urn u Ottawa L'Universitd canadienne Canada's university FACULTE DES ETUDES SUPERIEURES l^^l FACULTY OF GRADUATE AND ET POSTDOCTORALES u Ottawa POSTDOCTORAL STUDIES I.'University emiadienne Canada's university Charles Gyamera-Acheampong AUTEUR DE LA THESE / AUTHOR OF THESIS Ph.D. (Biochemistry) GRADE/DEGREE Biochemistry, Microbiology and Immunology FACULTE, ECOLE, DEPARTEMENT / FACULTY, SCHOOL, DEPARTMENT The Physiology and Biochemistry of the Fertility Enzyme Proprotein Convertase Subtilisin/Kexin Type 4 TITRE DE LA THESE / TITLE OF THESIS M. Mbikay TIRECTWRTDIRICTR^ CO-DIRECTEUR (CO-DIRECTRICE) DE LA THESE / THESIS CO-SUPERVISOR EXAMINATEURS (EXAMINATRICES) DE LA THESE/THESIS EXAMINERS A. Basak G. Cooke F .Kan V. Mezl Gary W. Slater Le Doyen de la Faculte des etudes superieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral Studies Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-59504-6 Our file Notre reference ISBN: 978-0-494-59504-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extra its substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada THE PHYSIOLOGY AND BIOCHEMISTRY OF THE FERTILITY ENZYME PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 4 Charles Gyamera-Acheampong Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry Department of Biochemistry, Microbiology, and Immunology Faculty of Medicine University of Ottawa January 2009 © Charles Gyamera-Acheampong, Ottawa, Canada, 2009 DEDICATION This thesis is dedicated to my deceased daughter, Harriet Ama Serwaa ii ABSTRACT This three-manuscript thesis focuses on the physiology and biochemistry of the fertility enzyme, Proprotein Convertase Subtilisin-Kexin type 4 (PCSK4). Efforts to develop effective, safe, and reversible methods of non-hormonal contraception especially for men have focussed either on sperm surface proteins involved in the functional maturation of sperm or on those responsible for sperm-egg interactions. PCSK4 is one of such proteins. It is a member of a family of serine endoproteinases involved in the proteolytic maturation of a wide array of inactive precursor proteins to their bioactive forms. It is primarily expressed in the gonads, and inactivation of its gene in mice causes male infertility and female subfertility. The first paper dealt with the reproductive function of PCSK4 by studying its subcellular localisation in testicular epithelium and on intact sperm, as well as its relevance for sperm acquisition of fertilising ability. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. Sperm from PCSK4-null mice underwent capacitation at a faster rate, they were induced to acrosome-react by lower concentrations of zona pellucida, and possessed egg-binding ability which was only half that of wildtype sperm. The second paper explored the molecular basis of the ability of PCSK4-null sperm to undergo capacitation at a much faster rate than WT sperm, as well as their reduced egg- binding ability. It focussed primarily on sperm protein tyrosine phosphorylation and the proteolytic processing of the sperm-egg ligands ADAM2 and ADAM3. During sperm capacitation, proteins undergo more tyrosine phosphorylation and more ADAM2 proteolytic processing in PCSK4-null sperm than in WT sperm. Thus, alterations in signal transduction iii and proteolytic processing during capacitation may underlie the fertilisation incompetence of PCSK4-null sperm. The third paper investigated the biosynthesis, maturation, and transport of PCSK4. Mouse PCSK4 was identified to be tightly bound through hydrophobic interactions to water- insoluble or detergent-soluble components of the plasma membrane overlying the acrosome. Also human proPCSK4, cloned from human embryonic kidney cells (HEK293), is slightly converted into its active mature form, and it is probably retained inside the ER where it associates with Glucose-regulated protein 78/Immunogen Binding Protein (GRP78/BiP). iv ACKNOWLEDGEMENTS I would like to express my profound gratitude to my supervisor, Dr. Majambu Mbikay, for his immense contribution towards the completion of this thesis. He devoted his time and energy, as well as sought for financial support for almost the entire duration of the program. I am also grateful to the remaining members of my thesis advisory committee: Dr. Mary Alice Hefford and Dr. Fraser Scott, for their comments and suggestions which have improved the quality of this thesis. In particular, I am indebted to Dr. Mary Alice Hefford for the time she took, amidst a very tight schedule, in editing the second draft of the thesis. Worthy of appreciation are Drs. Gunther Schmidt, Ajoy Basak, Janice Mayne, Ms. Francine Sirois, Mrs. Adrianna Gambarotta, Mr. Andrew Chen, and Ms. Nathalie Scamuffa for their valuable contributions. I am very grateful to Natural Sciences and Engineering Research Council of Canada for providing the financial support needed to carry out this study. I would like to extend my profound appreciation to my fellow graduate students in the Department of Biochemistry, Microbiology, and Immunology for the friendship and interactions we shared. I will particularly like to mention Hongbin Xu. My thanks further go to the support staff of BMI and the Chronic Disease Program of the Ottawa Hospital: Ms. Carol Ann Kelly, Ms. Nicole Trudel, Mrs. JoAnn McDonald, and Ms. Denise Joanisse for their extreme patience in dealing with all the needs and requests of graduate students. I would never forget the support and prayers of Pastor Andrew Aryee, Church/family members, friends, and loved ones both far and near. May the Lord richly bless you all for the great interest you showed in my studies. Last but not the least, to Mrs. Gyamera-Acheampong A., I say, a million thanks. As if having to support me spiritually, emotionally, and financially were not enough, you endured v the long hours I had to stay away from home so as to be in the laboratory. You might not be aware, but I want you to know that your love and encouragement contributed immensely to the successful completion of this thesis. I cannot, within the page limitations, thank everyone that has made a contribution to this thesis, but I do acknowledge in my heart, all contributions, both great and small. vi TABLE OF CONTENTS DEDICATION ii ABSTRACT iii ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF ABBREVIATIONS xi LIST OF FIGURES xv LIST OF TABLES xviii I. INTRODUCTION 1 1.1. NEED TO CONTROL WORLD POPULATION 1 1.2. HORMONAL CONTRACEPTION 3 1.2.1. Mode of action and efficacy of hormonal contraceptives 4 1.2.2. Side effects of hormonal contraception 4 1.3. NON-HORMONAL CONTRACEPTION 6 1.3.1. Utilised by women 6 1.3.2. Utilised by men 6 1.3.2.1. Vasectomy 7 1.3.2.2. Condoms and withdrawals 8 1.3.3. Experimental chemical contraceptives 8 1.3.3.1. Gossypol 8 1.3.3.2. NB-DNJ 10 1.3.4. Experimental immunocontraception 12 1.3.4.1. Anti-sperm antibodies 12 1.3.4.2. Search for contraceptive immunogens 13 1.3.4.3. Eppin 18 1.3.4.4. Recent progress in immunocontraception research 18 1.3.4.2.1. Fertilin a (ADAM1) 19 vii 1.3.4.2.2. Fertilin p (ADAM2) 20 1.3.4.2.3. Cyritestin (ADAM3) 20 1.3.4.2.4. Hyaluronidase (PH-20) 21 1.3.4.2.5. Lactate dehydrogenase C4 (LDH-C4) 21 1.3.4.2.6. Sperm Antigen 10 (SP-10) 22 1.3.4.2.7. Fertilisation Antigen-1 (FA-1) 22 1.3.4.2.8. Rabbit Sperm Autoantigens family (RSA family) 23 1.4. SPERM FUNCTIONAL MATURATION AND INTERACTION WITH EGGS 23 1.4.1. Protein phosphorylation 23 1.4.2. Sperm capacitation 24 1.4.3. Sperm-egg interaction 25 1.5. BIOCHEMISTRY AND BIOLOGY OF PCSK4 27 1.5.1. Limited endoproteolysis as a regulatory mechanism 27 1.5.2. Proprotein convertases 27 1.5.2.1. The PCSK family 27 1.5.2.2. PCSK functional domains 28 1.5.2.3. PCSK cleavage specificities 31 1.5.3. PCSK4: A Gonadal Convertase 32 1.5.3.1. Structure, expression, and activity 32 1.5.3.2.