Table Of ContentASPARTIC
PROTEINASES
Physiology and Pathology
Martin Fusek
Vaclav Vetvicka
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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
