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Gpi-Anchored Proteins in Reconstituted Lipid Bilayers

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Gpi-Anchored Proteins in Reconstituted Lipid Bilayers 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 O Marty T. Lehto, 2001 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON Kt A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre r4f8me Our file Notre réfdrence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfonq vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels May be printed or othenuise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. GPI-ANCHORED PROTEINS IN RECONSTITUTED LEPID BILAYERS: STRUCTURE, FUNCTION AND CLEAVAGE BY PI-SPECFIC PHOSPHOLPASE C 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<s ~hzrringiensis (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.
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