XDENTIFYING DOMAINS of SHIGA-Lim TOXIN 1 THAT
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XDENTIFYING DOMAINS OF SHIGA-LIm TOXIN 1 THAT ARE RESPONSIBLE FOR ITS MEMBRANE TRANSLOCATION by Mazen T. Saleh A Thesis Submitted in Conformity with the Requirements for the Degre of Doctor of Philosophy, Graduate Department of Medical Biophysics, in the University of Toronto Ocopyright by Mazen T. Saleh. 1997 National Library Bibliothéque nationale I*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, nie Wellington Ottawa ON KIA ON4 OttawaON K1AOW canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettaot à 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 microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othexwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT Identifyùig domains of shiga-like toxin 1 that are responsible for its membrane translocation Mazen T. Saleh Docmr of Philosophy.1997, Graduate Department of Medical Biophysics, in the University of Toronto Shiga-like toxin 1 (SLT-1).produced by pathogrnic strains of Escherchia coli. is a representative mernber of a superfamily of multimeric protein toxios known as the A/B family of toxins. SLT-1 is intemalized by cells via receptor-mediated endocytosis and localizes in various intracellular compartments. However, targets of these toxins are invariably Iocated in the cytoplasm. thus internalized SLT-I rnust translocate across a vesicular membrane in order to deliver its cytotoxic function. Earlier studies have revealed that toxins may exploit conditions in intracellular environments to facilitate translocation. For example, acidic pH of endocytic vesicles might dlow exposure of hydrophobic domains that associate with membranes, a necessary initial step in membrane translocation. SLT-1 was used to determine if this proposed mechanism was a general feature of all members of this family of toxins in tems of their mode of membrane insertion and uansIocation. Fluorescence spectroscopy has revealed that Ttp-34 in SLT-1 B subunit is perturbed at pH lcvels nomally found in endocytic and lysosomal vesicles. This effect was further characterized using circular dichroism. solid-phase receptor binding assays and gel permeation chromatography. The results support the conclusion that the çonformauond change observed with fluorescence studies is a local stmctunl effect and that the secondaiy and tertiary structures of the B subunit remain unaltered during a pH transition between pH 7 and pH 4. Moreover, studies carried out with the holotoxin indicated that the smcture of the toxh rernains largely unaffected by acidic pH in relation to neuual pH. The effects of other intraceUular microenvironments on SLT-1 translocation were investigated. Hydropathy profües of the A subunits of SLT-I revealed the presence of a hydrophobic domain between residues 220 and 246 flanked by positively charged amino acids that resembles the composition of signal sequences and trammembrane domains of integral membrane proteins. Studies with synthetic vesicles containing acidic lipids, such as phosphatidyl glycerol. have shown that synthrtic peptides corresponding to this region of the A 1 chain, can spontaneously insert into membrane vesicles. Our hypothesis is that this region may function in a similar way within the intact Al chah once localized in the ER. It is therefore concluded that acidic pH alone is not capable of eliciting a structurai change in internalized SLT-1. Rather. other intracellular processes, such as proteolytic processing and disulfide reduction, may be involved in exposing the hydrophobic C- terminus of the Al chain. The exposed domain may then insert into the lumenal side of the endoplasmic reticulum membrane as an initial step in membrane translocation. Furthexmore. factors endogenous to the ER. such as those involved in signal-sequence processing, protein folding and degradation, may play a role in the translocation of the A chah of SLT-1 across the ER membrane. ... lll ACKNO WLEDGMENTS This body of work is the result of the collective efforts of many individuals. I am grateful for the efforts and contributions of my supervisor, Dr. Jean Gariepy. The present frame of my scient& thinking was moulded by his relentless pursuit of excellence in reseuch. His patience provided me with the independence in the laboratory that 1 needed and his guidance ailowed me to successfully complete of my work 1 also wish to thank members of my student supervisory cornmittee, Dr. David Rose and Dr. Cheryl Arrowsmith for their concise encouragement, support and helpful discussions; my pst and present partners in the laboratory for making my stay stimulating and pleasant both at work and during evening discussions in bars. Without their efforts, 1 would have probably saved more money and my liver would be in a better state of health. They are : Beth Boyd, Bruce Carpick, Jim Ferguson, Francesca Bahr, Rajiv Ayra, Deming Liu, Kate Sheldon. Eric Lacasse, Mark Bray, Devender Singh. Reza Kiarasch, Wai-May Lim, Stuart Bisland, Kim Kawarnura, Ray Reilly and Nancy Stokoe, and a special thanks to John Simard. 1 also iake this opportunity to thank our collaborators, Dr. Ioan Boggs and Godha Rangaraj (University of Toronto), for the EPR work presented in chapter 4; Dr. Robert Hodges and Mr. Lome Burke (Department of Biochemistry, University of Alberta) for perfoming rnass spectrometry on the purified peptides and, Dr. Avi Chakrabartty for his help and access to his circular dichroism spectrometer and fluorometer. Finally. I would like to acknowledge Ms. Tanuja Chitnis and Dr. David Isenman for their help in the early stages of the work presented in chapter 1 as well as Drs. Penelope Stein and Randall Read for providing us with the crystûl coordinates for the B subunit of Shiga-like toxin 1. TABLE OF CONTENTS List of Tables List of Figures List of Abbreviations CHAPTER 1 Introduction .............................................................................. 1.1 The Role of Shiga-like Toxins in Disease ...................................... 1.2 The Structure of Shiga-like Toxin 1 ............................................. 1.2.1 The Smcture and Function of Shiga -1ike Toxin 1 A subunit ....... 1.2.2 The Structure of Shiga-like Toxin 1 B subunit ........................ 1.3 The Receptor of Shiga-like Toxin 1 .............................................. 1.4 Receptor-Mediated Endocytosis of Bound Toxin ............................. 1.5 Stnictural and Functional Homologies with other Toxins .................... 1.6 Goals and Objectives .............................................................. CHAPTER 2 Experimzntal Methods 2.1 Biochernicd Methods Purification of SLT-1 .................................................. Purification of SLT-1 B subunit ...................................... Purification of the synthetic peptides .............................. .. EIectroelution of SLT-1 A subunit .................................. Radiolabeling of SLT-1 and SLT-1 B subunit ....................... Gb3 solid phase binding assay ........................................ 2.1.7 Spin-labeling of ShTA(220-246) ..................................... 2.1.8 Preparation of lipid vesicles ........................................... 2.1.9 Binding of radiolabeled peptide to lipid vesicles .................... 2.1.10 Total Phosphorus Assay ............................................... 2.1.1 1 Amino acid analysis .................................................... 2.2 Characterization of Purified SLT-1 and its B subunit 2.2.1 Polyacrylamide gel electrophoresis ................................... 2.2.2 Western blot analysis ................................................... 2.2.3 Cytotoxicity assay ...................................................... 2.2.4 Cell-free protein synthesis inhibition assay .......................... 2.2.5 Gel permeaiion c hromatography ...................................... 2.2.6 Calculation of the molar absorption coefficient of SLT-I .......... 2.3 Immunological Methods 2.3.1 Generation of rabbit Anti-SLT-1 A subunit antisera ............... 2.3.2 Detection of rabbit anti-SLT-1 antibodies by ELISA ............... 2.4 Biophysical Methods 2.4.1 Fluorescence Spec troscopy ............................................ 2.4.2 Circular Dichroism ............................. .. ...........*...... 2.4.3 EPR measurements ..................................................... CHAPTER 3 Local Conformational Change in the B subunit of Shiga-like Toxin I at Endosomal pH 3.1 Introduction ........................................................................ 55 3.2 ResuIts and Discussion ..................................................... 59 3.2-1 The fluorescence of uyptophan-34 is perturbed at endosomal pH .......................... ........ ...-......-..-.-.... 3.2.2 The structure of the B-subunit is graduaily altered by pH conditions ranging from pH 2 to 4.5 ............................. 3.2.3 The tertiary