1
REVIEW
Subtilase-like pro-protein convertases: from molecular
specificity to therapeutic applications
F Bergeron, R Leduc and R Day
Département de Pharmacologie, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke,
3001, 12e Avenue Nord Sherbrooke, Québec, Canada J1H 5N4
(Requests for offprints should be addressed to R Day; Email: rday@courrier.usherb.ca)
ABSTRACT
Limited proteolysis of most large protein precursors
is carried out in vivo by the subtilisin-like
pro-protein convertases. Many important biological
processes such as peptide hormone synthesis, viral
protein processing and receptor maturation involve
proteolytic processing by these enzymes, making
them potential targets for the development of novel
therapeutic agents. However, the efficient develop-
ment of such molecules requires a better under-
standing of the molecular mechanisms of proteolytic
protein processing. Herein, we review the most
recent findings on the molecular aspects of
subtilisin-like convertase activity, such as the
INTRODUCTION
Proteolytic processing is a post-translational
modification by which the cell can diversify and
regulate the products of its genes. Just as a single
polycistronic bacterial gene can encode many
proteins with different functions, a mammalian
precursor protein can give rise, by hydrolysis of
selective peptide bonds, to several molecules with
different biological activities. Proteolytic processing
within the secretory pathway is crucial for the
activation or inactivation of many proteins and the
regulation of their cellular localization. Indeed,
processing is important in zymogen activation,
peptide hormone processing, complement acti-
vation, clot formation and lysis, angiogenesis and
tissue remodeling. Many precursors involved in
these important biological functions require cleav-
age at specific pairs of basic residues, such a
Lys-Arg. In mammalian species, this catalytic
function is now understood to be carried out by a
Journal of Molecular Endocrinology (2000) 24, 1–22
0952–5041/00/024–001  2000 Society for Endocrinology Printed in Great Britain
structural analysis of the proteases, the mechanisms
of enzyme/substrate specificity, their interaction
with other proteins such as 7B2, and the compara-
tive tissue and cellular distribution of the enzymes
and their substrates. These data are then used as a
background for the review of the known biological
functions of subtilisin-like pro-protein convertases,
the reported clinical cases involving proteolytic
processing defects and, finally, the ongoing develop-
ment of new therapeutic inhibitor molecules based
on this knowledge.
Journal of Molecular Endocrinology (2000) 24, 1–22
family of subtilisin-related pro-protein convertases,
known as the SPCs.
The discovery of the SPCs was based on initial
observations made in the mid-80s using yeast
genetics. A mutation in the Kex2 gene in
Saccharomyces cerevisiae prevented conjugation
because it did not permit proteolytic processing of
the alpha-mating factor precursor. Genetic com-
plementation with Kex2 revealed a deficiency in the
expression of a peptidase with specificity for
cleaving on the carboxyl (C) side of paired basic
residues (Julius et al. 1984). The Kex2 peptidase,
now known as kexin, is a serine protease of the
subtilisin family (Wells et al. 1983, Mizuno et al.
1988) that has been shown to be calcium-dependent
(Fuller et al. 1989a, Mizuno et al. 1989) and to
cleave correctly the pro-hormone precursors in
mammalian cells (Thomas et al. 1988). It was thus
logical to assume that a mammalian homolog could
exist. Shortly after the discovery of the yeast
enzyme, a sequence homology analysis (Fuller et al.
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2   and others · SPCs: molecular specificity to therapeutic applications
1989b) through the human genome database
identified a mammalian Kex2 homolog that was
encoded by the fur gene, on chromosome 15 (van
den Ouweland et al. 1989, 1990). Subsequently,
seven kexin-related mammalian enzymes were
identified. Although each enzyme has been
independently named by its discoverers, a simplified
nomenclature for the group of mammalian
processing proteases has been proposed (Chan et al.
1992), using the term SPC: SPC1 (furin or paired
amino acid converting enzyme (PACE)), SPC2
(PC2), SPC3 (PC1 or PC3), SPC4 (PACE4), SPC5
(PC4) SPC6 (PC5 or PC6-A) and SPC7 (LPC, PC7
or PC8).
The large number of mammalian SPCs now
discovered raises several intriguing questions as to
their potential overlapping or distinct functions.
Functional specificity could be determined by 1) a
high degree of substrate specificity by each enzyme,
and 2) a distinct cellular expression or specific
intracellular localization of each SPC. The future
development and successful application of SPC
inhibitors as potential therapeutic agents is highly
dependent on the availablility of this information.
Within the context of the intracellular environment,
will SPC inhibitors have to be designed that target
all SPC activities simultaneously, or will inhibitors
that target specific SPC activities be more effective?
Thus the knowledge gained from defining whether
SPCs have redundant or distinct functions, at the
level either of expression or of activity, will be
extremely useful. One approach to this problem is
to define the cellular localization of SPCs – that is,
to identify precisely the ‘cocktail’ of SPCs expressed
in any cell of interest. Indeed, this is an important
criterion, as the specificity of processing function is
not only dictated by the cleavage specificity of an
SPC, but also by its distinct localization. A second
approach is to define better the cleavage specificity
of each SPC and to define how SPCs are regulated,
for example at different levels including transcrip-
tional, translational, and through protein–protein
interactions (e.g. SPC2/7B2, see later). Finally,
the ability to ‘delete’ a specific SPC from the
cellular environment, such as has been accom-
plished with the creation of null mice, is especially
useful to define the unique functions of that SPC
and the possible compensatory contributions of
other co-localized SPCs.
MECHANISM OF SPC PRO-PROTEIN
PROCESSING
In general, SPCs appear to be highly specific
enzymes, cleaving pro-protein precursors at specific
Journal of Molecular Endocrinology (2000) 24, 1–22
positively charged amino acids, usually to produce
biologically active products. Other serine proteases,
such as trypsin or chymotrypsin, are far less
selective, often playing a role in protein degra-
dation. Peptide processing events occur in the
secretory pathway of eukaryotes at the carboxyl
terminal of Lys or Arg residues (occupying the P1
position). (The cleavage site of proteases is
described according to a nomenclature proposed by
Schechter & Berger (1967), whereby P1 is the first
residue on the amino (N)-terminal side of the
cleaved peptide bond, P2 the second residue, and so
on. The amino acids located on the C-terminal side
of the cleavage site are named P1 , P2 , P3 , etc.)
Typically, SPC cleavage sites feature Arg-Arg and
Lys-Arg motifs, but some are composed of
Arg-Lys, Lys-Lys, or even single Arg residues.
The substrate specificity of SPC1 has been the
object of numerous studies and is the best
understood, revealing the consensus recognition
sequence Arg-Xaa-Arg/Lys-Arg?, of which the P4
arginine is critical for cleavage (Hosaka et al. 1991,
Molloy et al. 1999). Analysis of a repertoire of more
than 40 precursor molecules cleaved at furin sites
also showed that 50% of the pro-proteins possessed
a serine residue in the P1 position. The mimi-
mal recognition sequence, Arg-Xaa-Xaa-Arg?, is
observed in all substrates found to be cleaved by
SPC1. This sequence was later confirmed in studies
using random substrate phage display. In these
experiments, virtually all clones coding for SPC1
substrates had an RxxR? motif, and many had Lys,
Arg, or Pro in the P2 position (Matthews et al.
1994a). The presence of a P4 Arg residue is not
mandatory for the activity of all convertases but,
generally, the presence of an Arg residue in P6, P4
or P2 has been shown to enhance the cleavage
efficiency by SPCs (Hosaka et al. 1991).
The general features of intracellular precursor
processing are shown schematically in Fig. 1.
Precursor processing can occur in both the
constitutive and the regulated pathways. It is
believed that SPC1 is important in the cleavage of
precursors in the constitutive pathway, whereas
SPC2 and SPC3 are important in the cleavage of
precursors in the regulated pathway. After SPC
cleavage, the newly exposed basic residues of the
released N-terminal peptide are removed by
carboxypeptidases specific for basic residues
(Kemmler et al. 1973). Recently, it has been shown
that this role can be carried out, not only by
carboxypeptidase E (CPE), but also by a structural
homolog known as carboxypeptidase D (CPD) (Xin
et al. 1997). CPE has been shown to be targeted to
secretory granules and appears to play this role in a
relatively endocrine-specific manner (Fricker 1991).
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 SPCs: molecular specificity to therapeutic applications ·   and others
 1. Processing of inactive precursors into bioactive peptides in constitutive
and regulated secretory pathways.
In contrast, CPD appears to have a role in
processing of proteins negotiating the constitutive
secretory pathway (Dong et al. 1999). Finally, after
removal of the basic residues, peptides having a
C-terminal glycine are often amidated by the action
of peptidyl-glycine-
-amidating mono-oxygenase.
This modification enhances the binding of neuro-
endocrine peptides to their receptor and increases
peptide stability in vivo (Mains et al. 1990).
Interestingly, the steps of this enzymatic cascade
involving the SPCs and carboxypeptidases appear to
be functionally linked. It has been observed that
carboxypeptidase activity enhances SPC2 process-
ing of pro-dynorphin, suggesting that the removal
of C-terminal basic residues prevents product
inhibition (Day et al. 1998). Hence, CPE activity
could play a regulatory role on SPC activity in vivo.
This notion is supported by studies on CPE-null
mice (fat/fat), which show generally reduced SPC
processing activity that could be due to the
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3
accumulation of C-terminally extended peptides
(Naggert et al. 1995).
STRUCTURAL ANALYSIS OF SPC ENZYMES
Substrate specificity, catalytic efficiency and intra-
cellular localization of each SPC are conferred by
the unique structural components of each molecule.
All the members of this enzymatic family have
similar N-terminal structures with common func-
tional domains, whereas their C-terminal domains
are variable. The more conserved N-terminal
domains are the signal peptide, the pro-segment,
the catalytic domain and the P domain, which
includes a conserved RGD motif (Fig. 2). The more
variable C-terminal domains include Cys-rich
regions, transmembrane and cytosolic domains,
amphipathic helices, and sorting domains.
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4   and others · SPCs: molecular specificity to therapeutic applications

 2. Structural comparison of the mammalian SPCs and their related yeast kexin (yKexin) and
bacterial subtilisin (Subt) BPN proteases.

Like other protein precursors, SPCs are first
synthesized as large inactive pro-proteins that
undergo proteolytic maturation in their transit
through the secretory pathway. The first domain to
be removed is the N-terminal signal peptide that is
required for the entry of the protein into the
secretory pathway. This peptide is cleaved by a
signal peptidase after the translocation of the
nascent polypeptide chain through the endoplasmic
Journal of Molecular Endocrinology (2000) 24, 1–22
reticulum (ER) membrane. The second processing
event is the removal of the 80–90 amino acid
pro-segment. This domain has two important
functions: as an intramolecular chaperone and as a
competitive inhibitor. Study of the bacterial
subtilisin model and other pro-proteins suggests
that N-terminal pro-peptides act as intramolecular
chaperones and are essential for proper folding of
the proteins to which they are attached (Eder et al.
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 SPCs: molecular specificity to therapeutic applications ·   and others
1993, Shinde & Inouye 1993). It has been
demonstrated that these pro-peptides are cleaved by
an intramolecular autocatalytic mechanism at the
ER level (Leduc et al. 1992, Creemers et al. 1993b,
Goodman & Gorman 1994, Matthews et al. 1994b,
Lamango et al. 1999), and this cleavage is important
for the correct sorting of the enzymes out of the ER
(Creemers et al. 1995, Zhou et al. 1995). Two
clusters of pairs of basic residues are found in the
pro-segment of each convertase. The excision of the
pro-peptide in the ER occurs at the C-terminal site,
for which the consensus sequence is Arg-Xaa-Arg/
Lys-Arg?. After this first cleavage, the pro-segment
most probably occupies the active site, acting as a
competitive inhibitor until it is released. In the case
of SPC1, a correct calcium and pH environment
found in the trans-Golgi network (TGN) enables
cleavage at the N-terminally located pair of basic
residues of the pro-peptide, thus releasing it from
the catalytic domain and activating the enzyme
(Anderson et al. 1997). Purified SPC3 pro-peptides
have been shown to inhibit SPC3 and SPC1 activity
in vitro (Boudreault et al. 1998), thus confirming the
role of the pro-peptide regulatory mechanism of
SPC activity. It is likely that this two-step
pro-segment processing is a common feature of all
SPCs. In most cases (with the exception of SPC2),
the first cleavage is an early event that is necessary
for the efficient folding and the sorting of the
protease in its appropriate cell compartment. The
second cleavage occurs later, when the enzyme has
reached its target compartment, where optimal
conditions of pH, Ca2+ concentration and inter-
actions with other proteins are found. The necessity
of a second cleavage for pro-peptide release insures
that the enzyme becomes activated only after
reaching its target cellular compartment.
The catalytic domain contains the active site of
the enzyme, with the typical catalytic triad of
subtilisin-related serine proteases, including the
Asp, His and Ser active site residues. SPCs use the
same enzymatic mechanism (i.e. the same catalytic
triad) as the trypsin-related enzymes, although no
sequence homologies exist between these two
families. Hence, SPCs are believed to have arisen
from a convergent evolutionary mechanism. Under-
standing the conformation of the catalytic domain of
SPCs is important, because this structure is
primarily responsible for substrate selectivity.
Although efforts are in progress to obtain three-
dimensional structures of SPCs, the only catalytic
domain model available at present is based on the
structure of bacterial subtilisin (Siezen et al. 1994,
Lipkind et al. 1995). Furthermore, various residues
that play a role in the substrate recognition of SPCs
have been identified by mutational analysis confirm-
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5
ing that negatively charged residues of the catalytic
pocket interact with positively charged residues of
the substrate (Creemers et al. 1993b, Roebroek et al.
1994, Rockwell & Fuller 1998). Interestingly, SPC
active site residues can be engineered to modify
substrate specificity (Rheinnecker et al. 1993), as is
the case, for example with furilisin (Ballinger et al.
1996), a bacterial subtilisin mutant that has been
re-designed to induce a substrate recognition
similar to that of mammalian SPC1. The active site
of SPCs also contains a conserved Asn residue
needed for the stabilization of the oxyanion hole
transiently formed during the hydrolysis reaction.
Curiously, SPC2 differs from the other SPCs, as the
Asn residue is replaced by an Asp. The significance
of this special feature of SPC2 is not clear, as an
Asp<Gln mutation at this residue has been
reported to produce minor effects on SPC2
activation and processing activity (Zhou et al. 1995).
Other studies report that the Asp residue could be
involved in the characteristic slow activation of
SPC2 and its acidic pH optimum (Scougall et al.
1998).
The P domain (also called HomoB domain) is
located C-terminally to the catalytic region of all
SPCs and is essential for the correct folding and the
stability of the enzyme. As the ternary structure of
SPCs is not yet understood, the exact conformation
of this particular domain remains hypothetical.
However, it has been proposed, on the basis of
secondary structure predictions, that the P domain
is an independently folded component consisting of
eight-stranded beta-barrels with well-organized
inner hydrophobic cores (Lipkind et al. 1998). This
compact subunit is believed to be essential for the
structural cohesion of SPCs, by establishing strong
hydrophobic interactions with the catalytic domain.
Mutational analysis and chimeric constructions in
the P domain of SPC3 demonstrate the importance
of this region in regulating the stability, calcium
dependence, and optimal pH of these enzymes
(Zhou et al. 1998). The importance of this domain
for the structural cohesion of the enzyme is also
demonstrated in human colon carcinoma LoVo
cells, in which a single nucleotide deletion in the
region covering the P domain of SPC1 completely
abolishes its activity (Takahashi et al. 1993, 1995b).
The P domain of SPCs also contains a conserved
RGD motif, the role of which remains unclear.
RGD motifs are typically found in extracellular
matrix proteins, such as fibrinogen, and are
involved in cellular adhesion via integrins. How-
ever, there is at present no evidence that the RGD
motif in SPCs is involved with integrin attachment,
either intracellularly or extracellularly (Seidah et al.
1994, Rovere et al. 1999). Mutational analysis of the
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6   and others · SPCs: molecular specificity to therapeutic applications
RGD motif in SPC3 produces confusing results,
with effects on substrate processing (pro-
opiomelanocortin, POMC), precursor processing
(pro-segment removal and C-terminal cleavage),
enzyme stability and intracellular sorting (Lusson
et al. 1997). From these same studies, it also appears
that modification of the P domain (i.e., within the
RGD motif) probably results in a structural
destabilization that could explain all other effects
observed. Other experiments with kexin P domain
mutants support the notion that such modifications
can have drastic effects on enzyme trafficking,
resulting in a failure to exit from the ER
(Gluschankof & Fuller 1994).
The regions located C-terminal to the P domain
are highly variable for each convertase. For
example, cysteine-rich regions are found in SPC1
(van de Ven et al. 1990), SPC4 (Kiefer et al. 1991),
and SPC6 (Lusson et al. 1993). The isoform of
SPC6, known as SPC6-B (see Fig. 2) has a
particularly extended Cys-rich region. Interest-
ingly, this domain has some homology with receptor
protein tyrosine kinase precursors. However, few
studies have examined the role of this domain,
which remains unknown. In contrast, the
C-terminal domain of SPC7 displays a unique
serine/threonine-rich region of unknown function
(Bruzzaniti et al. 1996, Meerabux et al. 1996,
Seidah et al. 1996c), reminiscent of the yeast Kex2
enzyme.
Other regions in the C-terminal part of SPCs
have been identified for which the functions are
better understood, such as those regions that
interact with lipidic membranes. Some SPCs
(SPC1, SPC7 and the variant isoforms, SPC4E and
SPC6B) have an integral transmembrane domain
followed by a cytoplasmic tail. This domain affects
cellular sorting and has been well studied for SPC1
(Molloy et al. 1999). As a constitutive pathway-
associated protease, SPC1 is a resident TGN
protein (Shapiro et al. 1997) that can enter a
recycling pathway through the plasma membrane
and the endosomes (Molloy et al. 1994). This
particular sorting pattern is made possible by its
transmembrane domain and the interactions of its
C-terminal tail with cytoplasmic proteins. An acidic
amino acid cluster important for cell sorting has
been identified within the SPC1 56 amino acid
cytosolic domain (Schafer et al. 1995). This motif
contains phosphorylated serine residues (Takahashi
et al. 1995a) that regulate the SPC1 intracellular
traffic by casein kinase II (Jones et al. 1995).
Dephosphorylation is carried out by PP2A phos-
phatase (Molloy et al. 1998). When phosphorylated,
the SPC1 cytoplasmic tail interacts with phospho-
furin acidic cluster sorting protein 1, a novel sorting
Journal of Molecular Endocrinology (2000) 24, 1–22
protein related to clathrin-associated molecules
(Wan et al. 1998). Other important cytoplasmic tail
sorting signals have been identified, namely a
tyrosine-based motif important for the interaction
with AP-1 Golgi-specific assembly proteins
(Teuchert et al. 1999) and, recently, hydrophobic
based motifs that could mediate internalization
(Stroh et al. 1999).
Other SPCs, such as SPC3 and SPC2, do not
have an integral membrane domain, but are believed
to have an amphiphatic helix that has been
hypothesized to mediate hydrophobic interactions
with membranes (Seidah et al. 1990). However, an
SPC2 deletion mutant lacking the putative
C-terminal amphipathic helix was nevertheless
membrane associated, suggesting that this domain
was not essential for attachment of SPC2 to
membranes (Shennan et al. 1991).
A COMPLEX CASE: SPC2 BIOSYNTHESIS
As for other SPCs, during its biosynthesis, SPC2
proceeds through a series of intermediate forms
(Guest et al. 1992, Lamango et al. 1999). After
pro-peptide removal by an autocatalytic mechanism
(Matthews et al. 1994b), the enzyme is further
processed by a truncation of its C-terminal domain
and glycosylation to yield a final product of 64 kDa
SPC2 (Shen et al. 1993). It is noteworthy that SPC2
and SPC3 pro-segment removal occurs at different
pH and calcium requirements, suggesting different
intracellular locations for these events (Shennan et
al. 1995). Finally, SPC2 is sorted to the regulated
secretory pathway (Guest et al. 1992, Taylor et al.
1998), where it fulfills its role in the production of
bioactive peptide hormones.
The biosynthesis of active SPC2 is tightly linked
to the expression of the neuroendocrine poly-
peptide, 7B2 (Jeannotte et al. 1997, Seidel et al.
1998). This protein was first isolated from pituitary
extracts (Hsi et al. 1982, Seidah et al. 1983) and its
cDNA was later cloned (Martens 1988, Mbikay et
al. 1989). It is a sulfated, non-glycosylated peptide
(Ayoubi et al. 1990, Paquet et al. 1994) with a
strictly neuroendocrine distribution. 7B2 expression
is required for the efficient transport, folding and
activation of SPC2 (Braks & Martens 1994, Martens
et al. 1994, Zhu & Lindberg 1995, Lamango et al.
1996). The two molecules are bound together at
their entry in the ER and are later cleaved by an
SPC1-like activity (Paquet et al. 1994) in their
transport through the secretory pathway (Braks &
Martens 1994). The SPC2 pro-segment is required,
but is not sufficient to confer 7B2 binding (Zhu
et al. 1998), and the interactions between these
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 SPCs: molecular specificity to therapeutic applications ·   and others 7

 3. Hypothetical model of 7B2 and SPC2 interactions. (A) Shortly after
biosynthesis, pro-SPC2 (75 kDa) specifically associates with pro-7B2 (27 kDa) within
the ER. (B) The complex exits from the ER and, within the TGN, SPC1 (scissors)
cleaves the C-terminal (C-T) peptide from pro-7B2 and also cleaves pro-SPC2
(75 kDa to 71 kDa form). (C) By an as yet unexplained mechanism, the 21 kDa 7B2
facilitates the conversion of pro-SPC2 to fully active SPC2 (68 kDa form). (D) The
C-T peptide inhibits the activity of SPC2 in late compartments (TGN or immature
secretory granules). Inhibition of SPC2 (68 kDa form) is released by cleavage of the
C-T peptide by SPC2 at KK residues (and after C-terminal basic residue trimming
by carboxypeptidases).

molecules have been used to explain the relatively
slow exit of SPC2 from the ER (Muller et al. 1997).
7B2 is considered to be a bifunctional molecule,
with its N-terminal domain behaving as a
chaperone-like protein, while its C-terminal peptide
domain is a specific SPC2 inhibitor (Martens et al.
1994, Lindberg et al. 1995, Zhu & Lindberg 1995)
having no effect on SPC3 activity (van Horssen
et al. 1995). This C-terminal region appears not to
be responsible for the observed binding of pro-7B2
with pro-SPC2 in the ER. A working model of 7B2
and SPC2 interactions based on these results is
summarized in Fig. 3.
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In vivo studies have attempted to establish the
role of 7B2. Whereas 7B2 and SPC2 are clearly
co-localized in rat brain neurons, 7B2 has also been
shown to be more widely distributed than SPC2
(Seidel et al. 1998), with many 7B2-positive neurons
showing a lack of SPC2 expression. What is the
function of 7B2 in these SPC2-negative neurons? It
remains to be established whether 7B2 specifically
binds another protein, maybe even an ‘SPC2-like’
molecule, in these neurons. These potential
additional functions of 7B2 have been highlighted
by recent gene knockout experiments (Westphal
et al. 1999). Although gene disruption of 7B2
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8   and others · SPCs: molecular specificity to therapeutic applications
 1. Overall distribution profile of the SPCs
Tissue distribution
Name
SPC1 Ubiquitous, but expressed at variable levels
SPC2 Neuroendocrine cells
SPC3 Neuroendocrine cells
SPC4 Endocrine and non-endocrine, widespread tissue expression
SPC5 Testicular germ cells
SPC6-A Endocrine and non-endocrine, widespread tissue expression
SPC6-B Digestive system and adrenal cortex
SPC7 Widespread, with higher levels in lymphoid-associated tissues
prevents activation of SPC2, other important effects
have been observed. 7B2-null mice display hypo-
glycemia, hyperproinsulinemia, and hypogluca-
gonemia. They also have increased circulating
adrenocorticotrophic hormone (ACTH) and corti-
costerone concentrations, with adrenocortical
expansion. 7B2-null mice do not survive more than
9 weeks, because of severe Cushing’s syndrome. In
these mice, pituitary intermediate lobe ACTH
hypersecretion results in a far more severe
endocrine disorder than is found in the SPC2-null
mice (Furuta et al. 1997). Thus it is highly likely
that 7B2 has functions additional to that of
activating SPC2.
TISSUE DISTRIBUTION AND CELLULAR
LOCALIZATION OF SPC ENZYMES
Numerous studies have been carried out to examine
the detailed tissue and cellular distribution of each
SPC (Day et al. 1992, 1993, Schafer et al. 1993,
Seidah et al. 1994, Beaubien et al. 1995, Dong et al.
1995, 1997, Seidel et al. 1998). Table 1 summarizes
the overall distribution profile. In general, it can be
stated that cells or cell lines do not express only one
SPC at a time, but rather express a distinct
‘cocktail’ of SPCs. Overlapping cellular expression
of SPCs raises the question of possible redundancy
of processing. This is best illustrated by studies
carried out in the rat brain, which examined
SPC mRNA distribution by in situ hybridization
(Fig. 4). Analysis of the brain localization shows
that SPCs have distinct distribution patterns,
suggesting that they also have distinct functions.
However, overlapping regional expressions are also
observed, which hints at some redundant functions
in the brain.
A summary of the analysis of the cellular
distribution of SPCs in the rat brain is given in
Table 2. In situ hybridization studies of brain tissue
at a cellular level are very useful when illustrated in
Journal of Molecular Endocrinology (2000) 24, 1–22
Intracellular localization
TGN, endosomes, cell surface
Secretory granules
Secretory granules, TGN
TGN, ?
?
Secretory granules, TGN
TGN
TGN
this way, as they show that some SPCs (SPC2,
SPC3 and SPC6) are exclusively expressed in
neurons, whereas others (SPC1, SPC4 and SPC7)
are expressed in both neurons and glial cells. In
contrast to glial cells, which possess only a
constitutive secretory pathway, neurons also have a
regulated secretory pathway. These data suggest
an association of SPC2, SPC3 and SPC6 with
the neuroendocrine phenotype and specialized
functions within the regulated secretory pathway.
Of the seven known mammalian SPCs, the
distribution of SPC5 is the most restricted, as it is
not expressed elsewhere than in testicular germ cells
(Nakayama et al. 1992, Seidah et al. 1992).
Although this unique expression pattern may reflect
a highly specialized processing function, no SPC
substrates have, as yet, been identified in germ cells.
The exclusive expression of SPC5 in germ cells also
suggests unique transcriptional regulation of this
gene. However, the germ cells also have the capacity
to express other SPCs, including SPC1 and SPC7.
A comparative in situ hybridization analysis of
adjacent sections of rat seminiferous tubules (Fig. 5)
reveals that both SPC5 and SPC7 are expressed
within germ cells, but in a mutually exclusive
pattern, some tubules containing greater levels of
SPC5 mRNA, the others being rich in SPC7. These
patterns of expression suggest distinct roles of SPC5
and SPC7 in the different stages of germ cell
maturation.
The extensive tissue distribution studies carried
out on the SPCs have revealed that each SPC could
have a distinct function in vivo, as no two SPCs
have identical patterns. These patterns of expres-
sion (whether at a cellular or intracellular level)
could compensate for some apparent common
cleavage specificities observed in vitro. A substrate
cleaved by an SPC in vitro may in fact never
encounter that SPC in vivo, because they either are
not co-expressed in the same cell or are simply
sorted to different intracellular compartments. Thus
overlapping functions of SPCs in vivo may in fact be
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 SPCs: molecular specificity to therapeutic applications ·   and others 9

 4. Photomicrographs of in situ hybridization of SPC mRNAs in adjacent rat

brain sagittal sections. Distinct distribution patterns are observed for SPCs. 3V, third
ventricle; 4V, fourth ventricle; Acb, accumbens nucleus; AD, anterodorsal thalamic
nucleus; AO, anterior olfactory nucleus; CA3, field CA3 of Ammon’s horn; Ce,
cerebellum; Cx, cerebral cortex; DG, dentate gyrus; Hy, hypothalamus; IC, inferior
colliculus; ICj, islands of Calleja; LS, lateral septum; LV, lateral ventricle; Olf,
olfactory bulb; PCL, Purkinje cell layer; PF, parafascicular thalamic nucleus; Pn,
pontine nucleus; S, subiculum; SC, superior colliculus; Th, thalamus; Tu, olfactory
tubercle.

minimized as a result of different regulatory gene
expression mechanisms of the enzyme and sub-
strate. Compensatory mechanisms have been
observed in SPC-null mice, but often represent only
a fraction of the processing activity of the SPC that
was inactivated. For example, in SPC2-null mice
(Furuta et al. 1997), the processing of pro-glucagon,
pro-somatostatin, and pro-insulin in the pancreatic
islets was shown to be severely impaired. However,
in the case of pro-insulin processing, a severe but
incomplete block in processing was observed,
suggesting that other convertases are able to provide
some compensatory cleavage at SPC2-favored sites
in pro-insulin. In SPC2-null mice, pancreatic
concentrations of insulin are still detectable, but
represent only about 20% of normal insulin
concentrations. Thus, whereas some substrates are
in fact cleaved by more than one SPC (at the same
cleavage site), it appears that in vivo compensatory
mechanisms are mostly limited events.
BIOLOGICAL FUNCTIONS OF SPC ENZYMES

 2. Cellular distribution of SPCs in rat brain A major objective of investigations on SPCs has
been to determine the specific biological functions
Neuron Glia
of each enzyme – that is, to establish specific target
Enzyme substrates within their biological context. Many
SPC1 + + studies have focused on the use of cellular
SPC2 +

co-infections using Vaccinia virus recombinants
SPC3 +

SPC4 + + (Thomas et al. 1988) to establish the cleavage
SPC6 +
properties of SPCs on target substrates. This
SPC7 + + approach has been useful in certain applications, for
example in the rapid production of recombinant
www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22
10   and others · SPCs: molecular specificity to therapeutic applications

 5. Comparative distribution of SPC5 and SPC7 mRNAs by in situ

hybridization in adjacent sections of rat testis. In the top panels, note the
complementary distributions, which show seminiferous tubules with high levels of
SPC7 (open arrows) or high levels of SPC5 (white arrows). In the bottom panels,
numbers indicate identical seminiferous tubles in adjacent tissue sections. Tubules
1–5 express high levels of SPC7, whereas tubules 6–9 express high levels of SPC5.

SPCs for in vitro studies. Also, with the use of a
cellular co-infection approach (co-infection of a
target precursor substrate with a particular SPC),
interesting results have been obtained regarding the
unique specificity of SPCs and their ability to cleave
precursors differentially (Thomas et al. 1988,
Benjannet et al. 1991, Leduc et al. 1992). However,
this method has its drawbacks, such as degradation
of the cellular environment induced by vaccinia
infection and the difficulty of carrying out compara-
tive kinetic studies of substrate cleavage. Taken
together, these conditions may explain certain
ambiguities that have been observed with the use
of co-infection, as for example in the cleavage of
pro-glucagon by SPC2. In this case, the Vaccinia
virus approach was unable to demonstrate the
specific formation of glucagon by SPC2 (even in the
presence of 7B2) (Dhanvantari et al. 1996), whereas
SPC2 antisense studies, gene knockout and gene
Journal of Molecular Endocrinology (2000) 24, 1–22
transfection studies (Rouille et al. 1994, 1995,
1997a, Furuta et al. 1997) all showed the obvious
involvement of SPC2 in the formation of glucagon.
Thus only a partial assessment of SPC biological
function is possible with co-expression or in vitro
studies. To complete the elucidation of the
biological role of SPCs, complementary in vivo
approaches must be considered, such as the study of
naturally occuring mutants (e.g. the LoVo cell line)
and SPC gene modulation using antisense (or gene
knockout) technologies. Another interesting method
by which to investigate the role of SPCs in vivo is to
study the co-localization of the enzymes with their
substrates at the tissue, cellular and intracellular
level. An example of such an approach is illustrated
in Fig. 6, showing the co-localization of POMC
mRNA with various SPC mRNAs in hypothalamic
neurons. The data show that all POMC neurons
contain SPC2 and SPC3, that some POMC neurons
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 SPCs: molecular specificity to therapeutic applications ·   and others 11

 6. Co-localization of SPCs and POMC in the hypothalamic arcuate nucleus

neurons, using double labeling in situ hybridization histochemistry. POMC neurons
were labeled with a digoxygenin-labeled cRNA probe and appear in each panel as dark
purple. SPC cRNA probes were radioactively labeled with [35S]UTP/[35S]CTP and
appear in each panel as autoradiographic grains (white dots). (A) Co-localization of
POMC and SPC3; all POMC neurons express SPC3 (black arrows). (B) Co-localization
of POMC and SPC2; all POMC neurons express SPC2 (black arrows). (C) POMC
neurons do not express SPC6 (open arrows). (D) Partial co-localization of POMC and
SPC4; black arrows show examples of co-localization and open arrows show examples
of POMC neurons that do not express SPC4.

contain SPC4, but that none can be shown to
express SPC6. These data reflect the established
role of SPC2 and SPC3 on in vivo POMC
processing, but also suggest that SPC4 could also
play a role in POMC processing in a subset of
arcuate neurons. This type of co-localization
approach is a powerful way to gather data on
cellular enzyme–substrate relationships and
potential SPC biological functions.
One of the SPCs with the best-defined biological
function is SPC1 (Denault & Leduc 1996,
Nakayama 1997, Molloy et al. 1999). In terms of
cell metabolism, SPC1 is believed to be vital, as it is
considered to be a ‘housekeeping’ convertase,
processing constitutively secreted proteins in all
cells. Its ubiquitous distribution, cellular localiz-
ation and substrate specificity support this notion.
In vitro and in vivo studies reveal that SPC1 is
able to cleave various constitutively secreted pro-
tein precursors, including insulin pro-receptor
www.endocrinology.org
(Mondino et al. 1991), pro-von Willebrand factor
(Wise et al. 1990), pro--nerve growth factor
(Bresnahan et al. 1990), pro-transforming growth
factor 1 (Dubois et al. 1995) and pro-endothelin-1
(Denault et al. 1995a). SPC1 does not appear to be
essential for survival in established cell lines, as the
absence of active SPC1 in LoVo or CHO RPE.40
cells is not lethal. Survival of these cells without
functional SPC1 may be due to the expression of
other SPCs, such as SPC4, in LoVo cells,
suggesting compensatory functions by other SPCs.
However, the essential role of SPC1 has been
revealed by in vivo studies demonstrating that SPC1
expression is essential for the normal development
of the mouse embryo (Roebroek et al. 1998).
Whereas SPC1 is ubiquitously expressed, other
SPCs, such as SPC2 and SPC3, have well-defined
patterns of expression that are mostly restricted to
the neuroendocrine system. This distribution is
consistent with their accepted role in pro-hormone
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12   and others · SPCs: molecular specificity to therapeutic applications
and pro-neuropeptide processing. Furthermore,
SPC2 and SPC3 are co-localized with peptide
hormones within dense-core secretory granules.
The specific role of SPC2 and SPC3 has been
shown in the processing of pro-insulin (Bennett
et al. 1992, Smeekens et al. 1992), pro-glucagon
(Rouille et al. 1995, 1997a,b) and POMC (Benjannet
et al. 1991, Thomas et al. 1991). These cleavage
specificity studies have been supported by gene
knockout (Furuta et al. 1998) and antisense studies
(Bloomquist et al. 1991, Rouille et al. 1994,
Rothenberg et al. 1996, Johanning et al. 1998).
These substrates possess multiple basic residues
that are differentially cleaved by SPC2 and SPC3,
yielding different products depending on the
relative expression of SPC2 and SPC3. In general,
it appears that SPC3 activity results in a more
limited precursor proteolysis, generating relatively
high-molecular-weight species, whereas SPC2
cleavage is more extensive, producing smaller forms
of mature products.
The issue of differential processing by SPC2 and
SPC3 can be exemplified with pro-dynorphin as a
model precursor. The interest in pro-dynorphin lies
in the fact that it contains multiple apparently
similar paired basic amino acid cleavage sites, in
addition to two potential single Arg cleavage sites.
Pro-dynorphin is also, along with POMC and
pro-enkephalin, one of three precursors involved
in the production of mammalian opioid peptides.
In vivo, differential processing of this 26 kDa
pro-protein is observed in different tissues (Day &
Akil 1989). For example, pro-dynorphin is ex-
pressed in anterior pituitary gonadotropes and in
magnocellular neurons of the hypothalmus. In the
anterior pituitary, pro-dynorphin is processed to
yield high-molecular intermediates of 8, 10 and
16 kDa, whereas, in the hypothalamus, final opioid
peptide products, including dynorphin A(1–17,)
dynorphin A(1–8), dynorphin B(1–13) and
-neo-
endorphin, are obtained (Fig. 7). In vitro studies
demonstrated that SPC3 is primarily responsible for
the production of the high-molecular-weight inter-
mediates. The 8 kDa intermediate requires cleavage
at a single Arg residue, a function that can be
carried out by SPC3 (Dupuy et al. 1994). In
contrast, SPC2 was shown to generate the smaller
opioid-active dynorphin A(1–17), dynorphin B(1–
13), and
-neo-endorphin products (Day et al.
1998). In common with SPC3, SPC2 was also able
to cleave at single Arg residues, generating the
C-peptide but, in addition, dynorphin A(1–8). The
differential cleavage patterns of pro-dynorphin
by SPC2 and SPC3 obtained in vitro reflect
the processing patterns observed in vivo. Co-
localization studies indicate that hypothalamic
Journal of Molecular Endocrinology (2000) 24, 1–22
magnocellular neurons express both SPC2 and
SPC3, whereas anterior lobe gonadotropes express
only SPC3. Thus complete processing of pro-
dynorphin to biologically active opioid peptide
products is dependent upon the expression of
SPC2. Additional support comes from studies with
SPC2-null mice, which showed impaired pro-
dynorphin processing, especially in the formation of
dynorphin A(1–8) (Day et al. 1998).
Compared with SPC1, SPC2 and SPC3, the other
members of the SPC family have been the object of
fewer studies and, thus, their biological roles are not
as well defined. The first enzymatic studies on
SPC4 activity revealed characteristics similar to
those of SPC1, but with some differences in
specificity (Rehemtulla et al. 1993). On the basis of
preliminary studies on tissue distribution and
enzymatic properties, SPC4 was first believed to
be very similar to SPC1. Moreover, the close
proximity of the SPC1 and SPC4 genes on
chromosome 15 suggested that these genes evolved
from a common ancestor by gene duplication
(Kiefer et al. 1991). However, more recent studies
on SPC1 and SPC4 in vitro activities have
highlighted the differences between these enzymes.
Compared with SPC1, SPC4 has a low sensitivity
for calcium chelators and dithiothreitol (Mains
et al. 1997), and it is not affected by the SPC1
inhibitor,
1-antitrypsin-portland (AT-PDX, see
below). In fact, several reports show that SPC4 is
closer to SPC6 than SPC1 in many respects. SPC6
is the enzyme with the greatest degree of sequence
homology with SPC4 (Lusson et al. 1993,
Nakagawa et al. 1993). In vitro enzymatic studies
of the recombinant enzymes revealed a close
relationship between SPC4 and SPC6 (Creemers
et al. 1993a). Finally, a distinct distribution has
been observed for the two enzymes, which is
complementary in some tissues (Dong et al. 1995,
Zheng et al. 1997). Despite the differences in the
in vitro properties of SPC1 and SPC4, all the
reported SPC4 substrates can also be cleaved by
SPC1 or other SPCs with an equal or superior
efficiency. Among the reported SPC4 substrates
are pro-brain-derived neurotrophic factor, pro-
neurotrophin 3 (Seidah et al. 1996a), the insulin
receptor (Alarcon et al. 1994), pro-nerve growth
factor (Seidah et al. 1996b), pro-somatostatin
(Brakch et al. 1995), human serum albumin (Mori
et al. 1999) and the HIV viral coat protein, GP160
(Hallenberger et al. 1992, Morikawa et al. 1993).
The cleavage of the GP160 precursor illustrates
the need to confirm in vitro data with in vivo
observations. Even though enzymatic assays with
recombinant proteins suggest that GP160 is cleav-
able by SPC1 and SPC4 (Decroly et al. 1994), this
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 SPCs: molecular specificity to therapeutic applications ·   and others 13

 7. Differential processing of pro-dynorphin by SPC2 and SPC3. Cleavage by SPC3

produces large intermediate molecules, whereas SPC2 generates smaller peptides. The
processing pattern obtained with SPC3 corresponds to that observed in vivo within the
anterior lobe gonadotropes. The processing pattern obtained with SPC2 corresponds to that
observed in vivo within the hypothalamus (see text for discussion).
NE,
-neo-endorphin;
Dyn, dynorphin.

processing is probably not physiologically relevant,
because it occurs with very low efficiency (Inocencio
et al. 1997). Moreover, SPC1-deficient cell lines
process GP160 as efficiently as do normal cells
(Ohnishi et al. 1994). Finally, it has been proposed
that a Ca2+-independent protease may in fact be
responsible for in vivo GP160 cleavage (Inocencio
et al. 1997). The role of SPC4 in vivo remains
unclear, as none of the above cited precursors
demonstrates a clear preferential susceptibility to
SPC4 activity.
www.endocrinology.org
The cloning of SPC6 cDNA revealed the
existence of two enzyme isoforms, named SPC6-A
and SPC6-B, which differ only in their C-terminal
end. These isoforms are believed to exert different
functions, as SPC6-A has a soluble C-terminal
domain and is targeted towards the dense-core
secretory granules in endocrine cells, whereas
SPC6-B has a transmembrane C-terminal domain
and is located in the vesicles of the Golgi apparatus
(De Bie et al. 1996). The SPC6 isoforms are
differentially expressed in the gastrointestinal tract,
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14   and others · SPCs: molecular specificity to therapeutic applications
and the expression of SPC6-A is affected by dietary
content (Udupi et al. 1997), suggesting a regulatory
role for the processing of gut peptides. Other
potential biological functions of SPC6 have been
identified. It may play a role in the cleavage of
pro-neurotensin/neuromedin N in human colon
cancer cells (Rovere et al. 1998). It may be also
involved in the process of sexual differentiation, by
activating pro-Müllerian substance (Nachtigal &
Ingraham 1996). In addition, the transfection of
SPC6 in COS cells induces cleavage of the
extracellular domains of the receptor protein
tyrosine phosphatases (Campan et al. 1996).
Enzymatic characterization of the recombinant
SPC7 reveals a substrate specificity largely similar
to that of SPC1 (Munzer et al. 1997), with which
it is highly homologous. Just like SPC1, SPC7 has
a C-terminal transmembrane domain and a cyto-
plasmic tail responsible for its subcellular localiz-
ation at the TGN level, possibly with an
endosomal recycling pathway similar to that of
SPC1. At the structural level, PC7 has a
C-terminal region rich in serine and threonine,
whereas SPC1 has a cysteine-rich domain.
Interestingly, SPC7 is not phosphorylated like
SPC1, but is palmitoylated in its cytoplasmic tail
(van de Loo et al. 1997). As this enzyme was only
recently discovered, more studies are needed to
determine its role in vivo.
CLINICAL ASPECTS OF SPC ACTIVITY
Theoretically, there are two ways of designing
therapeutic approaches targeting SPCs: restoring
the defective processing responsible for a disorder,
and blocking a processing event that is essential for
a given disease state.
With regard to the restoration of defective
processing, there is, at present, little evidence of
pathologies associated with altered protein process-
ing. Some autosomal dominant syndromes have been
identified in which a mutated precursor leads to an
incorrect processing pattern, inducing pathological
conditions. These diseases are related to the
impaired processing of pro-insulin (Peters 1987),
pro-albumin (Brennan 1989), and blood coagulation
factor IX (Bentley et al. 1986). However, these
diseases are not related to an impaired activity of
SPCs, but are caused by structural modifications in
the substrate. One interesting application of SPCs
in the restoration of hormone production is the
re-engineering of rat prepro-insulin-1 to introduce
SPC1 cleavage sites, thus allowing processing
into mature insulin by SPC1 in liver (Muzzin et al.
1997).
Journal of Molecular Endocrinology (2000) 24, 1–22
The first evidence of a metabolic disorder
induced by a defective enzyme involving precursor
cleavage came from the study of fat/fat mice. A
spontaneous mutation in the CPE gene of this strain
induced a single amino acid substitution in a highly
conserved domain of the exopeptidase, yielding an
inactive enzyme. Biochemical analysis of insulin-
like peptides in these animals revealed a high
content (approximately 80%) of unprocessed pro-
insulin, whereas the processed insulin chains still
had their C-terminal pairs of basic residues
untrimmed. However, an unexpected finding was
the detection of unprocessed pro-insulin. This
phenomenon may be explained by the observation
that SPC2 activity is inhibited by C-terminally
extended peptides, possibly by a product inhibition
mechanism (Day et al. 1998).
Recently, an isolated case of SPC3 deficiency has
been identified in a human patient. One allele of the
SPC3 gene has a Gly<Arg483 mutation that
prevents processing of pro-SPC3 and leads to its
retention in the ER. The mutation of the other allele
causes skipping of exon 5, leading to loss of 26
residues, a frameshift and creation of a premature
stop codon within the catalytic domain (Jackson et
al. 1997). These mutations seem to produce effects
similar to those observed in the fat/fat phenotype –
that is, obesity and pro-insulinemia, accompanied
by abnormal glucose homeostasis, hypogonado-
tropic hypogonadism and hypocortisolism. Such
inherited syndromes of normal proteolytic process-
ing are uncommon; they arise from the occasional
combination of two alleles containing random
mutations of an SPC gene. Recent advances in
gene transfer therapy (Knoell & Yiu 1998) raise the
hope that these rare inherited defects could be
corrected by the reintroduction of normal SPC
genes.
The other field of therapeutic application involv-
ing SPC activity is the inhibition of unwanted
proteolytic processing. Various pathogenic agents,
including bacterial toxins and viral coat proteins,
have been found to require proteolytic processing
by SPCs in order to produce their effects (Garten
et al. 1994, Gordon et al. 1995, Rott et al. 1995).
Blocking the proteolytic processing of such patho-
genic proteins opens the way to the development of
new therapeutic treatments based on enzyme
inhibition.
Blocking SPC activity may be deleterious, as
these enzymes are essential for the normal expres-
sion of important cellular proteins. Thus, for each
disease state, the enzyme specificity of patho-
genic precursors must be well understood and
the design of therapeutic SPC inhibitors should
be made as specific as possible, in order to avoid
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 SPCs: molecular specificity to therapeutic applications ·   and others
cross-inhibition of important cellular proteolytic
processing. Nonetheless, it cannot be ruled out that,
in certain pathological states, wide-spectrum SPC
inhibitors may also be useful.
SPC INHIBITION
Ideally, effective control of the production of biologi-
cal peptides involved in various disease states could be
achieved by specifically inhibiting the enzymatic ac-
tivity of SPCs. Although antisense technologies aimed
at decreasing the endogenous levels of SPC mRNA
have been considered, enzyme-based inhibition
achieved by designing a variety of molecular entities,
ranging from non-peptidic or peptidomimetic
molecules to small peptide substrates and to larger
proteins, would be the preferred choice.
The initial characterization of the enzymatic
specificities of SPCs spearheaded efforts in the
design, conception and testing of compounds aimed
at inhibiting these enzymes. A straightforward
approach has made use of the consensus cleavage
site sequence of SPCs for the synthesis of peptides
capable of acting as pseudosubstrates or transition
state analogues. For example, elucidation of the
SPC1 recognition sequence, Arg-Xaa-Xaa-Arg, has
led to the design of molecules such as the family
of chloromethylketones, which behave as tight-
binding irreversible inhibitors of SPC1 (Angliker
1995, Jean et al. 1995).
Structure–activity studies using peptidyl sub-
strates based on a region spanning the pro-region
and catalytic domain of mouse SPC3 (Molloy et al.
1992, Basak et al. 1995) or the cleavage sequence of
human pro-parathyroid hormone (Lazure et al.
1998), showed that inhibition constants in the
micromolar range can be attained. Some of the pepti-
domimetic agents irreversibly react with the active-
site serine, thus acting as suicide inhibitors. These
peptide-based molecules have proven quite useful in
ascertaining the role of SPCs in processing precur-
sors of viral glycoproteins (Hallenberger et al. 1992,
Volchkov et al. 1998), hormones (Lazure et al. 1998),
enzymes and other pro-proteins (Denault et al.
1995b, Jean et al. 1993, Ledgerwood et al. 1996).
They have the inherent advantage of being synthe-
sized on a large scale and having the propensity of
modified peptides to penetrate membranes, making
them well suited for therapeutic use as large-
spectrum, pharmacologically stable inhibitors. How-
ever, none of them has, as yet, demonstrated marked
specificity. It is clear that, because of the enzymatic
property of this family of proteinases of cleaving on
the C-terminal of basic residues, other consider-
ations will have to be taken into account in order to
www.endocrinology.org
15
synthesize compounds of higher specificity. One of
these is the availability of all SPCs, enabling re-
searchers to compare the inhibitory properties of a
given molecule in vitro. The use of these isolated
SPCs for the identification of specific SPC inhibitors
with high throughput methods such as peptide com-
binatorial libraries is already under way, as in the
case of SPC2 and SPC3 (Apletalina et al. 1998).
Because most enzymes possess natural inhibitors,
efforts have also been made to identify endogenous
molecules that could modulate SPC activity. As
discussed above, one of these, the dual function 7B2
that facilitates pro-SPC2 maturation (Muller et al.
1997), has also been found to be inhibitory. Indeed,
within its C-terminal peptide, the Lys171-Lys172
site is particularly important for the ability of 7B2
to inhibit SPC2 (van Horssen et al. 1995).
Pro-segments of SPCs can also be considered to be
modulators of enzymatic activity. Acting as a
multifunctional domain, the pro-peptide not only
acts as a steric chaperone, but also binds to catalytic
domains of SPCs and inhibits protease activity.
This mechanism has been observed in vitro and
in vivo for SPC1 and SPC3, whereby pro-segments
inhibit the enzymes by a slow, tight-binding
mechanism (Boudreault et al. 1998). In fact, the
pro-domains are quite potent inhibitors, as
demonstrated for SPC1, with IC50 values of 14 nM.
Serpins (SERine Protease INhibitors) are nat-
urally occuring proteins that play a role in the
regulation of serine proteases activity. Although
most serpins identified today specifically inhibit
trypsin-like serine proteases, a recently described
member of this family, called PI8, has been shown
to inhibit SPC1 by a rapid, tight-binding mech-
anism (Dahlen et al. 1998). PI8 is a 45 kDa
ovalbumin-type serpin that contains two SPC1
recognition sequences, Arg-Asn-Ser-Arg339 and
Arg339-Cys-Lys-Arg342 in its reactive site loop.
Once this loop enters the catalytic pocket, it forms
an SDS-stable serpin–enzyme complex, characteris-
tic of physiological serpin–proteinase interactions.
Attempts at abolishing SPC activity that were
based on redesigning the reactive site of known
inhibitors of serine proteinases to fit SPC recog-
nition sequences were initially performed using
turkey ovomucoid third domain. Its reactive site
was re-engineered by incorporating the SPC1
cleavage site, Arg-Xaa-Lys-Arg, in its reactive
pocket, yielding a molecule of Ka 1·110
7 M
1
(Lu et al. 1993). Similar modifications of the bait
region of the general protease inhibitor,
2
macroglobulin (Gly-Phe-Tyr-Glu686-Ser-Asp<
Arg-Ser-Lys-Arg686-Ser-Leu), also led to the
production of an intracellularly active SPC1
inhibitor (Van Rompaey et al. 1997).
Journal of Molecular Endocrinology (2000) 24, 1–22
16   and others · SPCs: molecular specificity to therapeutic applications
Using a similar approach of site-directed muta-
genesis of reactive centers, other existing poly-
peptides of the serpin family were targeted that
led to the development of a potent inhibitor for
SPC1. Indeed, changing residues corresponding to
the P4 and P1 residues of the reactive site loop of

1-antitrypsin to accommodate the specificity of
SPC1 (Ala-Ile-Pro-Met<Arg-Ile-Pro-Arg) led to a
molecule specifically inhibiting SPC1, with an IC50
of 0·6 nM (Anderson et al. 1993). Interestingly, this
serpin, AT-PDX, conserves the biochemical
properties of the native serpin, by rapidly forming
an irreversible complex with SPC1 that is
resistant to heat and denaturants. Kinetic analysis
of AT-PDX inhibition has revealed that it acts
through a slow, tight-binding mechanism. It also
behaves as a suicide substrate inhibitor, as
binding to the active site results in an equal
probability that proteolysis will ensue or that a
kinetically trapped SDS-stable complex with the
enzyme will be formed (Dufour et al. 1998, Jean
et al. 1998). Because of its high selectivity toward
SPC1 (Jean et al. 1998), this novel inhibitor
has already been used to assess the biological role
of SPC1 in processing different precursors
(Anderson et al. 1993, Denault et al. 1995b,
Watanabe et al. 1995, Decroly et al. 1996, Kayo
et al. 1997, Zarkik et al. 1997, Abrami et al. 1998,
Cui et al. 1998, Logeat et al. 1998). Other
naturally occuring substances have been tested for
their ability to inhibit SPCs. A recent study
reported the identification of substances (an-
drographolides) from the medicinal plant, An-
drographis paniculata, that possess low (>30 µM)
inhibitory potencies toward SPCs (Basak et al.
1999).
Lately, much focus has been put on develop-
ing approaches to abolishing SPC1-mediated
cleavage of viral and bacterial pathogens. However,
because not all these precursors are processed in
the same cellular compartment, the physicochemi-
cal properties of the inhibitory compounds must be
considered. For example, it is believed that
cleavage of the protective antigen of Anthrax is
performed by SPC1 found at the cell surface, that
proteolysis of exotoxin A of Pseudomonas would
take place in endosomal compartments, but that
viral glycoprotein precursors such as the Ebola
glycoprotein would occur in the TGN. This
signifies that inhibiting cell surface, and possibly
endosomal, SPC1 may be facilitated by its
accessibility to hydrophilic molecules such as
peptides and proteins. To reach the intracellular
TGN, more lipophilic compounds would be
needed. These issues must also be considered for
all other SPCs.
Journal of Molecular Endocrinology (2000) 24, 1–22
CONCLUSIONS
The mammalian SPCs comprise a complex family
of enzymes that carry out apparently similar
processing functions (cleavage at basic residues), yet
clearly have unique biological roles as a result of
their distinct catalytic specificities and localizations.
Although the clinical relevance of SPCs remains
unclear, the design of specific inhibitors will
undoubtedly lead to a better understanding of the
complex cellular mechanisms in which the SPCs
participate. A better understanding of the substrate
specificity of each SPC could lead to a rational
design of specific inhibitors. These substrate-
specificity studies must be focused on a more
relevant biological context than has been done
previously. Although the design of specific SPC
inhibitors is a major goal, targeting a single SPC
with a highly specific inhibitor may not have the
fully desired effect, because of some partial
compensatory mechanisms, thus the development of
wide-spectrum inhibitors may also be useful under
certain conditions. However, as several SPCs are
often co-expressed within a particular cell type, this
approach may prove to be particularly risky. The
knowledge of how these enzymes function and
interact is critical for the future successful appli-
cation of SPC inhibitors within a therapeutic
context.
ACKNOWLEDGEMENTS
We wish to thank the Medical Research Council of
Canada for supporting this work. R L and R D are
both scholars (Chercheurs-Boursiers) of the Fonds
de la Recherche en Santé du Québec. We also wish
to thank Mr Eric Dufour for helpful discussion.
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