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Identification and Characterization of a Homophilic Binding and Neuritogenic Site in the Cell Adhesion Molecule L1

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Identification and Characterization of a Homophilic Binding and Neuritogenic Site in the Cell Adhesion Molecule L1 Identification and Characterization of a Homophilic Binding and Neuritogenic Site in the Cell Adhesion Molecule L1 Xiaoning Zhao A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto O Copyright by Xiaoning Zhao, 1998 i National Library Bibliothèque nationale 1*1 ofCmada du Canada Acquisitions and Aquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellingtori OttawaON KIA ON4 OtiawaON K1AW canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnbute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fonne 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 substantid extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otheMrise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Xiaoning Zhao Department of B iochemistry University of Toronto 1998 Thesis Title: Identification and Characterization of a Homophilic Binding and Neuritogenic Site in the Ce11 Adhesion Molecule LI Abstract The neural ce11 adhesion molecule LI is an integral membrane glycoprotein which mediates cell-ce11 adhesion and promotes neurite outgrowth from neuronal cells. Mutations in the LI gene have been implicated in several neurological diseases. To investigate the mechanism and the relationship of these two intrinsic f'unctions of LI, studies were carried out to identify specific domain(s) and sequences involved in homophilic binding and the promotion of neunte outgrowth. Recombinant L 1 domain proteins were used to identiS the domain involved in L 1 homophilic binding. Results based on Covasphere binding assays indicate that the second Ig-like (Ig2) domain of L1 is sufficient and necessary for hornophilic interaction. Results from neurite outgrowth assays further demonstrate that the Ig2 domain of LI is a potent neuritogenic substrate. The colocalization of these two intrinsic activities of L1 suggests a close relationship between homophilic binding and the promotion of neunte outgrowth. Several HSAS/MASA (Hydrocephalus as a result of stenosis of the aqueduct of Sylviud mental retardation, aphasia, shuffling gai t and adducteci thumbs) mutations have been localized to L1 Ig2. The effects of two HSASlMASA mutations on the homophilic binding and neuritogenic activities of recombinant L1 Ig2 were assessed. The HSAS mutation R 184Q abolished both Ig2-associated activities, while the MASA mutation H210Q had only modest effects. The deleterious effects of these two mutations thus correlate very well with the severity of their respective clinical phenotype. The results aiso implicate a role for Arg- 184 in L1 homophilic binding The synthetic oligopeptide approach was used to identiQ the homophilic binding site in LI Ig2. The peptide containing sequences flanking Arg-184 (HIKQDERVTMGQNG) inhibited both L1-dependent ce11 aggregation and neunte outgrowth, whereas the peptide consisting of flanking sequences of His-210 had only minor effects. Further analysis using peptide analogues indicates that the charged residues as well as the hydrophobie residues irnrnediately adjacent to Arg-184 may also play an important role in L 1 homophilic binding. Peptides that inhibited L 1 homophilic binding also inhibited L 1-dependent neurite outgrowth, suggesting a direct link between these two intrinsic activities of L1. I would Iike to take this opportunity to extend my special thanks to my Ph.D. supervisor Dr. Chi-Hung Siu, for his guidance, encouragement, patience and enthusiasm. He taught not only science during my years in his laboratory, but aiso how to be a good scientist. He is the person who put confidence in me, which 1 will be gratefbl in my hiture career. 1 would aIso like to thank my graduate study committee membea, Dr. John Roder and Dr. A. Bennick, for their advice on my project and ail their help dong the way of my graduate training. I also thank them for reading my thesis and giving me vaiuable suggestions. I would like to thank dl members in this laboratory, past or present, for dl their help, their encourage, and their Company. They provided vaiuable advice and discussion for my project, and left me with wondemil memories of rny graduate years. 1 would like to thank my parents for their love and support. They guided me to the road of science, and provided me the strength and courage to go on. I would also like to thank my grandmother, who is the one who always has trusted in me and believed that 1 could have a doctoral degree since I was in elementary school. 1thank the Medial Research Council of Canada for their financial suppon. Finally, 1 would like to thank my dearest husband Qingyang Huang, for his patience when waiting for me in the lab for my late experiments, for his encouragement when I was frustrateci, and most importanily, for his love and support. TABLE OF CONTENTS Page Abstract ......................... ....... .................................................... ............................ 1 Acknowledgments ............................... .., ....................................................... iii Table of Contents ................................................................................................ iv ListofFigures ..................... ... ......................................................................... ...vi List of Tables .................................................... .......... ................ vrii List of Abbreviations ...... ................... ,. ......... ix Chapter 1: Introduction 1. Ce11 Adhesion. ............................................................ ..................... 2 . CeIl Adhesion Molecules ........................................................... 4 III. Cell Adhesion. Molecule L 1 ....................................... - ..-...... ........... 24 IV. Descnpuon of the Project .................................. ..................... 47 Chapter LI: Colocalization of the Homophiiic Binding Site aml the Neuritogenk Activity of the CeII Adhaion Moiecule L1 to its Second Ig-like Domain 1. Introduction ....................................... ,. 67 II. Experimental Procedures................................................................... 70 III, Results ................. ........ ....................................................................... 77 N. Discussion ........... .......................................................................... 85 Chapter III: DifZerenaal Effects of Two Hydrocephalus/MASA Syndrome Mutations on Che Cell Adhesion Molecule LI 1. Introduction ....................... ..., ................................................................ 1 15 II. Experimental Procedures ................ ......... .... ............. ............... 1 17 III. Results .........-..................-....., ....... ........ 120 IV. Discussion .............................................................................................. 124 Chapter N: Identification of the Homophüic Bindiog Site in ~mmunogiobulin-lüreDomain Two of Uie CeII Adhesion Molecule L1 1. Introduction .......... ..... ............................. .-... ... ..................... 142 II. Experimental Procedures.... .. ...... .. ..... ... .. .. .. .. ... .. .. .. 145 . Results ............................................................................................ 150 IV. Discussion .. ..-. .... .... ..... .. ..-..... .... ... ..... ......... .... -..... ...... .. ... ... .... ..... .... .... 157 Chapter V: Conclusions And Futnre Perspectives ........................................... 182 Appendu 1: Two-hybrid Screening for Proteins Interacting With Ceil Adhesion MoIecuie LI 1. Introduction ............................................. ............................................ 189 . Exprimenid Procedures ..................................................................... 191 III . Results ......................... .. ....................................................................... 196 IV . Discussion .................................................................................... 200 Reference ............................................................................................................ 215 Pages Chapter 1: Integrin Subunit Associations and Ligand Specifkities ...................... Schematic Structure of Integrin............................ .... ............................ Schematic Stnicture of Selectin ........................................................ Structural Organization of the Classicd Cadherins ..................... ........ Topology Diagrarns of Ig-iike Domains .............................................. Peptide and Domain Structure of Human Cell Adhesion Molecule Ll Alignment of Ig-like domains of Human L1 ......................... ....... List of Ll Mutations Implicated in Hydrocephalus and MASA syndrome ................................. ... .......................................................... Chapter 11: Construction and expression of GST fusion proteins ...............
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