Table Of ContentContributors to Volume 23
Article numbers are in parentheses following the names of contributors. Affiliations listed are current.
DRAHCIR G. ALLEN (11), Center of Research on Occupational and Environ-
mental Toxicology, Oregon Health Sciences University, Portland, Oregon
10279
H. ILLERAB (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Cen-
tre National de la Recherche Scientifique, 06560 Valbonne, France
KaY SENRAB (20), Department of Biochemistry and Molecular Biology,
University of Leeds, Leeds LS2 9JT, United Kingdom
K. ~IEHTJtERYEB (18), Center for Molecular Biology, University of Heidel-
berg, D-6900 Heidelberg, Germany
HaMaoi ATTESSUOB (9), Biochimie des Signaux R6gulateurs Cellulaires et
Mol6culaires, Universit6 Pierre et Marie Curie, F-75006 Paris, France
NACNUD J. ~UEBVMAC (19), St. Vincent's Institute of Medical Research,
Fitzroy, Victoria 3065, Australia
SIEW YEEN IAI-IC (22), Department of Medicine, University of Melbourne,
Austin Hospital, Melbourne, Victoria 3048, Australia
F. trELCEI-IC (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Cen-
tre National de la Recherche Scientifique, 06560 Valbonne, France
LUAP NEHOC (9), Biochimie des Signaux R6gulateurs Cellulaires et Mol6cu-
laires, Universit6 Pierre et Marie Curie, F-75006 Paris, France
~rEVw'aS R. YBSORC (8), School of Biomolecular Sciences, Liverpool John
Moores University, Liverpool L3 3AF, United Kingdom
P. Dauci-i (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Centre
National de la Recherche Scientifique, 06560 Valbonne, France
TREBOR YAD (2), Laboratory of Biochemical Neuroendocrinology, Clinical
Research Institute of Montreal, Montreal, Quebec, Canada H2W 7R1
V. DIw (21), CEN de Saclay, 91191 Gif s/s Yvette, France
EEtrAM-NNA ~rACNUD (19), St. Vincent's Institute of Medical Research,
Fitzroy, Victoria 3065, Australia
xi
CONTRIBUTORS OT VOLUME 32
DYOLL D. REKCIRF (13), Department of Molecular Pharmacology, Albert
Einstein College of Medicine, Bronx, New York 16401
S. J. FULLER (18), Department of Pathology, University of Melbourne,
Parkville, Victoria 3052, Australia
DLORAH RENIAG (10), Laboratory of Neurochemistry, National Institute of
Neurological Disorders and Strokes, National Institutes of Health, Be-
thesda, Maryland 20892
NOSILA M. NOSBIG (16), Medical Research Council, Neurochemical Pathol-
ogy Unit, Newcastle General Hospital, Newcastle Upon Tyne NE4 6BE,
United Kingdom
CRAM J. NAMSKCULG (17), Fishberg Research Center in Neurobiology,
Mount Sinai School of Medicine, New York, New York 92001
OADAS ARUMIK (14), Center for Biomedical Science, School of Medicine,
Chiba University, Chiba 260, Japan
ANEHTA SIDALK (19), St. Vincent's Institute of Medical Research, Fitzroy,
Victoria 3065, Australia
ENNA C. ECNERWAL (19), Department of Biology, Medawar Building, Uni-
versity College London, London WC1E 6BT, United Kingdom
ACCEBER A. WEL ,7( 12), Peptide Biology Laboratory, Baker Medical Re-
search Institute, Prahran, Victoria 3181, Australia
SIRI GREBDNIL (5), Department of Biochemistry and Molecular Biology,
Louisiana State University Medical Center, New Orleans, Louisiana 70112
KRAM O. YLEVIL (10), Department of Biochemistry, Bowman Gray School
of Medicine, Wake Forest University, Winston-Salem, North Carolina
75172
DRAHCIR E. SNIAM (6), Department of Neuroscience, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 50212
C. L. SRETSAM (18), Department of Pathology, University of Melbourne,
Parkville, Victoria 3052, Australia
Y. OUSAM (21), Takeda Chemical Industries, Ltd., Pharmaceutical Group,
Tsukuda 300-42, Japan
NHOJ R. TNOMREDCM (16), Medical Research Council, Neurochemical Pa-
thology Unit, Newcastle General Hospital, Newcastle Upon Tyne NE4
6BE, United Kingdom
CONTRIBUTORS TO VOLUME 32 xi
KCIREDERF A. O. NHOSLEDNEM (22), Department of Medicine, University
of Melbourne, Austin Hospital, Melbourne, Victoria 3048, Australia
ANAIRAM SIRROM (10), Department of Physiology and Pharmacology, Bow-
man Gray School of Medicine, Wake Forest University, Winston-Salem,
North Carolina 75172
TERRY J. OPGENORTH (14), Aging and Degenerate Disease Research, Abbott
Laboratories, Abbott Park, Illinois 46006
NHOJ E. RATNIP (3), Department of Neuroscience and Cell Biology, Univer-
sity of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medi-
cal School, Piscataway, New Jersey 45880
G. DEER (18), Department of Pathology, University of Melbourne, Parkville,
Victoria 3052, Australia
DEMAHOM MALOHR (9), Biochimie des Signaux R6gulateurs Cellulaires et
Mol6culaires, Universit6 Pierre et Marie Curie, F-75006 Paris, France
SEMAJ L. STREBOR (17), Fishberg Research Center in Neurobiology, Mount
Sinai School of Medicine, New York, New York 92001
NITRAM K.-H. ,g,HCS REF (2), Department of Anatomy and Cell Biology,
Phillips University of Marburg, D-35037 Marburg, Germany
LIBAN G. SEIDAH (1), Laboratory of Biochemical Neuroendocrinology,
Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W
7R1
RUHTRA SEKLUHS (15), Department of Surgery, University of Melbourne,
Melbourne, Victoria 3084, Australia
D. H. LLAMS (18), Department of Pathology, University of Melbourne,
Parkville, Victoria 3052, Australia
A. NAI HTIMS ,7( 12), Peptide Biology Laboratory, Baker Medical Research
Institute, Prahran, Victoria 813 l, Australia
ENNAILUJ KCATS (11), The Vollum Institute, Oregon Health Sciences Uni-
versity, Portland, Oregon 10279
YRAG SAMOHT (4), Vollum Institute, Oregon Health Sciences University,
Portland, Oregon 10279
LERUAL SAMOHT (4), Vollum Institute, Oregon Health Sciences University,
Portland, Oregon 10279
Xll ~176 CONTRIBUTORS TO VOLUME 32
YNOHTNA J. RENRUT (20), Department of Biochemistry and Molecular Biol-
ogy, University of Leeds, Leeds LS2 9JT, United Kingdom
YDUJ K. EKYLSNAV (4), Vollum Institute, Oregon Health Sciences Univer-
sity, Portland, Oregon 10279
B. TNECNIV (21), Institut de Pharmacologie Mol6culaire et Cellulaire, Cen-
tre National de la Recherche Scientifique, 06560 Valbonne, France
J. P. TNECNIV (21), Institut de Pharmacologie Mol6culaire et Cellulaire,
Centre National de la Recherche Scientifique, 06560 Valbonne, France
A. NNAMEDIEW (18), Center for Molecular Biology, University of Heidel-
berg, D-6900 Heidelberg, Germany
NUYHSNIJ R. Wu-WON6 (14), Aging and Degenerative Diseases Research,
Abbott Laboratories, Abbott Park, Illinois 60064
NIM GNEHZ (3), Department of Anatomy and Cell Biology, Columbia Uni-
versity College of Physicians and Surgeons, New York, New York 23001
YI ZHOU (5), Department of Biochemistry and Molecular Biology, Louisi-
ana State University Medical Center, New Orleans, Louisiana 70112
Preface
The generation of bioactive peptides from inactive precursor molecules
involves a series of highly ordered, enzyme-mediated processing events.
The posttranslational modifications can occur within the cell at the point of
secretion or postsecretion. The precise pattern of processing for any given
precursor also can vary, depending on the site of expression and/or stage of
development, reflecting the differential expression of processing enzymes.
The last decade or so has seen the characterization of both peptide products
and the majority of the processing enzymes involved in their production,
thus facilitating the development of various biochemical, immunological,
and molecular probes necessary to characterize these complex pathways in
greater detail.
The aim of this volume in the Methods in Neurosciences series is to
describe in a very practical way the "state-of-the-art" technology being
developed and applied in the field of peptidases and neuropeptide process-
ing. It is divided into three sections. The first, "Molecular Approaches for
the Study of Intracellular Processing Enzymes," covers strategies for the
molecular characterization of processing enzymes, including cloning, ex-
pression, localization by in situ hybridization, and the use of antisense
mRNA to block enzyme expression. The second, "Immunological and Bio-
chemical Approaches to the Study of Peptide-Processing Pathways," de-
scribes the combination of more classical approaches such as immunoas-
says, HPLC, and the use of specifically modified substrates to characterize
both the precise pattern of peptide products in a given tissue and the regula-
tion and distribution of the enzymes involved in their generation. Finally, the
last section, "Identification and Characterization of Extracellular Process-
ing Enzymes in the Central Nervous System," is designed to provide an
insight into, as well as strategies for, the investigation of this exciting and
developing area in which extracellular enzymes can generate, modulate, or
terminate peptide signals in the central nervous system.
In this book, like others in the series, the authors have been encouraged to
provide chapters that reflect the latest techniques being developed in their
laboratories, with their own specific scientific interests providing the practi-
cal application. Each chapter provides sufficient detail to allow the experi-
mental procedures to be easily duplicated, although, for practical reasons,
lengthy operating procedures for common laboratory equipment have been
omitted. Absolute conditions for any given experiment are inevitably deter-
mined empirically; however, it is hoped that this volume will provide both
iiix
xiv PREFACE
the student and experienced researcher a valuable starting point in develop-
ing strategies for the study of peptidases and neuropeptide processing.
I would like to express my appreciation to the Baker Medical Research
Institute for supporting the production of this work. Appreciation is also
expressed to my fellow authors for the high standard of their contributions
and for meeting their deadlines.
A. IAN SMITH
Methods in Neurosciences
Volume 1 Gene Probes
Edited by P. Michael Conn
Volume 2 Cell Culture
Edited by P. Michael Conn
Volume 3 Quantitative and Qualitative Microscopy
Edited by P. Michael Conn
Volume 4 Electrophysiology and Microinjection
Edited by P. Michael Conn
Volume 5 Neuropeptide Technology: Gene Expression and Neuropeptide Receptors
Edited by P. Michael Conn
Volume 6 Neuropeptide Technology: Synthesis, Assay, Purification, and Processing
Edited by P. Michael Conn
Volume 7 Lesions and Transplantation
Edited by P. Michael Conn
Volume 8 Neurotoxins
Edited by P. Michael Conn
Volume 9 Gene Expression in Neural Tissues
Edited by P. Michael Conn
Volume 01 Computers and Computations in the Neurosciences
Edited by P. Michael Conn
Volume 11 Receptors: Model Systems and Specific Receptors
Edited by P. Michael Conn
Volume 21 Receptors: Molecular Biology, Receptor Subclasses, Localization, and Ligand Design
Edited by P. Michael Conn
Volume 31 Neuropeptide Analogs, Conjugates, and Fragments
Edited by P. Michael Conn
Volume 41 Paradigms for the Study of Behavior
Edited by P. Michael Conn
Volume 51 Photoreceptor Cells
Edited by Paul A. Hargrave
Volume 61 Neurobiology of Cytokines (Part )A
Edited by Errol B. De Souza
Volume 71 Neurobiology of Cytokines (Part )B
Edited by Errol B. De Souza
Volume 81 Lipid Metabolism in Signaling Systems
Edited by John N. Fain
Volume 91 Ion Channels of Excitable Membranes
Edited by Toshio Narahashi
VX
xvi VOLUMES IN SERIES
Volume 02 Pulsatility in Neuroendocrine Systems
Edited by Jon E. Levine
Volume 12 Providing Pharmacological Access to the Brain: Alternate Approaches
Edited by Thomas R. Flanagan, Dwaine F. Emerich, and Shelley R. Winn
Volume 22 Neurobiology of Steroids
Edited by E. Ronald deKloet and Win Sutanto
Volume 32 Peptidases and Neuropeptide Processing
Edited by A. Ian Smith
Volume 42 Neuroimmunology (in preparation)
Edited by M. Ian Phillips and Dwight E. Evans
Volume 52 Receptor Molecular Biology (in preparation)
Edited by Stuart C. Sealfon
Volume 62 PCR in Neuroscience (in preparation)
Edited by Gobinda Sarkar
1 Molecular Strategies for Identifying
Processing Enzymes
Nabil G. Seidah
Introduction
In the early 1960s it was proposed that polypeptide hormones are first synthe-
sized as inactive precursors that require specific cleavage after pairs of basic
residues (such as LysArg-, ArgArg-, LysLys-, and ArgLys-) in order to
release the active hormone. Since then this model has been extended to
other precursors, as it is also applicable to progrowth factors, proneuro-
trophic factors, hormonal receptors, adhesion molecules, retroviral surface
glycoproteins, proenzymes, and even certain protoxins. The elaboration of
the structures of many precursors as well as their biosynthetic products also
revealed that processing C terminal to single basic residues such as Arg-
(and less frequently Lys-) as well as after multiple basic residues (three or
more) occurs in about 20% of the processed sites utilized in vivo. Therefore,
it was of great interest to identify the proteinase(s) responsible for such
proprotein processing and to define whether cleavage after monobasic resi-
dues and C terminal to pairs of basic residues was performed by the same
enzyme(s). The search for the physiologically important processing enzymes,
termed "proprotein convertases" or "PCs," was laborious and a number
of laboratories, including our own, participated actively in this hunt (1). The
major breakthrough came in 1984, with the molecular identification of the
convertase responsible for the activation of the yeast a-mating factor and
killer toxin. The proteinase identified by genetic complementation of a KEX2
mutant strain was found to be a subtilisin-like serine proteinase (2, )3 and
is now called "kexin." The search for the mammalian counterpart of kexin
took about 5 years, before it was realized by computer database searches for
sequence identity to kexin that a partial human genomic sequence encoding a
protein called furin had already been reported by Roebroek et al. in 1986 (4).
In the reported DNA sequence only the active site serine and the catalytically
important asparagine residue found in all subtilisin-like proteases were identi-
fied. The complete sequence of the 5' end of the gene was completed in 1989
and it comprised the other two active site residues, aspartate and histidine (5).
Discovery of 1CP and PC2
Alignment of the amino acid sequences of furin and kexin within their cata-
lytic domains revealed a number of segments exhibiting a high degree of
Methods in Neurosciences, Volume 23
Copyright (cid:14)9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
4 I MOLECULAR APPROACHES
hFurin SGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFD 174
(cid:12)9 Z .... J:ll" I "-'Jl I:l''J'llJ:: ~:'11 :J s :::lZl
yKexin PSFPGSDINVLDLWYNNITGAGVV~~D_CzLDYENEDLKDNFCAEGSWD 196
hFurin VNDQDPDPEPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGV 224
(cid:12)9 II.. I.II :.I: IIIIIIII:II .I..IIIII:III:I:I:
yKexin FNDNTNLPKPR. . . LSDDYHGTRCAGE IAAKKGNNFCGVGVGYNAKI SG I 243
@ AS @
hFurin RMLDGEVTDAVDARSLGLNPNH I H I YSASWGPDDDGKTVHGPARLAE EAF 274
I'I.I-'I.. I: II .. (cid:12)9 .III.IIII.III-"II. I...I:
yKexin RI LSGD I TTEDEAASL IYGLDVND IYS CSWGPADDGRHLQGPSDLVKKAL 293
II o~
hFurin FRGVSQGRGGLGS I FVWASGNGGREHDSCNCDGYTNS IYTLS I SSATQFG 324
(cid:12)9 :II.,II'-I.I'I'IIIIII I.II-IIIIIIII.:.I'..: (cid:12)9
yKexin VKGVTEGRDS KGAI YVFASGNGGTRGDNCNYDGYTNS I YS I T I GAI DHKD 343
(.-m I (..
hFurin NVPWYSEACSSTLATTYSSGNQNEKQ IVTTDLRQKCTESHTGTSASAPLA 374
I III'II..'I.IIIII .'. I ..I.'..'I.-II.IIII.IIII
yKexin LHPPYSEGCSAVMAVTYSSG.. SGEYIHSSDINGRCSNSHGGTSAAAPLA 391
.GIF 1 Alignment of the amino acid sequences of human furin and yeast kexin within
the catalytic domain. The active site residues Asp", His", and Ser a are emphasized,
as well as the catalytically important Asn e. The sense )S( and antisense (AS) oligonu-
cleotides used to identify human 2CP are shown, as well as the primers I( and II)
that were first used to identify mouse 1CP and PC2.1, Identical sequence; ,: highly
similar ni sequence.
sequence identity (Fig. .)1 In 1989, the partial sequence of furin (from the
catalytically important Asn* up to the C terminus) )4( and the full sequence
ofkexin )3( were known. Accordingly, on the basis of the concept of sequence
conservation around the active sites of serine proteinases, polymerase chain
reaction (PCR) amplification of mRNA (reverse transcriptase-PCR or RT-
PCR) allowed two laboratories simultaneously to isolate for the first time
other mammalian homologs of kexin, known as PC1 (6, )7 and PC2 (6, 8),
representing the first endocrine and neuroendocrine processing enzymes
molecularly characterized in mammalian tissues. Polymerase chain reaction
amplification of a cDNA synthesized from human insulinoma total RNA,
using degenerate oligonucleotides encoding the consensus sequence sur-
rounding the active site residues Asp" (oligo S; Fig. )1 and His" (oligo AS;
Fig. )1 in kexin and related subtilisins, gave a 150-bp probe. The latter was
used to screen a human insulinoma library and to isolate a full-length cDNA
encoding a novel convertase called PC2 (8). Independently, PCR applied
to cDNA obtained from mouse pituitary total RNA using oligonucleotides
encoding the sequence around the catalytically important Asn* and the active
site Ser u of human furin (oligos I and II, Fig. ,)1 allowed the isolation of a
260-bp probe (6, 7). Screening mouse pituitary and mouse insulinoma libraries
