI-PLC cleavage of 5'-NTase in phosphatidylcholine proteoliposomes Initial rates of cleavage of 5'-NTase by Bt-PI-PLC at 37 OC in vesicles of DOPC (+), egg PC (A), DMPC (i)and PMPC ( ). Data points represent the mean * range (n=2); where not visible, error bars are included within the symbols. yet the initial rate of cleavage was substantially lowered by >7-fold. Clearly, lipid chain length has a drarnatic effect on anchor cleavage by Bt-PI-PLC.
The hvo PC species with substantially higher initial rates of anchor cleavage were those with unsaturated acyl chains. Egg PC is composed of -45% mono-, di-, and polyunsaturated acyl chains of primarily 16 and 18 carbon atom (Avanti Polar Lipids,
Alabaster AL), whereas DOPC has two monounsaturated 18 carbon chains. The initial rates of cleavage were 27% and 37% cleaved/mU Bt-PI-PLClmin for egg PC and
DOPC, respectively (Table 3.1). This represents a -27-fold increase in the initial rate of cleavage for egg PC, and a -38-fold increase for DOPC, compared to DMPC, with completely saturated acyl chains. Thus, the presence of unsaturated acyl chains has a drarnatic effect on GPI anchor cleavage by Bt-PI-PLC by substantially increasing the initial rate of cleavage as compared to lipid bilayer vesicles with saturated acyl chains.
The initial rate of cleavage correlates quite well with the Tmof the lipids. The lipid with the highest initial rate, DOPC, had the lowest melting temperature (-20 OC) (Avanti Polar
Lipids, Alabaster, AL). This was followed by egg PC (Tm = -10 OC) (Avanti Polar
Lipids, Alabaster, AL,), DMPC (24 OC) (Figure 3.4), and PMPC (30.5 OC) (Figure 3.4).
Therefore, Bt-PI-PLC prefers lipids with a low Tm,and its rate of cleavage appears to be directly related to lipid fluidity.
3.4.3 Effect of lipid bilayer surface charge on the cleavage of 5'-NTase by PI-PLC
Previous work in our laboratory suggested that membrane surface charge may affect the ability of PI-PLC to cleave the GPI-anchor [268]. A goal of this study was to further investigate this phenomenon by rneasuring the initial rates of Bt-PI-PLC cleavage of 5'-NTase in DMPC bilayers containing charged species. 5'-NTase was
81 Temperature CC) Temperature (y)
Figure 3.4 DSC scans of various phospholipid bilayers Various lipid bilayer sarnples (1 mglml) were subjected to DSC analysis as descnbed in Section 3.3.6 to determine the phase transition temperature (Td. Samples were pre- warrned to 40 OC, cooled to 4 OC, and thennally analyzed at a scan rate of 1.5 OC/min. Duplicate scans of the sarne sample were essentially superimposable. reconstituted into DMPC bilayers containing either negatively charged 16 carbon dicetyl phosphate (DiCP) or positively charged 18 carbon stearyIarnine (SA). At 20 OC, when the lipid bilayer is in the gel phase, inclusion of negatively charged DiCP in the bilayer increased the rate of mchor cleavage by Bt4I-PLC by -2-fold, whereas, inclusion of the positively charged SA slightly reduced activity (Figure 3.5A; Table 3.2). In the liquid-crystalline phase (37 OC) similar trends were observed, although positiveIy charged SA led to a much larger (-4-fold) reduction in the initial rate of anchor cleavage
(Table 3.2).
Taken together, these resuIts initially suggest that membrane surface charge affects the ability of Bt-PI-PLC to cleave the GPI anchor. A positive surface charge reduces the efficiency of anchor cleavage whereas a negative siirface charge slightly increases the rate of anchor cleavage. Since the IBS of the related enzyme, Be-PI-PLC, possesses a Lys residue (Lys44) [lll], and Bt-PI-PLC has a Lys residue at the same position [158], the increase in rate in the case of DiCP might be expected based on eIectrostatic considerations. Bilayer packing may also play a role in this process because there is a difference in the degree of activation or inhibition depending on whether the bilayer is in the rigid gel phase (20 OC) or the fluid liquid crystalline phase (37 OC).
Figure 3.4 shows that DiCP and SA increase the Tmof DMPC bilayers and broaden the phase transition. The effects on Br-PI-PLC activity may thus be explained by changes in both packing and fluidity of the bilayer, as well as surface charge effects.
3.4.4 Effect of Iipid phase state on the cleavage of 5'-NTase by Bt-PI-PLC
The activity of Bt-PI-PLC was determined with respect to temperature for cleavage of 5'-NTase in porcine lymphocyte plasma membrane vesicles, purified 1 i 5%""'" DiCP
O 10 20 30 40 50 Time (min)
Figure 3.5 Effect of lipid bilayer surface charge on the initial rate of Bt-PI-PLC cleavage of 5'-NTase in DMPC proteoliposornes Purified 5'-NTase in DMPC ( 0 ), DMPC + 5% (w/w) dicetylphosphate (DiCP) ( I) and DMPC + 5% (w/w) stearylamine (SA) (A) were incubated with Bt-PI-PLC at 20 OC (A) and 37 OC (B). The cleaved enzyme was separated fiom the bound enzyme by TX-114 phase partitioning, and the 5'-NTase activity of the cleaved enzyme was detennined. Data points represent the mean * range (n=2); where not visible, error bars are included within the syrnbols. TabIe3.2 Effect of various components on the cleavage of 5'-NTase by Br-PI-PLC
5'-NTase reconstituted into bilayers containing different components was incubated with Bt-PI-PLC for 15 min at 20 OC or 37 OC. Data are presented as the mean & S.E.M (n=4).
Initial rate (% cleaved/mU Bt-PI-PLClmin)
DMPC/5% DiCP (w/w) 0.085 * 0.005 DMPCISYo SA (w/w) 0.034 0.002 DMPC/S% GM, (w/w) 0.083 * 0.009 DMPC/S% GT,, (w/w) 0.092 h 0.007 DMPC/S% GM,/22% 0.27 * 0.01 cholesterol (wlwlw) DMPC/10:1 Thy-1 0.027 * 0.002 Ww) 5'-NTase reconstituted into proteoliposomes of DMPC, PMPC and egg PC, and purified
5'-NTase solubilized in CHAPS. The data for each systern were summarized in
Arrhenius plots (Figure 3.6) and values for the activation energy of anchor cleavage, E,.,, were determined fFom the slopes of the various plots (Table 3.3). For cleavage of
5'-NTase in DMPC, there was a break in the Arrhenius plot at 24 OC (Figure 3.6) which corresponds to the melting temperature of DMPC as determined by differential scanning calorimetry (DSC) (Figure 3.4). The apparent E,,, in the lower temperature range (18-
24 OC) was 6-fold higher than the E,,, in the higher temperature range (26-37 OC), where the membrane is in the fluid liquid crystalline phase (Table 3.3). It is also interesting to note that there is a measurable discontinuity in the data at 24-26 OC, just above the Tm of the bilayer. The E,,, changes to a formally negative value in this range. Calculation of
E,,, using the Arrhenius equation (see Section 3.3.4) assumes that the constant A (which includes the activation entropy AS$) remains constant at different temperatures. Changes in AS' arising kom alterations in collisional fiequency and orientation effects at different temperatures may therefore contribute to deviations from the Arrhenius equation. The apparent E,,, value of 446 kJ/mol for DMPC in the gel phase is anornalously high, and is probably due to changes in AS$ arising from the lower collision fiequency expected in gel state lipid.
As shown in Figure 3.6, porcine lymphocyte plasma membrane vesicIes showed a break in the Arrhenius plot at 27 OC. The E,,, in the lower temperature range was 5-fold greater than in the higher temperature range (see Table 3.3). Differential scanning calorimetry of lymphocyte plasma membrane vesicles showed that there was no thermotropic transition in the temperature range used in these experiments (Figure 3.4), egg PC A DMPC i PM PMPC 27 OC A CHAPS
Figure 3.6 Arrhenius plots of the initial rate of BI-PI-PLC cleavage of 5'-NTase in different lipid systems Data for porcine lymphocyte plasma membrane vesicles (m), purified 5'-NTase in CHAPS (A) and reconstituted proteoliposornes of egg PC ( ), DMPC (A)and PMPC ()Data points represent the mean * SEM (n=4); where not visible, error bars are included within the symbols. Table 3.3 Activation energies for Br-PI-PLC cleavage of 5'-NTase in different lipid environments
Values for E,,, were calculated f?om the dopes of the lines in Figure 3.5. The correlation coefficient (r) indicates the goodness of fit to a straight line by linear regression analysis.
Tm Ternperature range Eact r ( "Cl (" C) (kJmol-') Porcine lymphocyte plasma membrane
DMPC 18-24 446 * 35 0.988 24-26 negative 0.982
26-37 73* 11 0.946
egg PC -10 20-37 64*7 0.974
CHAPS solution 20-37 44*8 0.945 so the break in the Arrhenius plot must arise from some other factor, possibly a change in
AS$.
The cleavage of 5'-NTase was investigated in bilayers of PMPC (Tm = 30.5 OC,
Figure 3.4) which differs fkom DMPC structurally in having one acyl chain of length increased by two carbon atoms. Surprisingly, PMPC showed a different Arrhenius plot pattern from DMPC. As shown in Figure 3.6, there is a break in the plot at 27 OC, and a discontinuity in the data fiom 27-30 OC,just below the Tm. In contrast to the situation observed for DMPC, the plot has a negative slope in the region of the discontinuity, leading to a calculated E,,, of 285 kJ/mol (see Table 3.3). The value of Ex, in the temperature range below 27 OC (gel phase) is 2-fold higher than that calculated above
30 OC (liquid crystalline phase).
Cleavage of sohbilized 5'-NTase in CHAPS, and 5'-NTase reconstituted into egg PC vesicles, showed no breaks or discontinuities in the Arrhenius pIots (Figure 3.6).
The E,,, values for these two systems were similar to those deterrnined in the higher temperature ranges for 5'-NTase in lymphocyte plasma membrane vesicles, and 5'-NTase reconstituted into DMPC (Table 3.3). The Tm of egg PC is -10 OC (Avanti Polar Lipids,
Alabaster, AL). Therefore, for the temperature range used in these experiments (20-
37 OC), egg PC is in the fluid liquid crystalline phase and no melting transition takes place.
Taken together, these results suggest that the phase state of the lipid bilayer modulates the catalytic properties of Bt-PI-PLC cleavage of 5'-NTase. For synthetic phospholipids, the E,,, of anchor cleavage is higher when the bilayer is in the rigid gel phase, and substantially lower when it is in the fluid liquid crystalline phase. This difference is larger for DMPC bilayers (6-fold) compared to PMPC bilayers (2-fold).
Cleavage of 5'-NTase in both lipids shows complex behaviour, with a discontinuity in the temperature range near the T, of the bilayer.
3.4.5 Effect of lipid raft components on the cleavage of 5'-NTase by Bt-PI-PLC
3.4.5.1 Effect of gangliosides on the cleavage of 5'-NTase by Bt-PI-PLC
Gangliosides are negatively charged glycosphingolipid components of lipid rafts
[237,269,277,345] that may, therefore, affect Bt-PI-PLC cleavage activity by virtue of both their CO-localization with GPI-anchored proteins and theIr negative charge. The negative charge of gangliosides may also affect the ability of Bt-PI-PLC to cleave the
GPI anchor. The goal of this study was to see how the addition of gangliosides to DMPC bilayers would affect the cleavage of 5'-NTase by Bt-PI-PLC. The addition of 5% (w/w)
GM, to DMPC bilayers caused a change in the slope of the Arrhenius plot as compared to
DMPC aIone (Figure 3.7). The break in the Arrhenius plot occurred at a similar temperature in both cases (24 OC), and the discontinuity of positive slope remained, but there was a significant reduction in E,,, in both the gel phase (18-24 OC; -3 fold) and the liquid crystalline phase (26-37 OC; -2.2 fold) (Table 3.4). Differential scanning calorirnetry showed that the addition of 5% GM, broadened the phase transition of
DMPC but did not alter T, substantially (Figure 3.4). The initial rate of Bt-PI-PLC cleavage at 37°C was 4.4-fold lower for 5'-NTase in DMPCI5% GM, as compared to
DMPC alone (Table 3.2). However, at 20 OC, the initial rate of cleavage was significantly higher in DMPC/S% GM, as compared to DMPC alone. The ganglioside
GM, has a negative charge due to a single sialic acid residue in the headgroup of the molecule, therefore, it will add a negative charge to the bilayer surface equal to that of
90
Table 3.4 Activation energies for the cleavage of 5'-NTase by BI-PI-PLC in proteoliposomes containing various components
Values for E,,, were calculated frorn the slopes of the lines in Figures 3.5, 3.6 and 3.7. The correlation coefficient (r) indicates the goodness of fit to a straight line by linear regression analysis.
Temperature range Ex, r
(O C) (kJ.moI-') DMPC vesicles 446 * 35 0.988 negative 0.982
73 * 11 0.946
DMPC + 5% GM, 151 * 15 negative
34 *12
DMPC + 5% GM, + 22% cholesterol
DMPC + 10: 1 (w/w) Thy-1
negative 0.998
117 * 16 0.962
SCRL 118*7 0.98 1
negative 0.988
53 * 22 0.8 11 5% DiCP, but Meraway fiom the surface. Yet 5% GMl enhanced anchor cleavage at
20 OC but reduced anchor cleavage at 37 OC, compared to the enhancement seen at both
temperatures for DiCP. This disparity could be due to differences in bilayer packing
between DMPC/S% GMl and DMPC/S% DiCP. To examine how gangliosides with
multiple negative charges affected Bt-PI-PLC cleavage, 5'-NTase was reconstituted into
DMPC vesicles containing 5% (w/w) of the trisialoganglioside, GT,,. As seen in Table
3.2, GT,, behaved very similarly to GM, in lowering the initial rate of cleavage in DMPC
bilayers at both 20 OC and 37 OC, indicating that more negatively charged gangliosides do
not further increase the inhibition of Bt-PI-PLC cleavage activity. These results suggest
that packing effects, rather than surface charge, probably account for the effects of
gangliosides on PI-PLC activity.
As mentioned above, GM, has been found in lipid rafts along with GPI-anchored
proteins. A cornrnon method used to determine whether Iipid bilayers form rafts, or
detergent-resistant membranes (DRMs), is to test their solubility in ice-cold TX- 1 00 as
descnbed in Section 3.4.5. RafVI2R.M fractions float at a characteristically low density,
and can be found in fractions 2-8 of a sucrose density gradient following cold TX-100 treatment. As seen in Figure 3.8B, DMF'C/S% GM, liposomes fail to form DRM's, as evidenced by the lack of 5'-NTase activity in fractions 2-8 of the sucrose density gradients. In contrat, 5'-NTase reconstituted into SCRL, a synthetic lipid mixture known to mimic lipid rafts [263], was recovered in fiactions 2-8 of the gradient after cold
TX-100 treatment (Figure 3.8D). These results suggest that localization of 5'-NTase into
DRM's is not responsible for the observed behaviour of Bt-PI-PLC when cleaving, the protein in bilayers of DMPC/S% GM,. 1 (A) DMPC 1
h(B) DMPC + 5% GM, -
- (C)DMPC + 5%GM,+ 22 % chol. T
O 2 4 6 8 IO 12 14 Fraction Number Figure3.8 Detergent solubility of 5'-NTase in porcine lymphocyte plasma membrane and proteoliposomes of various lipids Purified 5'-NTase in DMPC (A), DMPC+S%GM, (B), DMPC+S%GM,+22% cholesterol (C) and SCRL (D) or lymphocyte plasma membrane vesicles (E) were treated with 1% TX-100 at 4 OC for 30 min and subjected to sucrose density gradient centrifugation as described in Materials and Methods. Bars represent the 5'-NTase activity k S.E.M. (n=3) in the fractions collected fkom the sucrose gradients. Cholesterol, along with sphingolipids, is a necessary component for the formation of lipid rafts [39,274] and can also influence the melting temperature of lipid bilayers.
For these reasons, 22% (w/w) cholesterol was added to DMJ?C/5% GM, (w/w) bilayers to detennine the effect of cholesterol on Bt-PI-PLC cieavage of 5'-NTase. The addition of cholesterol completely eliminated the break in the Arrhenius plot at 24 OC observed for both DMPC and DMPC/S% GM, (Figure 3.7). In fact, no break or discontinuity was evident over the entire temperature range. Differential scanning calorimetry confimed that the phase transition of DMPC/S% GM, (w/w) was completely eliminated with the addition of 22% cholesterol (w/w) (Figure 3.4) The E,,, was substantially lower
(-2-fold) for DMPC/5%GMl/22% cholesterol (18-37 OC) as compared to the E,,, for
DMPC alone and DMPC/S% GM, (Table 3.4). The initial rate of cleavage of Bt-PI-PLC was increased for DMPC/S% GM,/22?40 cholesterol as compared to DMPC/5% GMl at both 20 OC (-3.3 fold) and 37 OC (-1.7 fold) (Table 3.2). The result is that the cleavage rate at 20 OC is only -30% lower than the rate at 37 OC, whereas in bilayers of DMPC alone, there is a 25-fold drop in the cleavage rate when moving f?om 37 OC (liquid crystalline phase) to 20 OC (gel phase). These results confirrn that the phase state of the bilayer directly modulates Bt-PI-PLC activity; the rigid gel phase seerns to be unfavourable for catalytic activity. Figure 3.8C shows that DMPC/S% GM,/22% cholesterol bilayers fail to forrn DRM's as evidenced by the lack of 5'-NTase activity in
Eractions 2-8 of the sucrose gradients. This indicates that the effect of cholesterol on
Br-PI-PLC cleavage of 5'-NTase in DMPC/S% GM,/22% cholesterol is not due to the formation of lipid rafts. 3.4.5.2 Effect of Thy-1 on the cleavage of 5WTase by Bt-PI-PLC
A recent study in our lab has shown that the GPI-anchored protein Thy-1 can slightly broaden the gel to liquid-crystalline phase transition of DMPC bilayen but has no effect on Tm [243]. To investigate the effect of Thy-1 on Bt-PI-PLC cleavage,
5'-NTase was reconstituted in DMPC bilayers containing 10: 1 (w/w) Thy-1. Thy-1 had no effect on the break and discontinuity in the Arrhenius plot as compared to DMPC alone (Figure 3.9). However, the addition of Thy-1 lowered the E,,, in the gel phase
(18-24 OC; -1.6 fold) while increasing the E,,, in the liquid-crystalline phase (26-37 OC;
-1.6 fold) (Table 3.4) suggesting that Thy-1 moderates the change occumng on rnelting of DMPC bilayers. The initial rate of cleavage of Bt-PI-PLC was sirnilar for both systerns at 20 OC, but was reduced by a factor of -1.8 for DMPC/lO:l Thy-1 at 37 OC when compared to DMPC bilayers alone (Table 3.2). This reduction in activity may be due to a stenc hindrance effect of surface-bound Thy-l restricting access to the 5'-NTase anchor, or competition for 5'-NTase anchor cleavage by the Thy-1 anchor.
3.4.5.3 Cleavage of 5'-NTase in SCRL vesicles by Bt-PI-PLC
Sphingolipid/cholesterol-nch liposomes (SCRL) are known to form DRM's when
îreated with ice-cold nonionic detergents [263]. In Section 3.4.1, it was shown that the initial rate of cleavage of 5'-NTase in SCRL was quite high (compared to the cleavage of
5'-NTase in DMPC), and approached that observed for native lymphocyte plasma membrane. Figures 3.8D and E indicate that both lymphocyte plasma membrane vesicles and SC=, respectively, forrn DRM's when treated with cold TX-100, as evidenced by the localization of 5'-NTase activity to fiactions 2-8 of the sucrose gradient. Figure 3.9 shows that Bt-PI-PLC cleavage of 5'-NTase in SCRL had a break point at 28 OC, which 96 A DMPC DMPC + 1011 Thy-1 i SCRL
, PM 27 OC I
Effect of lipid raft components on the initial rate of Bt-PI-PLC cleavage of 5'-NTase Arrhenius plots for cleavage of 5'-NTase in lymphocyte plasma membrane vesicles ( ) and purified 5'-NTase in SCRL (sphingolipid-cholesterol-rich liposomes) (m), DMPC (A), and DMPC + 10:l (w/w) Thy-1 (4). Data points represent the mean * SEM (n=4); where not visible, error bars are included within the symbols. is similar to that seen for lymphocyte plasma membrane. However, there was aIso a discontinuity of positive dope in the Arrhenius plot for SCRL at 28-30 OC, which was not seen in lymphocyte plasma membrane. This discontinuity is similar to that seen at
24-26 OC for the DMPC-based lipid systems (Figure 3.9). Differential scanning calorimetry indicates that SCRL shows a very broad melting transition starting at -28 OC
(Figure 3.4), suggesting that the break and discontinuity in the Arrhenius plot may be due to melting of this lipid mixture, as with DMPC. The E,,, for cleavage of 5'-NTase in
SCRL is lower in the gel phase (1 8-28 OC; -1.4 fold) as cornpared to lymphocyte plasma membrane, but the E,,, is similar in the liquid-crystalline phase for both systems (Tables
3.3 and 3.4).
3.5 Discussion
Release of GPI-anchored proteins by endogenous phospholipases may play an important roIe in regulation of their surface activity, and rnay also generate second messengers that initiate transmembrane signalling processes [78,162,254,257]. Bacterial
PI-PLC1s have the ability to cleave the GPI anchor of membrane-bound proteins in marnmals, and this activity is thought to be responsible for the pathogenesis of some bacterial strains [34,182,192]. By studying the activity of bacterial PI-PLC's towards
GPI-anchored proteins, some light can be shed on the potential consequences of the release of GPI-anchored proteins by endogenous phospholipases. Our approach in the present work was to use the purified GPI-anchored ectoenzze 5'-NTase, reconstituted into defined phospholipids, to determine the effects of membrane fluidity and acyl chain length, membrane surface charge, and the presence of lipid raft components, on the ability of Bt-PI-PLC to cleave the GPI anchor. Bt-PI-PLC shows a high rate of cleavage activity towards the GPI anchor of
5'-NTase in egg PC and DOPC bilayers as compared to DMPC and PMPC bilayers, indicating that membrane fiuidity is important for Bt-PI-PLC activity. The reduced temperature of a lipid is defined as the experimental temperature minus the Tmof the lipid bilayer, and is a measure of the fluidity of a bilayer (a high reduced temperature indicates a high fluidity). A plot of the activity of Bt4I-PLC versus the reduced temperature shows a high correlation between membrane fluidity and Bt-PI-PLC activity (Figure
3.10). Clearly, the higher the fluidity of the bilayer, the more active Bt-PI-PLC becomes towards the GPI-anchor of 5'-NTase. Binding of the enzyme to the surface of the bilayer may be enhanced by more fluid membranes. Interfacial activation of phospholipases is a comrnon phenomenon and depends on the physicochemicai nature as well as the organization and dynarnics of the interface. Interfacial activation has been studied quite extensively for phospholipase A2 (reviewed in [88,142]) and other phospholipase enzymes [249]. Multiple phospholipase A, enzymes appear to have a cornmon
'interfacial binding surface' (IBS) that is Iocated on a flat external surface surrounding the active site [265]. Bacterial PI-PLC displays interfacial activation towards both the membrane bound substrate PI [160,332] and the water-soluble substrate cIP [349]. It was suggested that binding to the membrane surface Ieads to allosteric activation of the enzyme. The crystaI structure of PI-PLC fkorn Bacillus cereus (Bc-PI-PLC), which is highly homologous to Bt-PI-PLC [333], was determined alone and in a complex with myo-inositol or a kagment of the GPI-anchor, and has shed sorne light on the possible residues that make up the IBS (see Figure 1.3B) [Ill,1 121. Helix 42-48 and loop
237-243 (coloured in red in Figure 1.3B) surround the nm of the active site pocket and DOPC *
IO 20 30 40 50
Reduced temperature (OC)
Figure 3.10 Effect of lipid bilayer fluidity on the activity of Bt-PI-PLC The initial rates of cleavage of 5'-NTase in different PC bilayers (fkom Table 3.1) were pIotted against the reduced temperature, which was defined as the temperature of the cleavage assay (37 OC) minus the Tm of the Iipid bilayer. Data points represent the mean k S.E.M. (n = 3); where not visible, error bars are included within the symbols. contain an unusually high number of hydrophobic aliphatic and aromatic amino acid residues exposed to solvent. The helix and loop are highly flexible in the crystal structure and they rnay adopt different conformations when bound to a membrane interface. Both the helix and the loop contain a Trp residue, and Trp fluorescence was show to increase upon binding of Bt-PI-PLC to PC bilayers [332] and micelles [349]. The hydrophobic and aromatic residues of the loop and helix of PI-PLC may penetrate into the bilayer and stabilize the membrane-protein complex, in a similar fashion to the mode1 proposed for phospholipase A, [286]. It may be easier for the hydrophobic residues of the Br-PI-PLC enzyme to penetrate into a more fluid membrane, or the increased fluidity may stabilize the enzymehilayer complex, thus accounting for the increase in activity. The results outlined in the present work are in agreement with this proposal. Also supporting this suggestion is the observation that Bt-PI-PLC is less active towards unilamellar vesicles of long-chah PI, which would be expected to be less fluid than short-chain PI [350].
Also, Bt-PI-PLC is more active on PC micelles rather than more tightly packed PC vesicles [349], and substrate presentation in micelles rather than a bilayer leads to a higher apparent rate of hydrolysis by phospholipases in general [250]. There was aIso less interfacial activation of Br-PI-PLC when the enzyme encountered tightly packed, cross-linked PC molecules in unilamellar vesicles, as opposed to PC molecules in micelles 13491.
In the present study, a high rate of Bt-PI-PLC cleavage was also associated with lipid bilayers and membranes that form rafts (SCRL, native plasma membrane). This rnay be explained by the higher Iocal concentration of substrate (5WTase) as a result of clustering into the lipid raft microdomains. It has been suggested that bacterial PI-PLC acts on substrate in the processive 'scooting mode' of interfacial catalysis, where the enzyme stays bound to the membrane for multiple catalytic turnover cycles, up to 40-50 catalytic cycles per binding event [332]. If the substrate is coccentrated into membrane rafts, more catalytic turnover cycles couId occur per binding event, due to a higher local concentration of substrate.
The effect of temperature on the cleavage of 5'-NTase by Bt-PI-PLC was assessed by constnicting Arrhenius plots of anchor cleavage in various lipid bilayers..
The breaks in the Arrhenius plots correlated with bilayer phase transitions in every case, except for lymphocyte plasma membrane. A change in AS$ might account for the observed break in the Arrhenius plot in this instance, and for the anornalously high values of E,,, at Iower temperatures when membrane bilayers are in the rigid gel phase. Since
AS' is a function of the collision fiequency Z (see Section 3.3.4), a radical change in Z might be expected in the gel phase, and may lead to high values of E,,,. The E,,, of anchor cleavage was higher in the gel phase for al1 the Iipid bilayers analyzed, suggesting that the rigid gel phase is unfavourable for catalytic activity of Bt-PI-PLC. As discussed above, it rnay be more difficult for the hydrophobic sequences of the IBS of Bt-PI-PLC to penetrate the ngid gel phase, since it has tighter packing density and low surface deforrnability. When cholesterol is added to the DMPC bilayer, it abolishes the phase transition, and has a dramatic effect on Bt-PI-PLC activity. In the presence of cholesterol, the cleavage rate at 20 OC is only -30% lower than the rate at 37 OC, whereas in bilayers of DMPC alone, there is a 25-fold drop in the cleavage rate when moving fkom 37 OC (liquid crystalline phase) to 20 OC (gel phase). Cholesterol both increases the fluidity of the gel phase, and decreases the fluidity of the liquid crystalline phase, thus evening out the changes in fluidity that would normally occur over this temperature
range. These results confirm the effects of lipid fluidity and bilayer phase state on
Bt -PI-PLC activity.
At 37 OC, negatively charged DiCP slightly increased the activity of Bt-PI-PLC,
whereas positively charged SA substantially reduced the activity of Bt-PI-PLC, in
DMPC bilayers. Electrostatics play a vital role in the activity of phospholipase A2 by
helping to stabilize the enzyme/membrane complex [88,286]. Electrostatic interactions of
cationic and aromatic residues in the IBS of this enzyme initially bring the protein to the
membrane surface where membrane penetration by hydrophobie interactions of aliphatic
and aromatic residues further stabilize the enzyme/membrane complex [284]. Although
the overall charge of Bt-PI-PLC at physioiogical pH is negative (Oxford Glycosciences
Inc., Bedford MA; [268]), the electrostatic potentiaI of the IBS of the related enzyme
Bc-PI-PLC (helix 42-48 and loop 237-243) is positive, due to Lys44 (electrostatic
potential calculated using Rasmol Ver. 2.7.1.1 using the crystal structure in [11 11; PDB
1GYM). Bt-PI-PLC has a Lys residue in the same position (Lys4-4) [158], which may
also be Iocated in the IBS of this protein. Interfacial binding of PI-PLC may, therefore'
occur through both eIectrostatic and nonpolar interactions of the IBS with the membrane.
Positively charged vesicles containing SA rnay inhibit the binding of the IBS due to its
positive electrostatic potential, whereas, a negatively charged bilayer surface containing
DiCP may enhance interfacial binding. Differences in the degree of activation at 20 OC
and 37 OC suggest that packinçJfluidity may also explain the effects of DiCP and SA. In
fact, there is a high correlation between the reduced temperatures of DMPC bilayers containing DiCP and SA and Bt-PI-PLC activity (Figure 3.10). Taken together, this suggests that the changes in Br-PI-PLC activity caused by DiCP and SA rnay aise fiom both charge effects and differences in packing/fluidity of the bilayer.
The fact that GM, (with one negative charge) had the same effect as GT,, (with three negative charges) on anchor cleavage by Bt-PI-PLC also supports the proposa1 that the magnitude of the surface charge does not seem to have an effect on the enzyme activity. Again, the effects of gangliosides on anchor cleavage rnay be due to differences in bilayer packing/fluidity. Gangliosides me lcnown to inhibit the activity of a variety of lipoIytic enzymes including non-specific PI-PLC fiom B. cereus [SOI and C. pefiingens
[20,21], Type I PLAz fiom porcine pancreas [19,180] and type EI PLA? f?om snake venom
[49]. The proposed mechanisrn of inhibition in these cases is thought to be through direct alteration of the adsorbed enzyme, and/or by altering the availability of substrate at the membrane surface through steric interactions [SOI. These factors rnay also contribute to inhibition of Bt-PI-PLC cleavage by gangliosides. E,,, is the sarne in îhe presence of gangliosides as in their absence (in both the gel and liquid crystalline phase), which indicates that the energy barrier for anchor cleavage is not affected by the presence of the glycolipids. This suggests that gangliosides do not affect the overall intrinsic mechanism of catalysis by Bt-PI-PLC. They rnay affect the adsorption of the Br-PI-PLC to the membrane surface via packing/fluidity effects. Gangliosides have long acyl chains, and are less fluid than the DMPC bilayer. Therefore, they rnay decrease the adsorption rate or increase the desorption rate of Bt-PI-PLC fiom the bilayer surface. Gangliosides rnay also have a steric effect, by virtue of their large oligosaccharide headgroups, decreasing the access of Bt-PI-PLC to the substrate in the membrane interfacial region. The presence of the GPI-anchored protein Thy-l in the lipid bilayer reduced the overall activity of Bt-PI-PLC. However, similar to the effects of gangliosides, the E,,, was unchanged, suggesting that the overall intrinsic mechanism of catalysis was not affected. The packing/fluidity of DMPC bilayers was not changed significantly in the presence of 10: 1 (w/w) Thy-2 [243], since the mole fraction will be very srnaIl compared to 5% GM, (w/w). The reduced activity rnay be due to steric effects, as suggested above for gangliosides, or competition for 5'-NTase anchor cleavage by the Thy-1 anchor. CHAPTER 4: PROXIMITY OF THE PROTEIN MOIETY OF THE GPI- ANCHORED PROTEIN PLAP TO THE MEMBRANE SURFACE: A FLUORESCENCE RESONANCE ENERGY TRANSFER STUDY 4.1 Abstract
Indirect evidence &om biochemical and modelling studies suggests that the GPI
anchor may hold the protein close to the plasma membrane. However, there is a lack of
direct information on the proximity of the protein portion of this class of proteins to the
membrane surface. The present study uses FRET to address this important problem. The
GPI-anchored ectoenzyme PLAP was purified ti-om a plasma membrane extract of
human placental microsornes without the use of butanol extraction. Pwified PLAP was
fluorescently labelled at the N-terminus with 7-dimethylaminocoumarin-4-acetic acid
succinimidyl ester (DMACA-SE) and Oregon Green 488 succinimidyl ester
(OG488-SE) and reconstituted by detergent dilution into defined lipid bilayer vesicles
containing increasing mole fractions of the fluorescent probes 7-nitrobenz-2-oxa- 1,3-
diazo 1-4-y1)- l,2-dihexadecano yl-slr-glycero-3 -phosp hoeole (NBD-PE) or
octadecyl Rhodarnine B (C,,RhoB), respectively. The fluorescence of DMACA-PLAP
and OG488-PLAP donors was quenched in a concentration-dependent marner by the
lipid acceptors NBD-PE and C,,RhoB. The energy transfer data were analysed using an approach that describes FRET between a uniform distribution of donors and acceptors in an infinite plane. The distance of closest approach between the protein moiety of PL@ and the lipid-water interfacial region of the bilayer was estimated to be 8-12 A. This indicates that the protein portion of PLAP is located very close to the lipid bilayer. possibly resting on the surface. This contact may allow transmission of structural changes fiom the membrane surface to the protein, which could influence the behaviour and catalytic properties of GPI-anchored proteins. 4.2 Introduction
There is little information on the proximity of the protein portion of
GPI-anchored proteins to the surface of the cell. However, biochemical and modelling
studies have suggested that they rnay be close to each other. Studies in our laboratory
[268] and that of others [24] have suggested that the protein portion of the GPI-anchored
ectoenzyme 5'-NTase may be in direct contact with the membrane bilayer. This was
suggested based on the fact that the enzyme exhibited a discontinuity in the Arrhenius
plot when the bilayer was converted from the solid gel phase to the fluid liquid crystalline
phase [267]. In addition, the glycan portion of the GPI anchor of Thy-1 was predicted to
lie either between the lipid surface and the protein in a tightly folded conformation [IO],
or in a carbohydrate-binding pocket within the protein itself [240]. Ln both rnodels, the
protein dornain of Thy-1 is visualized as being very close to, or in contact with, the
bilayer. Measurement of the distance between the protein portion of GPI-anchored proteins and the bilayer surface, using a biophysical approach, would aid in the
eIucidation of the nature of the association of this class of proteins with the membrane.
FRET has become a powerful biophysical technique since it was first proposed as a 'spectroscopic der' for rneasurhg distances in biological systems [299]. The technique has been used to measure inter- and intramolecular distances in proteins, and the distance between a defined site in a protein and the membrane surface. For the integral membrane transport protein, the Ca2'-ATPase, FRET was used to estimate the distance of the ~a"
and ATP binding sites from the membrane surface [100,209], the location of an active
site lysine residue relative to several probes within the bilayer [311], and the distances between several cysteine residues and the active site lysine 1961. FRET also determined that the a,p, and y subunits of heterotrirneric G-protein were located close to the bilayer
[244], whereas the binding site in the growth hormone receptor was distant fkom the bilayer [37]. Morc recently, FRET was used to elucidate the proximity of the active sites of the two nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface Cl681 and to each other [239].
The present study describes the application of FRET to measure the proximity of the protein portion of the GPI-anchored ectoenzyrne, PLAP, to the membrane surface.
Using a purified, reconstituted system, the protein portion of PLAP was shown to be quite close to the bilayer, possibly resting on the membrane surface.
4.3 Materials and methods
4.3.1 Materials
Egg PE and egg PC were supplied by Avanti Polar Lipids (Alabaster, AL).
CWS, L-histidyldiazobenzyIphosphonic acid (phosphonate) agarose, p-nitrophenyl phosphate (PNP), ethylaminoethanol and TX-114 were purchased fkom Sigma Chemical
Co. (St. Louis, MO). Extracti-Gel D detergent removing gel was purchased ffom Pierce
Chemical Co. (Rockford, IL). Con A-Sepharose was purchased from Amersham
Phannacia Biotech AB (Baie D'Urfé, QC). Marina Blue 1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolarnine (MBPE), 7-dimethylarnino-coumarin-4-acetic acid succinimidyl ester @MACA-SE), octadecyl rhodarnine B chIoride (C,,RhoB),
(7-nitrobenz-2-oxa- l,3-diazol-4-y1)- l,2-dihexadecanoyl-sn-glycero-3-phosphoethmol- amine (NBD-PE; tiethylammonium salt), and Oregon Green 488 succinimidyl ester
(OG488-SE) were purchased Çorn Molecular Probes (Eugene, OR). 4.3.2 Purification of placental alkaline phosphatase (PLAP)
PLAP was purified fiom human placenta by a modification of the method of
Hawrylak [108]. Human placenta was obtained within one hour of delivery and
irnrnediately placed on ice. The umbilical cord and connective tissue were removed and
the remaining tissue was rinsed in cold PBS to remove most of the blood, divided into
-100 g portions, and flash frozen in liquid nitrogen until ready for purification. Thawed
placenta (100 g) was combined with 100 ml homogenization buffer (50 mM Tris/HCVl
rnM MgC12/0.1 mM ZnCI,, pH 8.5), together with two tablets of a protease inhibitor
cocktail Pierce, Rockford, IL). The mixture was hornogenized in a Waring blender for 3
min on low speed followed by 3 min on high speed. The homogenized sarnple was
passed through two Iayers of cheesecloth to remove large debris and then divided into
two equal volumes. Plasma membrane vesicles were prepared fiom each half according
to the method of Maeda et al. [177]. Briefly, homogenized placenta (50 g) was layered
over a sucrose cushion (41% wlv), followed by ultracentrifUgation at 95 000 g. Plasma
membrane was solubilized in 50 mM CHAPS (final protein concentration 1.5 rng/ml) for
4 h at 4 OC, followed by ultracentrifugation at 38 000 g for 30 min. The aqueous layer was recovered and chromatographed on a Con A-Sepharose colurnn equilibrated with basic colurnn buffer (BCB; 20 mM Tris/HCVO.S M NaCVlmM MgCIJ1 rnM CaCI, and protease inhibitors as above; pH 8.5). The pH of the pooled enzyme fractions (eluted with 0.4 M methyl a-D glucoside in BCB) was adjusted to 6.0 by the addition of 100 mM MES, pH 5.0, and enzyme was then adsorbed to phosphonate agarose and eluted with 50 mM PNP in MES buffer (pH 6.0). The PLAP active fractions were combined, dialyzed extensively against 10 mM ammonium bicarbonate buffer (pH 7.4), lyophilized to dryness, and stored at -70 OC. 110 4.3.3 Assay for PLAP activity
PLAP activity was assayed in 96-well plates in 1.O M ethylaminoethanoVl.5 mM
MgCl, (pH 9.8) containing 10 mM PNP at 37 OC. The absorbance at 404 nm was monitored using a 96-well kinetic plate reader, and enzyme activity was reported as PNP released (pmovmidmg).
4.3.4 Absorption spectra and fluorescence excitation/emission spectra
Absorption spectra were recorded using a Perkin-Elmer Lambda 6 Whisible
spectrophotometer (Perkin-Elmer, Norwalk, CT) with both sample and reference cells at
22 OC. Fluorescence spectra were recorded on a PT1 Alphascan-2 spectrofluonmeter
(Photon Technology International, London, ON) with the ce11 hoIders thermostated at
22 OC. The excitation and ernission slit-widths were both set at 4 nm.
4.3.5 Fluorescent labeiing of PLAP
PLAP was labelled with DMACA-SE and OG488-SE based on the protocol outlined by the manufacturer of the fluorescent dyes (Molecular Probes, Eugene, OR).
Pure, lyophilized PLAP was resuspended in 20 mM PBS containing 0.1% TX-100 (pH
6.5). The pH of the buffer was selected to label specifically the N-terminal arnino acid residue of PLAP by reducing the reactivity of lysine side chain residues. The specific dye in DMSO was added to PLAP (20:l mole ratio dye:protein) and the sample was stirred at roorn temperature for 4 h. The reaction was terrninated by the addition of 50 mM hydroxylamine (1140 of the volume) and stirred for an additional 30 min at room temperature. Following chromatographie separation on a Sephadex G25 column or a
Micro Bio-spin column (Biorad Laboratories, Mississauga, ON), the labelled protein was fûrther purified by three rounds of TX-114 phase separation (see Section 2.3.5). The detergent phase was diluted 10-fold in 50 mM TrisMêVO.15 M NaCV0.25 M sucrose
(pH 7.5) and TX-114 detergent was removed by chromatography on an Extracti-Gel D detergent removal column equilibrated with the same buffer. The purified, fluorescentIy-labelled protein (DMACA-PLAP or OG488-PLAP) was made up to
2 mM CHAPS using 200 rnM CWSin 50 mM Tris/HCVO.15 M NaCV0.25 M sucrose
(pH 7.5) and then stored at -70 OC.
4.3.6 Preparation of reconstituted vesicles containing PLAP
DMACA-PLAP and OG488-PLAP were reconstituted into phospholipid vesicles by a detergent dilution method. Egg PC, egg PE, DOPC, NBD-PE and Cl,RhoB were stored at -20 OC in CH,Cl-MeOH (2: 1 v/v). DMACA-PLAP was reconstituted into egg
PC/egg PE vesicles containing increasing mole fiactions of NBD-PE, whereas
OG488-PLAP was reconstituted into DOPC vesicles containing increasing mole
Gractions of C 18RhoB. Egg PC (0.15 prnol) and a mixture of egg PE and the appropriate arnount of NBD-PE (total PE/NBD-PE of 0.15 pmol) were dispensed into a series of microfige tubes. Similarly, DOPC (0.3 pmol) and the appropriate amount of Cl,RhoB were treated in the same manner. The lipid mixtures were dried under a gentle stream of nitrogen, and then further drkd in a vacuum dessicator for 1 h. To the dried lipid was added 10 p1 25 rnM CHAPS/O.ZS M sucrose in 50 mM Tris-HCI buffer (pH 7.4) and the contents were vortexed and sonicated in a Sonogen sonicator (Branson Instruments, Inc.,
Starnford, CT) therrnostated at 37 OC. The mixture was chilled on ice and DMACA-
PLAP or OG488-PLU (6 pg in 30 pl 2 rnM CHAPS/0.25 M sucrose/Tns-HC1 buffer) was added. After incubation on ice for 30 min, the volume of each sample was then diluted to 1 ml with 0.25 M sucrose/Tris-HC1 buffer, and the resulting vesic!es were
112 resuspended with a fine-gauge needle. The final lipid concentration was 0.3 mM and the protein concentration was 6 pg/ml with a 1ipid:protein ratio of 50:1 (w/w). Control vesicles were prepared in the same manner using unlabelled PLAP.
4.3.7 Dynamic Iight scattering
The size of the reconstituted vesicles was detemined using dynamic light scattering (DLS). This technique involves the measurement of the scattering of light at
90" to the incident of a laser bearn directed at a sample of freely diffusing vesicles.
Fluctuations in the intensity of the scattered light generate an autocorrelation function that is directly related to the diffùsion coefficient, D, of the vesicles in solution. The radius of the vesicles is calculated f?om D using the Stokes-Einstein equation, assuming spherical, hollow particles.
4.3.8 Resonance energy transfer measurements
Fluorescence intensities were measured using a PT1 Alphascan-2 spectrofluorirneter (Photon Technology International, London, ON). The excitation wavelengths for DMACA-PLAP and OG488-PLAP were 377 and 495 nrn, respectively, while emission was measured at 469 and 525 nm, respectively, with 4 nrn slits.
Fluorescence intensities were corrected for light scattering using controls containing unlabelled PLAP, and the inner filter effect was corrected at both the excitation and emission wavelengths using the equation p56,167,226]:
e.co,= (4 - ~)10~-~~(~~'~~) (1) where Fi,,, is the corrected value of the fluorescence intensity, Fiis the experimentally measured fluorescence intensity, B is the background fluorescence intensity, b is the path length in centimeters, and A,, and A&, are the absorbances of the sample at the
excitation and emission wavelengths, respectively.
4.3.9 Determination of parameters for FRET analysis
The resonance energy transfer efficiency (E) between donor and acceptor can be
where F and Fo are the fluorescence intensities of the donor in the presence and absence
of the acceptor, respectively. FRET efficiency is related to the distance (R)between the
donor and acceptor by the following equation:
R = &(E-' - 11"~ (3)
where R, is the distance at which the efficiency of energy transfer is 50%. Ro is
calculated fiom
Ro = (9.8 x lo3)(JK'Q,~ 4 ) 1/6 (A)
where J is the spectral overlap integral between donor and acceptor in units of cm3 M-',
K' is the orientation factor (taken as 2/3 based on the assumption that donor and acceptor
dipoles are mobile; [48]), QDis the fluorescence quantum yield of the donor, and rz is the
refractive index of the medium between the chromophores which, for a dilute aqueous
solution, is equal to 1.33 [342].
The spectral overlap integral, J, is defined by :
where FD(il) and &,(A) are the donor emission and the acceptor rnolar extinction coefficients, respectively, at h. The fluorescence emission spectra of DMACA-PLAP and OG488-PLAP were recorded using excitation at 377 and 495 nm, respectively and the absorption spectra of NBD-PE and C,,RhoB were measured. The spectral data were used to calculate J using equation (5) with the aid of a computer program designed by Dr. Uwe
Oehler (Department of Chemistry and Biochemistry, University of Guelph).
The quantum yields, QD, of DMACA-PLAP and OG488-PL@ ir? reconstituted vesicles were determined relative to standards, using polarkers set to O" in the excitation beam and 54.7" (rnagic angle) in the emission bearn. The fluorescence emission spectnim of DMACA-PLAP was compared to the emission spectrum of a standard soIution of qiiinine sulfate in 0.1 N H,SO, (both sample and standard had the same absorbame of where Qquin,,is 0.5 1 in 0.1 N H,SO, [93] and FDMAa-pwand F,,,,, are the integrals of the fluorescence of DMACA-PLAP and quinine sulfate in the wavelength range 375-675 nm, respectively. The quantum yield of OG488-PLAP was calculated in the same manner, using fluorescein in O. 1 N NaOH as the standard with Q,,,,,,,i, equal to 0.9 1 [93], over an integral wavelength range of 505-700 nrn (excitation = 495 nrn). Background scattenng was corrected with reconstituted vesicles containing unlabelled PLAP at the same protein concentration. 4.3.10 Analysis of the distance between donor and acceptor Since the distance of closest approach (L)between the donor and the acceptor in the FRET expenments was assumed to be much less than R,, the data were analyzed using the approach derived by Wolber and Hudson [340]. The solution calculates the distance of closest approach between a uniform population of randomly distributed 115 donors and acceptors on an infinite plane and is described by a simple series approximation: F / F, = ~,e-'" + A,e-kzc where: The parameter c describes the surface density of acceptors measured in number per A'. The value of c was calculated by dividing the mole ratio of acceptor/membrane lipids by the average area of the headgroups of phospholipids, 80 A'. The values of A,, k,. Ar and k, were taken fiom the tables of the exact solution to the series approximation by Wolber and Hudson for different ratios of L& [340]. 4.4 ResuIts 4.4.1 Purification of PLAP Human PLAP was successfully purified using a hvo-step affinity chromatography procedure, first on Con A-Sepharose (which isolates a-D-rnannose-containing glycoproteins), then on agarose conjugated to a covalently bound non-hydrolysable substrate analog, phosphonate (Table 4.1 ). The plasma membrane starting material was treated with the zwittenonic detergent CHAPS, which was able to solubilize -80% of the total membrane-bound protein from the lymphocyte plasma membrane. After purification on the Con A and phosphonate columns, a fold-purification of 30-fold was obtained relative to the plasma membrane starting matenal. SDS-PAGE analysis of highly purified PLU resulted in a major band with an apparent M, of -74 kDa (Figure 4.1, lane 4). TX-114 phase partitioning revealed that >95% of PLAP partitioned into the detergent phase, indicating that it had retained a GPI anchor (data not shown). Table 4.1 Purification of PLAP frorn human placenta Samples from various stages of the purification procedure were assayed for PLM activity as descnbed in the Materials and Methods section. One unit of activity corresponds to 1 pmol PNP hydrolyzed/min at pH 9.8 and 37 OC. Data are presented as the mean +: S.E.M. (n = 3) Stage of Protein Total activity Specific activity Fold purification purification (mg) (V x 1O-)) (U/mg> Plasma 144 st 1 70.5 f 14.9 0.49 * O. 1 1 1 membrane CHAPS 112k2 404 * 61 extract Con A colurnn 21.5 i OS 181 * 13 eIuate Phosphonate 3.3 =t 0.1 48.1 k 3.4 14.7 * 1.6 colurnn eluate Figure 4.1 Purification of PLAP from human placenta Plasma membrane fiom hurnan placenta (lane 1, 10 pg of protein), CHAPS-solubilized placenta1 plasma membrane (lane 2, 10 pg), the glycoprotein fraction eluted fiom the Con A-sepharose colurnn (lane 3, 10 pg protein) and purified PLAP (lane 4, 2 pg protein) were subjected to SDS-PAGE analysis in a 10% polyacrylarnide gel, followed by staining with silver. DMACA-PLAP (lane 5, 5 pg) and OG488-PLAP (lane 6, 5 pg) were subjected to SDS-PAGE in a 10% polyacrylamide gel and viewed on a transilluminator. The fluorescent bands were photographed with black and white Kodak film. The position of molecular-mass markers is indicated on the left, and the PLAP band is indicated by an arrow. In this work, PLAP was purified fkom the plasma membrane &action isolated from placenta1 microsomes. This method was adopted to eliminate the butanol extraction procedure that is cornmonly used as a first step in alkaline phosphatase purification [123]. Isolation of the plasma membrane as a first step resulted in a fraction that contained alrnost exclusively GPI-mchored PLAP, as cornpared to butanol extraction, which can isolate non-GPI-anchored PLAP depending on the pH of the extraction [201]. Also, the human placental plasma membrane provides a starting material that is more enriched in PLAP than the butanol extract, as can be seen by the fact that only a 30-fold e~chrnent from plasma membrane was needed to achieve homogeneous PLAP in this study (Table 4. l), compared to -500 to 2000-fold e~chmentrequired from the butanol extraction in other reports [31,20 11. 4.4.2 Fluorescent labelling and reconstitution of PLAP PLAP was specificaIly labelled on the amino group of the N-terminal arnino acid residue by adjusting the pH of the IabeIling reaction to 6.5, which has the effect of protonating Lys residues (and thus reducing their activity) while leaving the N-terminus partially deprotonated (Molecular Probes, Eugene, OR). The results of SDS-PAGE analysis of the labelled protein showed a single highly fluorescent band for DMACA-PLAP (Figure 4.1, lane 5), and OG488-PLAP (Figure 4.1, lane 6). DMACA-PLAP and OG488-PLAP were the donors in the FRET studies, and they were reconstituted into lipid bilayers containing fluorescently labelled lipids that acted as the acceptors. DMAC-4-PLAP was reconstituted into bilayers of egg PC/egg PE (1:l mole ratio) containing NBD-PE as the acceptor, and OG488-PLAP was reconstituted into DOPC bilayers containing C,,RhoB as the acceptor. Both of these labelled lipids have been cornrnonly used as FRET acceptors [37,41,168,212,348]. DLS analysis of the reconstituted vesicles showed that they compnsed a relatively homogeneous population of large vesicles (probably unilamellar) with a mean diarneter of -0.3 Fm (Figure 4.2). 4.4.3 Resonance energy transfer To assess the relative distance between the fluorescent label on PLAP and the lipid bilayer, energy transfer must take place between the DMACA- or OG488-labelled protein donors and the fluorescent lipid acceptors in the membrane. Energy transfer requires that there be a significant amount of overlap between the fluorescence emission spectrum of the donor and the absorbance spectrum of the acceptor. Figure 4.3A shows the spectral overlap of the emission of DMACA-PLAP and the absorbance of NBD-PE, and Figure 4.3B shows the spectral overlap of the emission OG488-PLAP and the absorbance of C,,RhoB. The spectral overlap for both pairs is quite large (see Table 4.2 for the spectral overlap integral, J) and the value of R, was calculated to be 3 1.5 A for the DMACA-PLAPMD-PE combination and 48 A for the OG488-PLAP/C,,RhoB combination. The large values of R, indicated that these two donor-acceptor pairs are highly suitable for FRET studies. In the case of NBD-PE labelled in the headgroup, the fluorophore is known to be located in the interfacial region of the bilayer, in the vicinity of the glycerol backbone [42]. The fluorescent probe on C,,RhoB is also known to be positioned in the polar headgroup region of the bilayer (Molecular Probes, Eugene, OR). Figure 4.4A shows a progressive decrease in the fluorescence emission of DMACA-PLAP, and a progressive increase in the sensitized fluorescence emission of NBD-PE, when the protein was reconstituted into vesicles with increasing mole fractions Vesicle Radius (pm) Figure 4.2 Size distribution of reconstituted vesicles containing PLAP Egg PC:egg PE vesicles (1:l mol ratio) containing PLAP were prepared by detergent dilution using CHAPS as descnbed in Section 4.2.2. The lipid to protein ratio was 50:l (w/w) (-650:l mol/mol). DLS measurement of size distribution was canied out as descnbed in Section 4.3 -7. Wavelength (nm) 400 450 500 550 600 650 700 750 Wavelength (nm) Figure 4.3 Overlap of the fluorescence emission spectra of the donors with the W-visible absorption spectra of the acceptors (A) Fluorescence emission spectnim of DMACA-PLAP (A,, = 345 nm,right scale) and the UV-visible absorption specmim of NBD-PE (left scale). (B) Fluorescence emission spectnun of OG488-PLU (le,= 495 nm, right scale) and the UV-visible absorption spectrum of C,,RhoB (left scale). Table 4.2 Spectral parameters for donor and acceptor pairsa FRET donor FRET acceptor donor qumtum yield, overlap integral, k Q, J (cm3 M-') (A) DMACA-PLAP NBD-PE O. 139 5.61 x IO-'' 31.5 OG488-PLAP C,,RhoB O. 194 5.07 x 1O-l3 48 .O " The refiactive index, n, was taken as 1.33, and the orientation factor, 2,was assumed to be 2/3 (see Section 4.3.9). 400 450 500 550 600 Wavelength (nm) 1.2 1 1 Wavelength (nm) Figure 4.4 Fluorescence resonance energy transfer between donors and acceptors Proteoliposomes of egg fE:egg PC (mole ratio of 1:l) (A) or DOPC (B) containing increasing mole fiactions of either NBD-PE or C,,RhoB, respectively, as indicated below. (A) FRET between DMACA-PLAP and increasing mole fractions of the acceptor NBD-PE; (a) zero; (b) 0.001; (c) 0.005; (d) 0.0075; (e) 0.01; (f) 0.025; (g) 0.05. The total phospho1ipid:PLAP ratio was 50: 1 (w/w) (-650: 1 mole ratio). (B) FRET between OG488-PLAP and increasing mole fractions of the acceptor C,,RhoB; (a) zero; (b) 0.005; (c) 0.0075; (d) 0.01; (e) 0.035. The emission spectrum in (B) has not been corrected for the contribution of fiee 06488 dye. The ratio of total phospho1ipid:PLAP was 50: 1 (w/w) (-650: 1 mole ratio). Ernission bandwidths were 4 m. of NBD-PE, and excited at the excitation wavelength of the donor (377 nrn). Similady, the fluorescence emission of OG488-PLAP decreased in the presence of increasing mole fiactions of C,,RhoB in the bilayer, accompanied by sensitized fluorescence ernission by C,,RhoB upon excitation at the excitation wavelength of the donor (490 nm) (Figure 4.4B). Clearly, energy @ansferis occurring between the donor fluorophore on the protein and the acceptor fluorophore in the lipid bilayer. The results of the quenching of donors are surnrnarized in Figure 4.5, which shows the relative fluorescence of the donor with respect to the mole ratio of acceptor. The fluorescence emission of both OG488-PLAP and DMACA-PLAP was efficiently quenched with increasing mole fractions of the acceptors (Figure 4.5A and B). This quenching cm be seen to be highly efficient by comparison to the quenching curves deterrnined for the situation where the donor and acceptor are both lipids CO-reconstituted in the sarne bilayer, Marina Blue-PE (MB-PE) and NBD-PE (Figure 4.5B; open circles). The high efficiency of quenching indicates that the donor and acceptor are in close proximity, such that the ratio of the distance of closest approach, L, to the value of R, is srnall, Le. LI& + O. In support of this view, the experimental data could not be fitted to the rnodels of Dewey and Harnmes [58] or Koppel et al. [154], both of which assume that the donor fluorophore is some distance away fiom the membrane containing the acceptor fluorophore (Figure 4.5). In the situation where LIR, -+ 0, the data can be fitted to a simple series approximation derived by Wolber and Hudson [340] which describes energy transfer between a uniform distribution of donors and acceptors on an infinite plane (see Section 4.3.10). With this approximation, F/Fo depends only on the acceptor surface density (c) and R,; it does not depend on L. In Figure 4.6, the data were fitted to 0.00 0.01 O.02 0.03 0.04 0.05 0-06 Mole Fraction of Acceptor Figure4.5 Curve fitting of the FRET data from the different donors and acceptors (A) Fitting of the FRET data for OG488-PLAP and C,,RhoB (a) to the models of Dewey and Hamrnes (-) and Koppel et al. ( - - - ). (B) Fitting of the FRET data for DMACA-PLAP and NBD-PE (a) to the modeIs of Dewey and Hammes (-) and Koppel et al. f - - - ). Data for energy transfer between MB-PE and NBD-PE in the sarne bilayer (0).Data points represent the mean k S.E.M. (n = 3). Figure 4.6 Curve fitting of the FRET data from the different donors and acceptors (A) Fitting of the FRET data for OG488-PLAP and C,,RhoB to the approximation of Wolber and Hudson (see Section 4.3.10) where L = O A ( -);~=12A(--- ); L = 24 A(....-.); L = 38 A (--- ); L = 48 A( - *-- ). (B) Fitting of the FRET data for DMACA-PLAP and NBD-PE to the approximation of Wolber and Hudson where L = O A(-);~=8 A(--- ); L = 16 A (-.--.a); L = 25 A (- O- ); L = 31.5 A ( - **- ). Data points represent the mean * SEM. (n=3). the exact solutions derived by Wolber and Hudson 13401 for values of UR,. In Figure 4.6A, the quenching of OG488-PLAP by C,,RhoB fits quite well to values of L between O (solid line) and 12 A (dashed line) and does not fit when L approaches 24 A (dotted line). The data for DMACA-PLAP and NBD-PE fits to values of L between O (solid line) and 7.9 A (dashed line), but not to a value of L of 16 A (dotted line) (Figure 4.6B). Thus, results fiom the donor-acceptor pairs both indicate that the fluorescent labels on PLAP are quite close to the interfacial regions of the bilayer, with a maximum distance of closest approach of -12 A. These results suggest that the GPI anchor is in a conformation that holds the attached protein very close to the membrane surface. 4.5 Discussion Little is known about the proximity of GPI-anchored proteins to the surface of the plasma membrane. One possibility is that the protein moiety of a GPI-anchored protein is located some distance fkom the membrane, as a result of the GPI anchor being in an extended conformation ('lollipop' model). Altematively, if the GPI anchor folds up into a compact conformation, or lies along the membrane surface, the protein may 'flop' down on to the membrane, where its activity could be modulated by the properties of the bilayer. In the case of 5'-NTase, the catalytic properties of the enzyme were affected by the phase state of the bilayer, strongly suggesting that it may contact the membrane [267]. Our approach in the present study was to use energy transfer to measure the distance between a GPI-anchored protein and the interfacial region of the bilayer. The FRET donor consisted of purified, fluorescently labelled PLAP, reconstituted into lipid bilayer vesicles containing a headgroup-labelled membrane lipid probe as the acceptor. PLAP was purified to homogeneity (see Figure 4.1) using a rnodified procedure that eliminated the butanol extraction step cornmonly employed to puri@ the protein [123]. By isolating the plasma membrane as a first step in the re-designed purification procedure, a larger fraction of the contaminating proteins were eliminated as compared to the butanol extraction approach, and >95% of the final purified protein retained a GPI anchor. For energy transfer experiments, purified PLAP labelled with DMACA or 06488 was reconstituted into lipid bilayers containing varying arnounts of the labelled membrane probes NBD-PE or C,,RhoB, respectively. Fluorescence rneasurements revealed that DMACA-PLAPNBD-PE and OG488-PLAP/C,,RhoB were highly suitable donor and acceptor pairs for FRET experiments, with a substantial degree of overlap between donor emission and acceptor absorbance, and high R, values. There was a hi@ degree of donor quenching in the presence of increasing arnounts of the acceptor in both cases, indicating that the donor and acceptor were close. The dependence of donor quenching on acceptor concentration did not fit the models of Koppel et al. [154] or Dewey and Hamrnes [58] (see Figure 4.5A and B), which have been used successfully to measure the distance between specific sites in various proteins and the membrane surface, including P-glycoprotein [168], the EGF receptor [37], and the Ca2+-ATPase[100,209]. In al1 these cases, the estimated distances were quite large, on the order of 1-1.5 x R,, so that acceptor mole fractions up to 0.4-0.6 were necessary for >90% quenching of the donor fluorescence. In the present study, donor quenching was almost complete at 0.05 mole fiaction of the lipid acceptor (see Figure 4.5A and B). The quenching data obtained in the present study fitted quite well to the series approximation of Wolber and Hudson 13401 which is intended for application to cases where the separation distance is small reIative to R,. The distance of closest approach between the fluorescent label on the PLAP protein and the surface of the bilayer was estimated to be in the range of 8-12 A. Distance values this small are approaching the lower limit that can be reliably measured by FRET [156]. For cornparison, distances of closest approach between small fluorescent lipid labels CO-reconstituted into the sarne lipid bilayer are -10 A, as measured by FRET [58,86] (see Figure 4SB). This analysis assumes that the PLAP-bound donor transfers energy to acceptors in only one leaflet of the bilayer. Since the lipid-bound fluorophore is located -7.5 A below the bilayer surface, in the interfacial region, there will be a very small contribution to quenching of the donor fkom acceptors in the opposite leaflet, in the case of OG488-PLAP and C,,RhoB & = 48 A). The transbilayer quenching contribution will be negligible in the case of DMACA-PLAP and NBD-PE & = 31.5 A). Taken together, the results of the present study indicate that the protein moiety of PLAP is positioned very close to the bilayer, possibly resting on the membrane surface. The X-ray crystal structure of PLAP has recently been solved at 1.8 A resolution (PDB 1EW2; Figure 1.5) [157]. The structure confirms the accurnulated biochemical evidence that PLAP is a dimer [68] and identifies several groups of residues that may contribute to the allosteric properties of the protein. However, missing kom the X-ray structural mode1 is a large C-termina1 portion, residues 480-5 13, and the GPE anchor that woüld be attached to the C-terminal Asp residue [242]. The inability to obtain structural data for this C-terminal region indicates that it has a high degree of flexibility. The distance between Pro479 (the last visible C-terminal residue in the crystal structure) and the N-terminal Ile is 51.5 A (as measured by RasMol Ver. 2.7.1). However, the high degree of flexibility in the rather large unstructured C-terminal region would allow for a wide variation in this distance. The dimenc structure presented indicates that the N-terminus of one monomer is much closer to the C-terminus of the second monomer, than to the C-terminus of the same monomer (see Figure 1S). The results of the curent study are consistent with the PLAP dimer resting on the surface of the membrane with the N-terminus of each monomer in proximity to the bilayer surface. In Chapter 2, we demonstrated that cleavage of the GPI anchor of 5'-NTase resulted in catalytic activation of the enzyme, and showed that the degree of activation depended on the nature of the lipid bilayer into which the protein was reconstituted [159]. Our faboratory aIso previously reported that reconstituted 5'-NTase demonstrated a decrease in activation energy when the bilayer was converted fkom the solid gel phase to the fluid liquid crystalline phase [267]. In addition, 5'-NTase fiom rat enterocytes displayed a break point on Arrhenius plots, which coincided with a lipid thermotropic transition [24]. Taken together, these results indicate that the protein portion of 5'-NTase rnay also be in direct contact with the lipid bilayer, which can modulate the catalytic properties of the enzyme. Modelling studies of the GPI-anchored Iyrnphocyte antigen Thy-1 suggestcd that this rnay be the case for this protein as well. The glycan portion of the GPI anchor of Thy-1 is predicted to lie either between the lipid surface and the protein in a tightly folded conformation [IO], or in a carbohydrate-binding pocket within the protein itself [240], and it may thus impose a particular conformation on the protein. In both models, the protein domain of Thy-1 is visualized as being very close to, or in contact with, the bilayer. If this is true of GPI-anchored proteins in general, there are several implications for the structure and function of this class of proteins. Such contact would provide a mechanism for transmission of structural changes from the membrane surface to the protein. First, changes in the fluidity of the membrane, might modulate the catalytic properties of al1 GPI-anchored proteins, as has been observed for 5'-NTase [24,159,267]. In addition, such close contact between the protein and the membrane, as a result of GPI anchor insertion, may affect the catalytic properties of the protein, independent of the physical properties of the membrane. As shown in Chapter 2 for 5'-NTase, the turnover nurnber of the enzyme is reduced when the GPI anchor is membrane inserted, and this results in catalytic activation when the anchor is cleaved by PI-PLC and the protein is released from the membrane surface. Last, close association of the protein and its GPI anchor with the membrane may affect the ability of PI-PLC to approach and cleave the anchor. CWAPTER 5: SUMMARY AND CONCLUSIONS 5.1 Summary and conclusions The properties of a large nurnber of diverse eukaryotic proteins may be modulated by anchorage to the surface of the ce11 by GPI. The GPI-anchored protein rn2y have increased lateral rnobility, may be targeted to specialized regions in polarized epithelial ceils, or may be clustered into specialized membrane microdomains called lipid rafts that are involved in signalling. It is clear that the membrane environment influences the organization of GPI-anchored proteins on the surface of the cell. However, there is little information on the effects of specific lipids on the functions of these proteins. The presence of the GPI anchor also makes this class of proteins susceptible to cleavage by phospholipases, which may influence their overall biological role. As a result of this cleavage, the properties of the newly released protein may be modulated, depending on how the protein interacted with specific lipids in the bilayer. Also, specific membrane lipids may influence the susceptibility of the GPI anchor to cleavage, by modulating phospl-iolipase activity. The objectives of this study were to explore the relationship of the membrane bilayer to both the structure and functional n~odulationof GPI-anchored proteins, and to the GPI-anchor cleavage activity of PI-specific phospholipase C. Two mode1 systems were used in this work. The first system employed the purified GPI-anchored ectoenzyme, 5'-NTase, reconstituted into defined lipid bilayers. Our studies exarnined the effects of specific lipids on enzyme activity and determined how this activity is modulated upon cleavage by Bt-PI-PLC. The same mode1 system was used to explore the effect of membrane lipids on the activity of Bt-PI-PLC towards GPI-anchored 133 proteins. The second mode1 systern, (the puified ectoenzyme, PLAP, reconstituted into lipid bilayers), was used to measure directly the location of the protein portion of the GPI-anchored protein relative to the bilayer surface, using a FRET approach. In Chapter 2, 5'-NTase was isolated in highly purified form &om porcine lymphocyte p lasma membrane. 5'-NTase did not show allosteric behaviour in detergent-solubilized or membrane-bound form in this study, even though the enzyme is dimeric. Bt-PI-PLC was used to cleave the anchor of 5'-NTase to determine if the catalytic properties of the enzyme are modulated upon anchor cleavage. Contrary to published reports for other GPI-anchored proteins, there was no pool of PI-PLC-resistant 5'-NTase evident in any situation examined in this study, suggesting that the GPI anchor of 5'-NTase is not covalently rnodified with additional acyl chains. An important result reported in Chapter 2 was the finding that 5'-NTase was catalytically activated following cleavage of the GPI anchor. This resulted &om a reduction in the catalytic efficiency of the enzyme when the anchor was inserted into a bilayer (possibly caused by a restriction on the enzyme) that is relieved upon anchor cleavage. Surpnsingly, the degree of catalytic activation depended on the Iipid composition of the membrane bilayer that the anchor was inserted into pnor to anchor cleavage. This suggests that different lipids affect the activity of 5'-NTase to different degrees, presumably by altering the conformation of the protein. This alteration in conformation couId be directly transmitted f?om the membrane to the protein solely through the GPI anchor. Altematively, the membrane may influence the protein portion of the enzyme through direct contact with the bilayer surface, with the GPI anchor folded up within the protein or lying flat on the membrane surface. The concentration of Bt-PI-PLC required to release 50% of 5'-NTase (EC,,) varied over a range of 200-fold, depending on the type of lipid used for the reconstitution of 5'-NTase. For example, 5'-NTase in native membrane, egg PC and SCRL was easily cleaved by Bt-PI-PLC (low EC,,) whereas, cleavage of 5'-NTase in DMPC, egg PE and SM proved to be more difficult @gh EC,,). The cleavage ability of Bt-PI-PLC towards 5'-NTase appeared to be directly influenced by the properties of the lipid acyl chims, as opposed to headgroup charge and structure. The EC,, value was a rather crude measurement of Bt-PI-PLC cleavage ability, so a more precise kinetic approach was used in Chapter 3 to explore further the nature of lipid effects on Bt-PI-PLC activity. In Chapter 3, we used the 5'-NTase mode1 system to further investigate how the properties of the host membrane cm affect GPI anchor cleavage by PI-PLC. This represents the first study of PI-PLC to use a rigorous kinetic approach to examine how the bilayer properties affect its cleavage of GPI-anchored proteins. The initial rate of Bt-PI-PLC was linear at the temperature, substrate concentrations, and time intervals used in this study. This indicates that Br-PI-PLC does not show the characteristic lag phase in activity that some interfacial enzymes exhibit on membrane-bound substrates. Br-PI-PLC cleavage activity of 5'-NTase in lipid bilayers was highly dependent on the chain length and unsaturation of the constituent phospholipids. The rate of anchor cleavage was directly proportionaI to the fluidity of the lipid bilayers. The higher the fluidity of the bilayer, the higher the activity rneasured for Bt-PI-PLC, possibly due to the fact that non-polar residues in the IBS of the enzyme can more easily penetrate highly fluid bilayers. High rates of cleavage were also oliserved in lymphocyte plasma membrane and in a lipid mixture that forms rafts. The sequestration of 5'-NTase into rafts 135 rnay increase the activity of Bt4I-PLC by providing a higher local concentration of GPI-anchored substrate. The effect of temperature and choiesterol content on Bt-PI-PLC activity confirrned the importance of lipid fluidity on anchor cleavage. The rigid gel phase is unfavourable for catalytic activity of Br-PI-PLC compared to the fluid Iiquid crystalline phase. Differences in membrane surface charge had little effect on the activity of Bt-PI-PLC. The effects of negatively charged DiCP and positively charged SA correlated quite well with membrane fluidity effects, rather than charge, and the negatively charged gangliosides GM, and GT,, showed that the magnitude of the surface charge does not influence anchor cleavage activity. The GPI-anchored protein Thy-1 had only a small effect on Bt-PI-PLC activity, which may be explained by steric interactions or cornpetition for substrate. Taken together, these results suggest that lipid fluidity and packing are the most important modulators of Bt-PI-PLC activity on GPI anchors. This study may aid in the identification of the important residues present in the IBS of PI-PLC that allow the enzyme to bind to the surface of the membrane bilayer for catalysis to take place. The results suggest that bulky non-polar residues of the BS, such as Trp or Ile, are more important for membrane interaction as opposed to charged residues. The results fiom Chapter 2 indicated that the activity of 5'-NTase was influenced by the lipid composition of the membrane bilayer. To date, there is little information about the proxirnity of the protein moiety of GPI-anchored proteins to the surface of the plasma membrane. The influence of the membrane on 5'-NTase may be transrnitted solely through the GPI anchor if the GPI anchor is in an extended conformation, like the 'lollipop' model pictured in Figure 5.1A, where the protein is located some distance fiom the bilayer surface. Alternatively, the protein portion may be influenced by direct contact with the membrane bilayer surface if the GPI anchor folds into a compact conformation, or lies dong the membrane surface (Figure S.1B). In Chapter 4, we zttempted to distinguish between these two models by directly measuring the distance between the protein portion of purified PLU and the membrane bilayer surface, using a FRET approach. This study represents the first direct measurement of the location of the protein portion of GPI-anchored proteins relative to the membrane surface. Purified PLAP was fluorescently labelled with DMACA or 06488 donors and reconstituted into membrane bilayer vesicles containing NBD-PE or C,,RhoB acceptors. Energy transfer was observed between DMACA-PLAP and M3D-PE, or OG488-PLAP and C,,RhoB, as demonstrated by reduced donor fluorescence and sensitized acceptor emission fluorescence. There was a high degree of donor quenching in the presence of increasing concentrations of acceptors, indicating that the donor and acceptor were in close proximity. The FRET quenching data were analyzed using an approach that is cornrnonly used to estimate distances behveen closely positioned donors and acceptors. The estimated distance of the PLAP-bound probe fiom the interfacial region of the bilayer was 8-12 A, which is approaching the distance calculated between small fluorescent lipid labels CO-reconstituted into the same bilayer. This suggests that the protein portion of PLAP is very close to the lipid bilayer, possibly resting on the membrane surface. Thus, it appears that the model in Figure 5.1B best describes the disposition of the GPI-anchored protein PLAP with respect to the bilayer. Interaction of the protein with the bilayer surface may be responsible for modulation of the fünction of GPI-anchored proteins in different membrane environments. 5.2 Suggestions for future work In this thesis, insertion of the GPI anchor into a membrane bilayer was found to reduce the catalytic efficiency of 5'-NTase, and the activity was restored when the GPI anchor was cleaved with Bt-PI-PLC. Catalytic activation upon anchor cleavage has been noted for other GPI-anchored enzymes, but in this study, we found that there was a difference in the degree of activation depending on the composition of the membrane bilayer. The properties of the membrane bilayer can, therefore, modulate the catalytic properties of 5'-NTase to different degrees. It is important to determine whether this result is restricted to 5'-NTase, or if it occurs for other GPI-anchored enzymes as well. We have improved upon a method for purifjmg miIligram quantities of PLAP, so this enzyme is an obvious candidate for testing whether the membrane bilayer can also modulate its catalytic properties and activity. Alterations in the activity of GPI-anchored enzymes may mise fi-om conformational changes. The GPI-anchored protein Thy-1 displays different reactivity towards antibodies upon treatment with PI-PLC, suggesting that a structural change takes place in the protein moiety upon anchor cleavage. Determination of the interaction of 5'-NTase (and PLAP) with specific antibodies before and after anchor cleavage would help to determine if the observed catalytic activation is due to a change in protein conformation. A change in inûinsic tryptophan fluorescence is often associated with a change in the conformation of a protein, and this property could also be monitored to determine if protein structural changes occur upon anchor cleavage. The IBS of phospholipase A? has been studied extensively by site-directed mutagenesis to identie the residues that are important in the membrane-binding process. Although the proposed IBS for PI-PLC has been elucidated from the crystal structure, little work has been done on identifjmg the important residues in the IBS that are responsible for membrane binding and interfacial activation. Site-directed mutagenesis studies are needed to confimi the results of the present study, which support the proposa1 that the residues of the PI-PLC IBS that penetrate the membrane bilayer are bulky non- polar residues, as opposed to charged residues. An interesting experiment would be to introduce one or more charged residues into the IBS of PI-PLC, and determine if the enzyme is activated towards anchor cleavage on lipid bilayer membranes of opposite surface charge. FRET studies on PLAP indicate that the protein rnay interact with the membrane bilayer surface. An interesting follow-up study could look at FRET between Trp residues in PLAP and fluorescent labels in the bilayer. One potential problem with this approach is that there are several Trp residues in PLAP that may contribute to fluorescence, but the average distance of the Trp residues fkom the bilayer would at least confirm that the protein is close to the bilayer. There is a Trp residue near the N-terminus of PLAP (Trpl2) that is conserved in alkaline phosphatases from many species. Since our study suggests that the N-terminus of PLAP is close to the bilayer surface (possibly in contact with it), monitoring of Trp fluorescence could determine if there is an interaction with the bilayer, since Trp fluorescence increases upon entering a non-polar environment. Experiments could be conducted by reconstituting PLAP into bilayers with different compositions to see if the Trp fluorescence changes, indicating an interaction of the protein with the bilayer. 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