ASPARTIC PROTEINASES Physiology and Pathology Martin Fusek Vaclav Vetvicka Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRCPress Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2019 by CRC Press © 1995 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. 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Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. A Library of Congress record exists under LC control number: Publisher's Note The publisher has gone to great lengths to ensure the quality oft his reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-0-367-20058-9 (hbk) ISBN 13: 978-0-429-26039-1 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com The authors wish to dedicate this book to the memory of Ivo Fusek, MD THE AUTHORS Martin Fusek, Ph.D. is a member of the Department of Biochemistry of the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences in Prague, Czech Republic. He graduated in 1984 from the Prague Polytechnique Institute with B.Sc (Honours) degree in organic chemistry and then obtained his Ph.D. degree in biochemistry in 1988 from the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences in Prague. Dr. Fusek spent three years (1988 - 1989 and 1992 - 1994) in the laboratory of Professor J. Tang in the Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma. During the period 1992 - 1994 he was a member of the laboratory of protein crystallography. In 1991 Dr. Fusek joined, for one year, the laboratory of Dr. P. Metcalf at the European Molecular Biology Laboratory in Heidelberg, Germany. Dr. Fusek is a member of the Czech Chemical Society and the Czech Biochemical Society. He has published more then 40 research papers and review articles. His current research is connected to the involvement of aspartic proteinases in human physiology and pathology. His work is focused mainly on the role of cathepsin D in human cancer and on the function of aspartic proteinases of Candida yeasts. Vaclav Vëtvicka, Ph.D. is an Assistant Professor at the Department of Pathology, Division of Experimental Immunology and Immunopathology of the School of Medicine, University of Louisville, Louisville, Kentucky. Dr. Vetviõka graduated in 1978 from Charles University in Prague, Czech Republic with a doctorate degree in biology and obtained his Ph.D. degree in 1983 from the Czechoslovak Academy of Sciences, Institute of Microbiology, Prague. He is a member of the Czech Immunological Society, American Association of Immunologists, and the International Society of Developmental and Comparative Immunology. In 1984, he was awarded the Distinguished Young Scientist Award of the Czechoslovak Academy of Sciences. During 1984 to 1985 and 1988 he spent 18 months as a Research Associate in Professor Kincade's laboratory in the Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma. He is the author or co author of more than 130 original papers, several review articles, and three patents. He is a co-author of the books Evolution of Immune Reactions and Immunology of Annelids and co-editor of the books Immunological Disorders in Mice and Immune System Accessory Cells, all recently published by CRC Press, Inc. Dr. Vètvioka's current major research interests include the role of macrophages in regulation of the immune responses, the role of complement receptors in NK cytotoxicity, and the phylogenic aspects of defense reactions. PREFACE Aspartic proteinases are enzymes important for many aspects of human life, from physiologic functions of digestive enzymes to participation of Plasmodium specific proteases on patho-physiology of malaria and from renin blood pressure control to processing of viral polyproteins by HIV specific proteinases. In addition, several industrial processes also use aspartic proteinases, such as in the production of cheese where bovine chymosin is the key element. In all these events and in many others, aspartic proteinases play a crucial role. The information gathered over the years of research on aspartic proteinases represents a large data set, and it is difficult to get a condensed overview of the field. The example of human renin can be used to explain the diversity of approaches to aspartic proteinases. From the point of view of human physiology, renin is an important enzyme which participates in processing of angiotensinogen. Cellular biology may see renin as a model for studies of cellular sorting and secretion. Biochemists must be puzzled by its extreme specificity. A similar description can be done for all aspartic proteinases. In our book we tried to select and bring together the information on enzymes which participate in processes important for human physiology and/or patho-physiology. The book is for every reader seeking prompt information on these enzymes without preliminary knowledge of the field. We did not include methodological comments on the discussed proteases since a specialized book appeared in the series of Methods in Enzymology. We hope that our book will be a useful tool in further research of aspartic proteinases. We would like to express our deep gratitude to all authors who kindly supplied their original photographs used in our book. Finally, we would like to conclude with a major acknowledgment to our wives Nina and Jane, for their never ending support and forbearance, sympathy, and understanding. Louisville - Prague April 1995 TABLE OF CONTENTS CHAPTER I Introduction 1 References 4 CHAPTER II Aspartic Proteinases - an Overview I. Aspartic Proteinases 7 A. Family of Aspartic Proteinases - Characteristic Properties ....7 1. LowpH of Action 7 2. Inhibitors Specific for Aspartic Proteinase 8 3. Sequences of Aspartic Proteinases 10 4. The Members of Aspartic Proteinase Family 11 B. Evolution of Aspartic Proteinases - Their Primary, Secondary, and Tertiary Structures 15 1. Primary Structures 15 2. Secondary and Tertiary Structures 17 a. Symmetry of N- and C-Terminal Lobes of Eucaryotic Aspartic Proteinases as a Consequence of Evolution of Aspartic Proteinases by Gene Duplication 17 3. Three-Dimensional Structure of the Active Site of Aspartic Proteinases 22 C. Catalytic Mechanism of Aspartic Proteinases 24 D. Specificity of the Proteinase Action 26 E. Inhibition of Aspartic Proteinases 29 1. Low Molecular Weight Inhibitors 29 2. High Molecular Weight Protein Inhibitors of Aspartic Proteinases 32 F. Propeptides of Aspartic Proteinases and Activation of Zymogens 32 1. Activation of Aspartic Proteinases 33 2. Involvement of Propeptides in Folding and Sorting of Zymogens 35 G. Concluding Remarks 35 References 36 CHAPTER III Pepsin A and Gastricsin and their Zymogens I. Introduction 53 A. Nomenclature 53 II. Localization of Pepsin A and Gastricsin and their Physiological Role 54 A. Localization of Pepsinogen A and Progastricsin in the Human Body 54 B. Intracellular Synthesis of Pepsinogen A and Progastricsin 59 C. Secretion of Pepsinogen A and Progastricsin 60 D. Activation of Zymogens 62 E. Age Dependent Changes in Gastric Enzymes 62 F. Seminal Progastricsin 63 III. Sequence and Expression 63 A. Sequences - Pepsinogen A and Progastricsin 63 B. Polymorphism of Pepsinogen A and Progastricsin 65 1. Pepsinogen A 65 2. Progastricsin 66 IV. Three-Dimensional Structure 67 A. Overall Shape - Pepsinogen A 68 B. Phosphoserine 68 70 C. Active Site 70 D. Comparison of Pepsin and Pepsinogen Structures 70 V. Proteolytic Activity 72 VI. Pepsin A and Gastricsin and Patho-Physiological Conditions 74 A. Determination of Serum Pepsinogen and Gastricsin 74 B. Normal Levels 75 C. Serum Pepsinogen A and Progastricsin and Patho physiological States 75 1. Ulcer Disease 76 2. Gastritis 76 3. Gastric Cancer 77 4. Helicobacter pylori and Gastric Proteinases 78 VII. Concluding Remarks 78 References 79 CHAPTER IV Renin I. Introduction 101 II. Localization, Secretion and Physiological Function of Renin 102 A. Localization of Renin andProrenin 103 1. Localization of Renin by Kidney. 103 2. Production of Prorenin by Tissues other than Kidney 104 B. Mammalian Secretory Pathways and Regulation of Renin Secretion 106 1. Morphology of Renin Secretion 107 2. Regulation of Renin Secretion 107 C. The Proteolytic Cleavage of Angiotensinogen - Basic Physiological Function of Renin 108 III. Sequence and Posttranslational Modifications 109 A. Gene Structure 110 B. Primary Structure 110 C. The Posttranslational Modifications 110 IV. Three-Dimensional Structure 112 A. Overall Shape 112 B. Active Site 113 V. Specificity of Renin Catalysis 115 VI. The Clinical Potential of Renin Inhibition 116 A. Introduction 116 B. Renin Specific Antibodies 117 C. Synthetic Renin Inhibitors 118 D. Pharmacology of Renin Inhibition 120 VII. Concluding Remarks 123 References 124 CHAPTER V Cathepsin D I. Introduction 143 II. Physiological Functions 143 A. Intracellular Catabolism 143 B. Processing of Antigens and Peptide Hormones 144 C. Tissue Remodeling 148 III. Sequence and Regulation of Expression 149 A. Sequence of Cathepsin D 149 B. Regulation of the Expression 151 IV. Cellular Targeting, Processing, and Localization of Cathepsin D 152 A. Targeting of Cathepsin D to the Lysosomes 152 B. Posttranslational Modifications of Cathepsin D 155 C. Localization of Cathepsin D 157 1. Endosomal and Lysosomal Localization 157 2. Attachment to Intracellular Membranes 157 3. Extracellular Localization 160 V. Three-Dimensional Structure 161 A. Overall Shape, Processed Loop 161 B. Targeting, Recognition Signal 162 C. The Active Site 164 VI. Catalytic Properties 164 A. Characterization of Subsite Preferences 166 VII. Patho-Physiological Involvement of Cathepsin D 169 A. Involvement of Cathepsin D in Neurodegenerative Diseases 169 B. Involvement of Cathepsin D in Neoplastic Diseases 170