ASPARTIC PROTEINASES Structure, Function, Biology, and Biomedical Implications

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY

Editorial Board:

NATHAN BACK, State University of New York at Buffalo

IRUN R. COHEN, The Weizmann Institute of Science

DAVID KRITCHEVSKY, Wistar Institute

ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research

RODOLFO PAOLETTI, University of Milan

Recent Volumes in this Series

Volume 357 LACTOFERRIN: Structure and Function

Edited by T. William Hutchens, Sylvia V. Rumball, and Bo Lonnerdal

Volume 358 ACTIN: Biophysics, Biochemistry, and Cell Biology

Edited by James E. Estes and Paul J. Higgins

Volume 359 TAURINE IN HEALTH AND DISEASE

Edited by Ryan J. Huxtable and Dietrich Michalk

Volume 360 ARTERIAL CHEMORECEPTORS: Cell to System

Edited by Ronan G. O'Regan, Philip Nolan, Daniel S. McQueen, and David J. Paterson

Volume 361 OXYGEN TRANSPORT TO TISSUE XVI

Edited by Michael C. Hogan, Odile Mathieu-Costello, David C. Poole, and Peter D . Wagner

Volume 362 ASPARTIC PROTEINASES: Structure, Function, Biology, and Biomedical Implications

Edited by Kenji Takahashi

Volume 363 NEUROCHEMISTRY IN CLINICAL APPLICATION

Edited by Lily C. Tang and Steven J. Tang

Volume 364 DIET AND BREAST CANCER

Edited under the auspices of the American Institute for Cancer Research; Scientific Editor: Elizabeth K. We is burger

Volume 365 MECHANISMS OF LYMPHOCYTE ACTIVATION AND IMMUNE REGULATION V: Molecular Basis of Signal Transduction

Edited by Sudhir Gupta, William E. Paul, Anthony DeFranco, and Roger Perlmutter

A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher.

ASPARTIC PROTEINASES Structure, Function, Biology, and Biomedical Implications

Edited by

Kenji Takahashi The University of Tokyo Tokyo, Japan

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

Llbrar~ of Congr(lss Cat~loglng-l n-PubllcUlon au,

Aspar t IC pro.eln, ses s , rUCture, f unc.IDn, blDlog~ , and blo.ed le.1 l.pl.Catlon. I edIted b~ Ken jl takahashI ,

p. C.. (Advances (n e.porl.anta l . odleln, and biology v . 362)

Proceodlngs of the 5t In.erna • • onal Con f erence on Aspar. IC Pro telnosn, held Sept. 19-24 , 1993 at t he "11110 Museu. of Phl r . ,ceu, lc.1 SCience .nd InduStr~, In K • • ashl •• -eho. Clfu Prefectur e , J>pan.

Incluoes t>lb'logr.phlc.1 refer ences Ind Index.

1. Asplrtlc prote lnues--Congrnsn . I. takahashI . KenJ I .

II. Intorn.t lon.1 CDnf,rence on AspartiC Protelnases 15th 1993 Na i t O Museu' of Ph.ruceutlc.l SCi ence Ind Indust r ~) III . SerIes.

I(lN...M, I. Aspart Ie Prote In>ses--ph~s I 0 )cgv--ecngrusas . .,1 AD5S9 v, 362 1994 I QU 136 A8376 1994 1 DP609.A86A88 1994 612'.01516--dc20 J)'I,LM/DLC fcr Library of Congre ss

Proceedings of the Fifth International Conference on Aspanic Proteinases, he ld September 19--24, 1993, in Gifu, Japan

ISBN 978-1-4613-576 1-2

C 1995 Springer Science+Business Media New Yortc. Originally published by Plenum Press, New York in 1995 Softcover reprint of the hardcover 1st edition 1995

10987654321

All rights reserved

94-43762

'"

No part of this book may be reproduced. stored in a relTieval system, or lTansmittcd in any form or by any means, elec\ronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

ISBN 978-1-4613-5761-2 ISBN 978-1-4615-1871-6 (eBook)DOI 10.1007/978-1-4615-1871-6

This work is dedicated to the loving memory of Tomoko and Matsue Takahashi

PREFACE

The 5th International Conference on Aspartic Proteinases was held on September 19 through 24, 1993, at Naito Museum of Pharmaceutical Science and Industry, Kawashima­cho, Gifu Prefecture, Japan, about 15 miles northwest of Nagoya City. About 100 scientists attended the conference, including 52 from 14 countries outside Japan, and 32 papers were presented by invited speakers, and 58 papers as posters. The purpose of this conference was to present and discuss new information on the structure, function, and biology, and related topics, including biomedical implications, of aspartic proteinases, and this book is a collec­tion of nearly all the papers presented at the meeting.

Aspartic proteinases belong to one of the four major classes of proteinases, the others being serine, cysteine, and metalloproteinases, and are so called since they have two catalytic aspartic acid residues in common in their active sites. Most of them are optimally active at acidic pH, hence the long-used name "acid proteinases," which, indeed, was the major title of the first conference of this series. However, some of them are active at around neutral pH, indicating their physiological roles in a wider range of pH than hitherto considered.

The previous history of this series of international conferences and related additional meetings was summarized in some detail in the preceding book by Ben Dunn (3). The first conference was organized by Jordan Tang and held at Norman, Oklahoma, in 1976. The second conference was organized 8 years later by Vladimir Kostka and held in Prague, Czechoslovakia in 1984. After 4 years, the third conference, organized by Bent Foltmann, was held at Elsinore, Copenhagen, Denmark in 1988. This was followed 2 years later by the fourth conference, organized by Ben Dunn and held at Sonoma County, California, in 1990. The proceedings of the first, the second, and the fourth conferences were published (1-3). In addition, two small meetings (workshops) were held. One was organized by Tom Blundell and John Kay and held at the Birkbeck College, London, England, in 1982, and the other was organized by Michael Samloff, sponsored by Chugai Pharmaceutical Company of Japan, and held in Tokyo, Japan in 1985. Thus, the fifth conference was indeed the first major meeting of this series organized and held in Asia, which further demonstrated that the conference was really international.

On the occasion of the Sonoma conference, several people suggested that Japan might be the candidate for the site ofthe next conference. Through occasional communications and discussions with Ben Dunn and Jordan Tang during 1991 to 1992, I finally decided to organize the fifth conference and to hold it somewhere in Japan in 1993. Although the previous conferences had not been numbered, I dared to use the term "The 5th" for this conference just to demonstrate the continuity of this important series of conferences.

In this book, the papers are arranged, except for some papers, in the order as they were presented at the conference, essentially following the style of the previous volume (3). The presentations were grouped into four categories, i.e., gastric, non-gastric, retroviral, and microbial aspartic proteinases. The grouping is not always very strict, and the latter two groups

vii

viii Preface

are treated separately from the non-gastric proteinases group. Each group is devided into two sections, i.e., plenary lectures and poster reports, corresponding to oral and poster presenta­tions, respectively. As can be seen from the book, significant new advances have been made these few years in various aspects of aspartic proteinases. They include X-ray structural studies on progastricsin, renin, cathepsin D, etc.; molecular dynamics; protein engineering by site-di­rected mutagenesis; and design and chemical syntheses of new inhibitors and even new enzymes. Further, several new aspartic proteinases have been found and characterized, and notably a greater number of papers than before were presented this time on intracellular aspartic proteinases, including cathepsins D and E, novel processing proteinases, several plant protei­nases, plasmodium proteinases, etc., thus expanding further the realm of aspartic proteinases and simultaneously demonstrating their wider range of biological importance. Much attention was also paid from the biomedical point of view to several reports on aspartic proteinases related to diseases, such as renin, HIV and related retrovirus proteinases, Candida proteinases, and malaria parasite proteinase, and on the clinical application of serum pepsinogens. Thus, aspartic proteinases appear to become increasingly important in the biomedical area.

I deeply acknowledge Eisai Company, Ltd., for generously providing us with the beautiful and comfortable site and facilities for the conference and lodging, and thank people in the Eisai Industrial Park and Kawashima Co., Ltd., for their kind and warm accomodations. This also made it possible for all participants to enjoy more intimate academic discussions and communications until late every evening after the sessions. I am especially grateful to Professor Ben Dunn for helpful advice and suggestion about the organization of the conference, selection of speakers and editing of this book, and assistance in raising funds. My sincere thanks also go to Drs. Jordan Tang, John Kay, and Alexander Wlodawer for helpful suggestions and advices about selection of speakers, and Drs. Shin-ichi Ishii, Teruhiko Beppu, and Kazumasa Miki for help in raising funds. I am thankful to all my colleagues, including Drs. Takashi Kageyama, Akihiko Moriyama, Masaru Tanokura, Hideshi Inoue, Tomonari Muramatsu, Masao Ichinose, Shinko Tsukada, and others and to the 13 graduate students in my laboratory, for assistance in the organization and operation of the conference. Drs. H. Inoue and T. Muramatsu especially devoted much time to this work. Further, thanks are due to Mrs. Hisako Kamioka for skillful assistance as a secretary for the conference and for preparation of this book, and Mrs. Masako Taguchi for help with several aspects of the secretary work. I also express my thanks to Mss. Mary Safford and Eileen Bermingham and Messrs. Thomas L. Flood and Brian P. Halm of Plenum Publishing Corporation for helpful suggestions and cooperation for the preparation of this book.

Finally, I express my sincere thanks to International Science Foundation for coop­eration as the coorganizer ofthis conference and to many groups that have provided finantial support for the conference, without which this successful meeting would not have been realized. Their names are listed in a preceding page.

It is hoped that this book will serve as one of the milestones for further advances in studies on this unique class of enzymes.

REFERENCES

Kenji Takahashi Tokyo, Japan

1. "Acid Proteases : Structure, Function, and Biology," Jordan Tang, ed., Advances in Experimental Medicine and Biology, Vol. 95, Plenum Press, New York (1977).

Preface

2. "Aspartic Proteinases and Their Inhibitors: Proceedings of the FEBS Advanced Course No. 84/07," Vladimir Kostka, ed., Walter de Gruyter, Berlin (1985).

3. "Structure and Function of the Aspartic Proteinases. Genetics, Structures, and Mechanisms," Ben M. Dunn ed., Advances in Experimental Medicine and Biology, Vol. 306, Plenum Press, New York (1991).

ix

ACKNOWLEDGMENTS

Financial Support for the Conference was Provided by the Following Sponsors:

Ajinomoto Co., Inc. Applied Biosystems Japan, Inc. DAINABOT Co., Ltd. Eisai Co., Ltd. Funakoshi Co., Ltd. Glaxo Group Research, Ltd. Japan Energy Corporation Kato Memorial Bioscience Foundation Kikkoman Corporation Kirin Brewery Co., Ltd. Meito Sangyo Co., Ltd. NEC Corporation Nippon Chemiphar Co., Ltd. Osaka Pharmaceutical Manufacturers Association Roche Products, Ltd. Seikagaku Corporation, Tokyo Research Institute SmithKlein Beecham Seiyaku K. K. Suntory Institute for Biomedical Research Terumo Life Science Foundation The Asahi Glass Foundation The Kajima Foundation The Mitsubishi Foundation The Naito Foundation The Pharmaceutical Manufacturers' Association of Tokyo The Tokyo Biochemical Research Foundation Werner-Lambert / Parke-Davis Pharmaceutical Research Yakult Honsha Co., Ltd.

xi

CONTENTS

Gastric Aspartic Proteinases: Plenary Lectures

1. Comparison of the Active Site Specificity of the Aspartic Proteinases Based on a Systematic Series of Peptide Substrates ............ . ............... . .

Ben M. Dunn, Paula E. Scarborough, W. Todd Lowther, and Chetana Rao-N aik

2. The Molecular Structure of Human Progastricsin and Its Comparison with that of Porcine Pepsinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11

Michael James, Stanley Moore, Anita Sielecki, Maia Chemaia, and Nadezhda Tarasova

3. A New Way of Looking at Aspartic Proteinase Structures: A Comparison of Pepsin Structure to Other Aspartic Proteinases in the Near Active Site Region. . . .. 19

Natalia S. Andreeva, Alexey Bochkarev, and Igor Pechik

4. Rearranging Pepsinogen and Pepsin by Protein Engineering . . . . . . . . . . . . . . . . . . 33

Xinli Lin and Jordan Tang

5. Comparative Investigations on Pig Gastric Proteases and Their Zymogens .... . . 41

Bent Foltmann, Kenneth Harlow, Gunnar Houen, Peter K. Nielsen, and Per Sangild

6. Non-Mammalian Vertebrate Pepsinogens and Pepsins: Isolation and Characterization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53

Kenji Takahashi, Masao Tanji, Etsuko Yakabe, Akira Hirasawa, Senerath B. P. Athauda, and Takashi Kageyama

7. Transcription Regulation of Human and Porcine Pepsinogen A ............... 67

G. Pals, P.H.S. Meijerink, J. Defize, J.P. Bebelman, M. Strunk, F. Arwert, A. Timmerman, and W.H. Mager

xiii

xiv Contents

Gastric Aspartic Proteinases: Poster Reports

8. A Comparative Study on Amino Acid Sequences of Three Major Isoforms of Human Pepsin A ................................................ 77

Nadezhda Tarasova, Nancy D. Denslow, Benny F. Parten, Nha Tran, Hung P. Nhuyen, Arwyn Jones, Norman B. Roberts, and Ben M. Dunn

9. Molecular Mass Determination by Electrospray Mass Spectrometry of Human Pepsins, Gastricsin, and Porcine Pepsin A Variants .... . . . . . . . . . . . . . . . .. 83

A.T. Jones, B.N. Green, S.P. Wood, and N.B. Roberts

10. Evidence for Electrostatic Interactions in the S2 Sub site of Porcine Pepsin ...... 91

Chetana Rao and Ben M. Dunn

11. Protein Engineering of Surface Loops: Preliminary X-Ray Analysis of the CHY155-165RHI Mutant ......................................... 95

Raj R.Y. Dhanaraj, Jim E. Pitts, Phil Nugent, Poonsook Orprayoon, Jon B. Cooper, Tom L. Blundell, Janna Uusitalo, and Merja Penttilii

12. Seminal Progastricsin ................................................. 101

Pal Bela Szecsi and Hans Lilja

13. Effects of Hydrocortisone on the Pepsinogen-Producing Cells in Rat Stomach Mucosa ........................................................ 107

Masao Ichinose, Shinko Tsukada, Kazumasa Miki, Nobuyuki Kakei, Masashi Matsushima, Naohisa Yahagi, Satoshi Ishihama, Yasuhito Shimizu, Kiyoshi Kurokawa, Hiroshi Fukamachi, Satoshi Yonezawa, Senarath B.P.Athauda, Takashi Kageyama, and Kenji Takahashi

14. Effects ofOmeprazole, a Proton Pump Inhibitor, on Pepsinogen-Producing Cells, with Special Reference to Neonatal Development ...................... 115

Nobuyuki Kakei, Masao Ichinose, Shinko Tsukada, Masae Tatematsu, Noriaki Tezuka, Naohisa Yahagi, Masashi Matsushima, Satoshi Ishihama, Yasuhito Shimizu, Masahiro Kido, Kazumasa Miki, Kiyoshi Kurokawa, Kenji Takahashi, and Hiroshi Fukamachi

15. Transcription of Embryonic Chick Pepsinogen Gene Is Affected by Mesenchymal Signals through Its 5 '-Flanking Region .............................. 125

Kimiko Fukuda, Hidetoshi Saiga, and Sadao Yasugi

16. Serum Pepsinogen Values as Possible Markers for Evaluating the Possibility of Peptic Ulcer Recurrence under HrBlocker Half-Dose Maintenance Therapy .............. . ........................................ 131

Masashi Matsushima, Kazumasa Miki, Masao Ichinose, Nobuyuki Kakei, Naohisa Yahagi, Masahiro Kido, Yasuhito Shimizu, Satoshi Ishihama, Shinko Tsukada, Kiyoshi Kurokawa, and Kenji Takahashi

Contents xv

17. The Clinical Application of the Serum Pepsinogen I and II Levels as a Mass Screening Method for Gastric Cancer ... .. .......... .. .............. 139

Kazumasa Miki, Masao Ichinose, Nobuyuki Kakei, Naohisa Yahagi, Masashi Matsushima, Shinko Tsukada, Satoshi Ishihama, Yasuhito Shimizu, Takehisa Suzuki, Kiyoshi Kurokawa, and Kenji Takahashi

18. A Minute Gastric Cancer Detected by aNew Screening Method Using Serum Pepsinogen I and II .............................................. 145

Naohisa Yahagi, Kazumasa Miki, Masao Ichinose, Nobuyuki Kakei, Masashi Matsushima, Masahiro Kido, Yasuhito Shimizu, Satoshi Ishihama, Shinko Tsukada, Kiyoshi Kurokawa, and Kenji Takahashi

19. Two Cases of Early Colorectal Cancer Associated with Gastric Adenoma Detected by Serum Pepsinogen Screening Method .................. . .. 149

Yasuhito Shimizu, Nobuyuki Kakei, Tomonori Wada, Naohisa Yahagi, Masahiro Kido, Satoshi Ishihama, Shinko Tsukada, Masashi Matsushima, Masao Ichinose, Kazumasa Miki, Kiyoshi Kurokawa, and Kenji Takahashi

Non-Gastric Aspartic Proteinases: Plenary Lectures

20. Comparisons of the Three-Dimensional Structures, Specificities and Glycosylation of Renins, Yeast Proteinase A and Cathepsin D ......... . .. 155

C. F. Aguilar, V. Dhanaraj, K. Guruprasad, C. Dealwis, M. Badasso, J. B. Cooper, S. P. Wood, and T. L. Blundell

21. Discovery ofInhibitors of Human Renin with High Oral Bioavailability ........ 167

Dennis 1. Hoover, Bruce A. Lefker, Robert L. Rosati, Ronald T. Wester, Edward F. Kleinman, Jasjit S. Bindra, William F. Holt, William R. Murphy, Michael L. Mangiapane, Gregory M. Hockel, Ian H. Williams, Ward H. Smith, Michael 1. Gumkowski, Richard M. Shepard, Mark 1. Gardner, and Mark R. Nocerini

22. Structure of Human Cathepsin D: Comparison ofInhibitor Binding and Subdomain Displacement with Other Aspartic Proteases ........ .. .. .... 181

John W. Erickson, Eric T. Baldwin, T. Narayana Bhat, and Sergei Gu1nik

23. Cathepsin D Crystal Structures and Lysosomal Sorting ...................... 193

Peter Metcalf and Martin Fusek

24. Isolation and Characterization of Human Gastric Procathepsin E and Cathepsin E .................................................... 20 I

Senarath B.P. Athauda, Takashi Kageyama, Takayuki Takahashi, Hideshi Inoue, Masao Ichinose, Masanori Ukai, and Kenji Takahashi

xvi Contents

25. Isolation, Characterization, and Structure of Pro cathepsin E and Cathepsin E from the Gastric Mucosa of Guinea Pig ... . .......................... 211

Takashi Kageyama, Masao Ichinose, Kazumasa Miki, Akihiko Moriyama, Satoshi Yonezawa, Masao Tanji, Senarath B. P. Athauda, and Kenji Takahashi

26. Cathepsin E and Cathepsin D: Biosynthesis, Processing, and Subcellular Location 223

Kenji Yamamoto

27. Glycoproteins of the Aspartyl Proteinase Gene Family Secreted by the Developing Placenta .... . ..... . ..... . ... .. . . . . .. . ...... .. .. ... ... 231

R. Michael Roberts, Sancai Xie, Robert J. Nagel, Boon Low, Jonathan Green, and Jean-Fran90is Beckers

28. Structure and Possible Function of Aspartic Proteinases in Barley and Other Plants ..... ... ... .. . . . . . . ... .. .. . . .. .. .. . .... ........... .... . .. 241

Jukka Kervinen, Kirsi Tormakangas, Pia Runeberg-Roos, Kunchur Guruprasad, Tom Blundell, and Teemu H. Teeri

29. Aspartic Proteinases (Cyprosins) from Cynara cardunculus spp.flavescens cv. Cardoon; Purification, Characterisation, and Tissue-Specific Expression .. . 255

P.E. Brodelius, M.e. Cordeiro, and M.S. Pais

Non-Gastric Aspartic Proteinases: Poster Reports

30. Acid-Activation of Rat Prorenin following Non-Proteolytic Alteration ......... 267

F. Suzuki, N. Tanaka, K. Takeuchi, Y. Muramoto, T. Inagami, K. Murakami, and Y. Nakamura

31. Human Procathepsin D: Three-Dimensional Model and Isolation . .. ... . ... .... 273

Gerald Koelsch, Peter Metcalf, Vaclav Vetvicka, and Martin Fusek

32. Identification of Five Molecular Forms of Cathepsin D in Bovine Milk ...... .. . 279

Lotte Bach Larsen and Torben Ellebcek Petersen

33. Site-Directed Mutagenesis of a Disulfide Bridge in Cathepsin D: Expression, Activation, Purification, and Characterization . ... . .. . .... . .......... . . 285

Brian M. Beyer and Ben M. Dunn

34. Expression of Rat Cathepsin D cDNA in Saccharomyces cerevisiae: Intracellular Sorting of Cathepsin D to Yeast Vacuole .. . ....... . .......... . ...... . 289

Yukio Nishimura, Hideaki Fujita, Keitaro Kato, and Masaru Himeno

35. Molecular Cloning and Immunocytochemical Localization of Jasmonic Acid Inducible Cathepsin D Inhibitors from Potato ..... . ... .. . .. .. . . .. .... . 293

Borut Strukelj, Maja Ravnikar, Pika Mesko, Mateja Poljsak-Prijatelj, Jose Pungercar, Gregor Kopitar, Igor Kregar, and Vito Turk

Contents xvii

36. Purification and Characterization of an Acid Proteinase from Dirofilaria immitis Worms ........................................................ 299

Kumiko Sato, Yutaka Nagai, and Mamoru Suzuki

37. Inhibition and Entrapment of Aspartic Proteinases by urMacroglobulin ........ 305

Senarath B.P. Athauda, Hideo Arakawa, Takayuki Takahashi, Masaaki Nishigai, Eiji Ido, Hiroyuki Kyushiki, Yoshiyuki Yoshinaka, Atsushi Ikai, Jordan Tang, Masanori Ukai, and Kenji Takahashi

38. Recombinant Human Cathepsin E ...................................... . 315

Jeffrey Hill, Douglas Montgomery, and John Kay

39. Expression of Human Cathepsin E in Methylotrophic Yeast, Pichia pastoris ..... 319

Masayuki Yamada, Takao Matsuba, Takeshi Azuma, Hideo Suzuki, Kenji Yamamoto, and Hitoshi Hori

40. Purification and Characterization of Recombinant Human Cathepsin E ......... 325

Hiroshi Iida, Takao Matsuba, Masayuki Yamada,Takeshi Azuma, Hideo Suzuki, Kenji Yamamoto, and Hitoshi Hori

41. Comparison of Biochemical Properties of Natural and Recombinant Cathepsin E 331

Takayuki Tsukuba, Yamada Masayuki, Hitoshi Hori, Takeshi Azuma, and Kenji Yamamoto

42. Increased Expression and Specific Localization of Cathepsins E and D in Vulnerable Brain Regions of Aged and Postischemic Rats ............... 335

Hiroshi Nakanishi, Kazuyoshi Tominaga, and Kenji Yamamoto

43. Characterization ofCathepsins E and D Accumulated at Early Stages of Neuronal Damage in Hippocampal Neurons of Rats ............................ 341

Kazuyoshi Tominaga, Hiroshi Nakanishi, Motoyuki Yajima, and Kenji Yamamoto

44. Functional Aspects of Cathepsin E: Is It an Embryonic or Fetal Type of Aspartic Proteinase? ..................................................... 345

Satoshi Yonezawa, Masao Ichinose, Shinko Tsukada, Kazumasa Miki, and Takashi Kageyama

45. Tissue- and Cell-Specific Control of Guinea Pig Cathepsin E Gene Expression .. 349

Shinko Tsukada, Masao Ichinose, Kazumasa Miki, Nobuyuki Kakei, Masashi Matsushima,Naohisa Yahagi, Satoshi Ishihama, Yasuhito Shimizu, Masahiro Kido, Hiroshi Fukamachi, Kiyoshi Kurokawa, Satoshi Yonezawa, Takashi Kageyama, and Kenji Takahashi

46. Cathepsin E Is Expressed in Fetal Rat Glandular Stomach Epithelial Cells in Primary Culture in the Absence of Mesenchymes ..... . . ... .. . ......... 357

Hiroshi Fukamachi, Masao Ichinose, Satoshi Ishihama, Shinko Tsukada, Chie Furihata, Satoshi Yonezawa, and Kazumasa Miki

xviii Contents

47. Cathepsin E Expressed in Pancreatic Cancer .............................. 363

Takeshi Azuma, Masayuki Yamada, Hajime Murakita, Yasuyuki Nishikawa, Yoshihiro Kohli, Kenji Yamamoto, and Hitoshi Hori

48. Plant Aspartic Proteinases from Cynara cardunculus spp.jlavescens cv. Cardoon; Nucleotide Sequence of a cDNA Encoding Cyprosin and Its Organ-Specific Expression .... . ............................... . ... 367

M.C. Cordeiro, Z.-T. Xue, M. Pietrzak, M.S. Pais, and P.E. Brodelius

49. Cardosin A and B, Aspartic Proteases from the Flowers of Cardoon ............ 373

Carlos Faro, Paula Verissimo, Yingzhang Lin, Jordan Tang, and Euclides Pires

Retroviral Aspartic Proteinases: Plenary Lectures

50. Mechanism of Autoprocessing ofa Mini-Precursor of the Aspartic Protease of Human Immunodeficiency Virus Type 1 ............................. 379

Edward Co, Gerald Koelsch, Jean A. Hartsuck, and Jordan Tang

51. Mutants of HIV-l Protease with Enhanced Stability to Autodegradation ........ 387

Alfredo G. Tomasselli, Ana M. Mildner, Donna 1 Rothrock, Jean 1. Sarcich, June Lull, Joseph Leone, and Robert 1. Heinrikson

52. Identification of Amino Acid Residues of the Retroviral Aspartic Proteinases Important for Substrate Specificity and Catalytic Efficiency ..... . .. . .... 399

C. E. Cameron, H. Burstein, D. Bizub-Bender, T. Ridky, I. T. Weber, A. Wlodawer, A. M. Skalka, and J. Leis

53. Inhibitor-Resistant Mutants of the HIV-l Aspartic Protease ........ . .......... 407

Bruce D. Korant

54. Design and Synthesis of HI V Protease Inhibitors Containing Allophenylnorstatine as a Transition-State Mimic ...................... . ................ 413

Yoshiaki Kiso

55. Breaking the Shackles of the Genetic Code: Engineering Retroviral Proteases through Total Chemical Synthesis .................................. 425

Stephen B.H. Kent, Manuel Baca, John Elder, Maria Miller, Raymond Milton, Saskia Milton, lK.M. Rao, and Martina Schnolzer

Retroviral Aspartic Proteinases: Poster Reports

56. X-Ray Structure of a Tethered Dimer for HIV-l Protease . ... . ............... 439

T.N. Bhat, E.T. Baldwin, B. Liu, Y.-S.E. Cheng, and J.w. Erickson

Contents xix

57. Structure of HI V-I Protease with KNI-272: A Transition State Mimetic Inhibitor Containing Allophenyinorstatine ................................... 445

Eric T. Baldwin, T. Narayana Bhat, Sergi Gulnik, Beishan Liu, Yoshiaki Kiso, Hiroaki Mitsuya, and John W. Erickson

58. Molecular Dynamics of HI V- I Protease in Complex with a Difluoroketone-Containing Inhibitor: Implications for the Catalytic Mechanism .. .. .... . .... . ..... . .......... . . .... ....... .. . . ... ... 451

Abelardo M. Silva, Raul E. Cachau, Eric T. Baldwin, Sergei Gulnik, Hing L. Sham, and John W. Erickson

59. Activated Dynamics of Flap Opening in HIV-l Protease ..................... 455

Jack R. Collins, Stanley K. Burt, and J. W. Erickson

60. Computer Simulation and Analysis of the Reaction Pathway for the Decomposition of the Hydrated Peptide Bond in Aspartic Proteases . .. ... . 461

R.E. Cachau, LA. Topol, S.K. Burt, A.M. Silva, and J.W. Erickson

61. Activities of Precursor and Tethered Dimer Forms ofHIV Proteinase . .. ....... 467

Lowri H. Phylip, Jonathan T. Griffiths, John S. Mills, Mary C. Graves, Ben M. Dunn, and John Kay

62. Site-Directed Mutagenesis of HI V-I Protease: Generation of Mutant Proteases with Increased Stability to Autodigestion ..................... . ....... 473

Alfredo G. Tomasselli, Ana M. Mildner, Donna J. Rothrock, Jean L. Sarcich, June Lull, Joseph Leone, and Robert Heinrikson

63 . Molecular Modeling of the Structure of FIV Protease . . . .. ... . .... .. .... .. .. 479

Alia Gustchina

64. Processing, Purification, and Kinetic Characterization of the Gag-Pol Encoded Retroviral Proteinase of Myeloblastosis Associated Virus Expressed in E. coli . . . ..... . . . ... . ..... . . . .. ... .... .. .. .. . . ..... .. .. .. ........ 485

Jifi Brynda, Milan Fabry, Petr J. Tichy, Magda Horejsi, and Juraj Sedilicek

Microbial Aspartic Proteinases: Plenary Lectures

65. Extracellular Aspartic Proteinases from Candida Yeasts . ..... .. ... ... .... . .. 489

Martin Fusek, Elizabeth Smith, and Stephen L Foundling

66. Tyrosine 75 on the Flap Contributes to Enhance Catalytic Efficiency of a Fungal Aspartic Proteinase, Mucor pusillus Pepsin ...... . ..... . ... . .......... 501

Teruhiko Beppu, Young-Nam Park, Jun-ichi Aikawa, Makoto Nishiyama, and Suheharu Horinouchi

67. Aspartic Proteinases from the Human Malaria Parasite Plasmodium Jalciparum .. 511

Colin Berry, John B. Dame, Ben M. Dunn, and John Kay

xx Contents

68 . Yeast and Mammalian Basic Residue-Specific Aspartic Proteases in Prohormone Conversion ................................................... . . 519

Y. Peng Loh, Niamh X. Cawley, Theodore C. Friedman, and Le-Ping Pu

69. Pepstatin-Insensitive Carboxyl Proteinases ............ ... .. . ...... . ....... 529

K. Oda, S. Takahashi, T. Shin, and S. Murao

Microbial Aspartic Proteinases: Poster Reports

70. The Three-Dimensional X-Ray Crystal Structure of the Aspartic Proteinase Native to Trichoderma reesei Complexed with a Renin Inhibitor CP-80794 .543

Jim E. Pitts, Mark D. Crawford, Phil G. Nugent, and Ron T. Wester

71. A Quantum Mechanical Model of the Hydration and Acidity of the Active Site in Aspartic Proteases ...... ..... . . .. .. .... . ........... .... ... . ...... 549

I. A. Topol, R. E. Cachau, S. K. Burt, and J. W. Erickson

72. Site-Directed Mutagenesis of Rhizopuspepsin: An Analysis of Unique Specificity 555

W. Todd Lowther and Ben M. Dunn

73. Site-Directed Mutagenesis Revealed Role of Sub site Residues of Mucor pusillus Pepsin in Catalytic Function . ...................................... 559

Young-Nam Park, Makoto Nishiyama, Sueharu Horinouchi, and Teruhiko Beppu

74. A Novel Intracellular Acid Proteinase from the Plasmodia of a True Slime Mold, Physarum polycephalum . ................. . .............. ... . . .... 565

Kimiko Murakami-Murofushi, Takayuki Takahashi, Hiromu Murofushi, and Kenji Takahashi

75. Characteristics ofYAP3, a New Prohormone Processing Aspartic Protease from S. cerevisiae .. . ................. .. .............. . ... . ........... 569

A.Y. Azaryan, T.C. Friedman, N.X. Cawley, and Y.P. Loh

76. Candida albicans Aspartic Proteinase: cDNA Cloning and Comparison among Strains ........................................................ 573

Somay Yamagata Murayama, Osamu Takeda, Hiroyuki Mukai, Kazutoh Takesako, Eiko Sono, Ikunoshin Kato, and Hideyo Yamaguchi

77. Molecular Cloning and Sequence Analysis ofa Gene Encoding an Aspartic Proteinase from Aspergillus oryzae ................................. 577

M. Takeuchi, K. Ogura, T. Hamamoto, and Y. Kobayashi

78. Aspergillus niger var. macrosporus Proteinase B. cDNA Cloning, Expression, and Activation of the Proenzyme . . ..... . ..... . ...... . ... .. .... .. . .. .... 581

Hideshi Inoue, Jing-Fang Lu, Senarath B. P. Athauda, Kwang-Hoon Kong, Takaharu Hayashi, Takao Kimura, Osamu Makabe, and Kenji Takahashi

Contents xxi

79. Expression and Secretion of Recombinant Aspartic Proteinases by Bacillus brevis 589

Takaharu Hayashi, Hideshi Inoue, Masashi Kato, Shigezo Udaka, and Kenji Takahashi

80. Expression in E.coli of Aspergillus niger var. macros porus Proteinase A, a Non-Pepsin Type Acid Proteinase ................................... 597

Naofumi Kagami, Hideshi Inoue, Takao Kimura, Osamu Makabe, and Kenji Takahashi

81. X-Ray Crystallographic Study of a Non-Pepsin-Type Acid Proteinase, Aspergillus niger Proteinase A ............................................... 605

Hiroshi Sasaki, Masaru Tanokura, Tomonari Muramatsu, Atsushi Nakagawa, So Iwata, Toru Hamaya, Toshio Takizawa, Toshiaki Kono, and Kenji Takahashi

82. Conformation Analysis of Non-Peps in-Type Acid Proteinase A from the Fungus Aspergillus niger by NMR ................... .. .............. ... .. 611

Masaki Kojima, Masaru Tanokura, Yutaka Muto, Hiroshi Miyano, Ei-ichiro Suzuki, Toru Hamaya, Toshio Takizawa, Toshiaki Kono, and Kenji Takahashi

Author Index .................................. .. ....................... 617

Subject Index ........................................................... 621

COLOR PLATES

C hapter I, Dunn t! al.: Figurt 1. Ribbon diagram representatioll of the stru<:1Ure of all archetypal Ispanie proteinase. The N-termillal domain is 011 the right ill this view. The side chaills of the two Catal)1ic residues are represented by red stick struC1Ures, alld the two oxygen atoms arc illdicaled as ~all dcr Waals surfaces, seen as red dots. The positions of tWO imponallt Glu residues ill porcille pepsill (13 alld 281) are also given by while Slick struclures, with the oxygen atoms of these illdicated as white ~an der Waals surfnces.

Chapler 21, HOO"er el al.: Figurt 2. Predicted bindillg of a morpholine-containing illhibitor to humall renill.

Inhibitor CP-S I ,2&1 (Mor -CO-PiIt· Nle· DifluoroC·StatoncNH Me, endOlhiaptpsin-bound X ·ray coordinates [30]), modelled illio the X-ray coordinates of recombinant human renin (from the reninICP-85,339 crystal structure [31]. View shows the solvent-accessible (Connoly) surface of renin (green) and the $OlvclltoCxposcd N-terminus of the illhibitor (yellow. with morpholinc oxygen in red). The N-tenninus of II"Ic inhibitor is solvellt-cxposed.

•

, •

• • • '. • • . . .. .' .. ". ', '" . ". -,,\ .... ..

" ~ .......... a . . . ".... ..... . ,,; :" ., " .~:;':-... "; ";." . '-, '. '. ,. ,." "

' •• , ~ ' I ' ~ .. ",. , .. . "'" ",." ... ~ ": ~....--.: ;}Ii-'·: . ~ ... .. ".' ~ "" . ." ... . .. '.. .. . " . ,. .. ... .. . : : ',: ", : '::. - :'.:",-0 ,'::' :,: '

.' ... '.' ~ ........ , ...... e'. .. ..... ,. . ... . . ". " ".. .... • ... .e.. ~.' .. ,," ... • •• , ' •. - I,et·,"" .. '.... ./.. .' .. .. ... • '... t ...... _. .. ..• " .. ,.# .", ••. ",. ..."'.... .. .. '.. .. .. '-.~.

Chapter I, Dunn et al.: FIgure 4. Representation oflhe acidic (AsP/Glu) and hydrophobic residue distribution within the active site clefts of (A) porcine pepsin and (8) human cathepsin D. Asp and Glu side chains are colored red in these space-filling diagrams, Hydrophobic residues are colored purple, and hydrophobic residues (e.g., Ser, Thr, Asn, Gin) are C(llored green. Residues outside the active site cleft are shown as cyan spheres. Both structures are oriented allhe same angle.