(Received for publication, July 27, 1994; and in revised form, October 31, 1994)
From the
PC2 and PC3, which is also known as PC1, are subtilisin-like
proteases that are involved in the intracellular processing of
prohormones and proneuropeptides. Both enzymes are synthesized as
propolypeptides that undergo proteolytic maturation within the
secretory pathway. An in vitro translation/translocation
system from Xenopus egg extracts was used to investigate
mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent
rapid (t < 10 min) processing of the 88-kDa propolypeptide
at the sequence RSKR to generate the 80-kDa active form of
the enzyme. This processing was blocked when the active site aspartate
was changed to asparagine, suggesting that an autocatalytic mechanism
was involved. In this system, processing of pro-PC3 was optimal between
pH 7.0 and 8.0 and was not dependent on additional calcium. These
results are consistent with pro-PC3 maturation occurring at an early
stage in the secretory pathway, possibly within the endoplasmic
reticulum, where the pH would be close to neutral and the calcium
concentration less than that observed in later compartments. Processing
of pro-PC2 in the Xenopus egg extract was much slower than
that of pro-PC3 (t = 8 h). It exhibited a pH optimum of
5.5-6.0 and was dependent on calcium (K
= 2-4 mM). The enzymatic properties of
pro-PC2 processing were similar to that of the mature enzyme. Further
studies using mutant pro-PC2 constructs suggested that cleavage of
pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results
were consistent with pro-PC2 maturation occurring within a late
compartment of the secretory pathway that contains a high calcium
concentration and low pH.
PC2 and PC3 are members of the eukaryotic family of
subtilisin-like proteases that are involved in the intracellular
processing of propolypeptides within the secretory pathway (Smeekens
and Steiner, 1990; Seidah et al., 1990; Smeekens et
al., 1991). Expression of PC2 and PC3 is restricted to
neuroendocrine cells, which is compatible with their role in the
processing of proneuropeptides and prohormones. Both enzymes require
pairs of basic amino acids at their cleavage site. Both enzymes are
synthesized as larger precursor molecules that undergo proteolytic
maturation within the secretory pathway. PC2 is synthesized as a 69-kDa
prepropolypeptide. The NH-terminal signal sequence is
removed during segregation within the endoplasmic reticulum, where
glycosylation generates a 75-kDa propolypeptide. When expressed in Xenopus oocytes, pro-PC2 undergoes cleavage at the sequence
KRRR
to generate a 71-kDa intermediate that is further
cleaved at the sequence RKKR
to generate the 68-kDa mature
enzyme. Processing of the 75-kDa propolypeptide can also occur directly
by way of cleavage at RKKR
(Shennan et al.,
1991b; Matthews et al., 1994). A similar pattern of processing
was shown to occur in GH4 cells (Benjannet et al., 1992) and
in islets of Langerhans (Guest et al., 1992). Maturation of
pro-PC2 has been shown to occur by way of an autocatalytic reaction
(Matthews et al., 1994).
PC3 is synthesized as an 88-kDa
propolypeptide that undergoes cleavage at the sequence RSKR to generate an 80-kDa active form of the enzyme (Benjannet et
al., 1992; Zhou and Lindberg, 1993). Further cleavage occurs at
the COOH terminus to generate 75- and 66-kDa forms (Zhou and Lindberg,
1993; Rufaut et al., 1993; Benjannet et al., 1993).
Processing of pro-PC3 occurs very rapidly in its biosynthesis (Zhou and
Lindberg, 1993; Benjannet et al., 1993), while pro-PC2
processing is very slow (Shennan et al., 1991b; Guest et
al., 1992).
Here we describe in detail the calcium and pH dependence for pro-PC2 and pro-PC3 maturation and show that optimal conditions for processing are very different for the two enzymes. The results suggest that pro-PC3 may undergo autocatalytic processing within the endoplasmic reticulum or early secretory compartment where the pH is near neutral and the calcium concentration in the micromolar range, while pro-PC2 processing occurs in a late secretory compartment where the pH is low (5.5) and the calcium concentration within the millimolar range.
The wild-type and mutant PC2 and PC3 constructs used in the
present study are shown in Fig. 1. PC2 and PC3 contain a highly
conserved subtilisin domain with a catalytic triad of Asp, His, and Ser
residues. In addition, PC2 has an Asp at residue 285 while PC3 has an
Asn residue at 282 that are thought to stabilize the transition
complex. PC3M2 contains a mutation of the sequence RSRR to
SSGR
thereby removing a potential cleavage site. PC2M3 and
PC3M3 contain mutations that remove the propolypeptide processing site, i.e. deletion of RKKR
in PC2M3 and R83S in PC3M3.
PC2M4 contains a change of Asp
to Asn, which renders the
enzyme catalytically inactive, while PC3M4 contains the equivalent
mutation of Asp
to Asn. PC2M5 contains a mutation of
Asn
to Asp, while PC2M7 has a major deletion of the
entire P-domain and C-terminal sequences. All cDNAs were subcloned into
the expression plasmid SP64T, and mRNA was generated by in vitro transcription. mRNAs were then translated in a cell-free system
prepared from Xenopus eggs. This system has previously been
shown to cleave signal peptides, segregate proteins within membranes,
perform some glycosylation and phosphorylation events, and to assemble
polypeptide chains of multisubunit proteins (Matthews and Colman,
1991).
Figure 1: Schematic representation of wild-type PC3 (A) and PC2 (B) and the mutants used in this study. The figure indicates the propeptide, the subtilisin domain (dark area), and the P-domain (hatched area) as well as the propeptide cleavage sites, the Asp-His-Ser of the catalytic site, and the Asp/Asn of the oxyanion hole.
To investigate PC3 propolypeptide processing, mRNA encoding
PC3 was translated in the egg extract in the presence of
[S]methionine for 30 min. RNase was then added
to inhibit further translation and the reactions chased for periods of
time (Fig. 2). An 88-kDa polypeptide was observed that underwent
partial processing within the 30-min pulse labeling period. Complete
processing of the 88-kDa polypeptide was observed within a
30-40-min chase period. Mutagenesis of the putative processing
site RSRR
to SSGR had no effect on processing to the
80-kDa form (Fig. 3), while mutation of the alternative
potential processing site RSKR
to RSKS prevented
processing of the 88-kDa precursor at the 3-h time point, suggesting
that processing was occurring at this site and was compatible with
proteolytic cleavage of an NH
-terminal propeptide. Some
processing to an intermediate 84-kDa form did occur after prolonged
chase (Fig. 3, track 6), which may be the result of
cleavage at the site RSRR
by an endogenous extract
protease. Maximal processing of the 88-kDa pro-PC3 polypeptide occurred
at pH 7.0 (Fig. 4). Over the pH range 6.5-8.0, a broad
peak of activity was observed between pH 7.0 and 8.0 with little
activity above pH 8.0 (data not shown). Processing was not dependent on
added calcium over the range 0-10 mM (Fig. 5).
The mutant PC3M4, in which the Asp of the catalytic triad had been
altered to an Asn, remained unprocessed over the 24-h time period (Fig. 6), suggesting that pro-PC3 maturation was autocatalytic.
Figure 2:
Time course of pro-PC3 processing. PC3
mRNA was translated in the egg extract for 30 min, and translation was
stopped by the addition of RNase (40 µg/ml final concentration).
Aliquots were taken at various times, membranes were prepared as
described under ``Materials and Methods,'' and samples were
analyzed by SDS-PAGE and fluorography. Track M denotes C-labeled protein markers, with the molecular mass in kDa.
These bands were quantified by laser scanning densitometry and plotted
to show the percentage processing (ratio of 88- to 80-kDa bands)
against time.
Figure 3:
Identification of the processing site of
pro-PC3. PC3M2 and PC3M3 mRNAs were translated in the egg extract for 1
h, RNase was added, and the reactions were chased for 0, 3, and 24 h.
Membranes were prepared as described under ``Materials and
Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Track M denotes C-labeled protein markers, with
the molecular mass in kDa.
Figure 4:
pH dependence of pro-PC3 processing. PC3
mRNA was translated in the egg extract for 30 min, and RNase was added
to inhibit further translation. Aliquots were then made 0.1 M with respect to buffer at different pH using MES for pH
5.0-6.5 and TES for pH 7.0. Reactions were then chased for 15
min, membranes prepared as described under ``Materials and
Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Track M denotes C-labeled protein markers, with
the molecular mass in kDa. Bands were quantified by laser scanning
densitometry and plotted to show the percentage processing (ratio of
88- to 80-kDa bands) against pH.
Figure 5:
Effect of calcium on pro-PC3 processing.
PC3 mRNA was translated in the egg extract for 30 min, and RNase was
added to inhibit further translation. Aliquots were then adjusted to
the calcium concentrations shown by addition of a 10-fold stock of
calcium chloride. Reactions were then chased for 15 min, membranes
prepared as described under ``Materials and Methods,'' and
samples analyzed by SDS-PAGE and fluorography. Arrows indicate
positions of C-labeled protein markers, with the molecular
mass in kDa. Bands were quantified by laser scanning densitometry and
plotted to show the percentage processing (ratio of 88- to 80-kDa
bands) against calcium concentration.
Figure 6:
Processing of pro-PC3 is autocatalytic.
PC3 and PC3M4 RNA were translated in the egg extract for 1 h, RNase was
added, and the reactions were chased for 0, 3, and 24 h. Membranes were
prepared and analyzed by SDS-PAGE and fluorography. Track M denotes C-labeled protein markers, with molecular
mass in kDa.
We have previously shown that the maturation of pro-PC2 involves
cleavage of a 75-kDa glycosylated propolypeptide at the sequence
RKKR to generate the 68-kDa mature form of the enzyme
(Matthews et al., 1994). To investigate the pH optimum for
pro-PC2 processing, PC2 mRNA was translated in the egg extract for 2 h
followed by a chase period of 6 h in different pH buffers (Fig. 7). Processing occurred within a narrow pH range of
5.5-6.0. The effect of calcium on pro-PC2 processing was
determined by translating PC2 mRNA for a pulse period of 2 h followed
by a chase period of 20 h at different calcium concentrations (Fig. 8). Pro-PC2 processing was stimulated by calcium in the
millimolar range with processing being nearly complete within 20 h in
10 mM calcium. Using these optimum conditions of pH and
calcium, the time course of pro-PC2 processing was investigated (Fig. 9). Processing of the 75-kDa pro-PC2 polypeptide to the
mature 68-kDa polypeptide was slow (t = 8 h).
Figure 7:
pH dependence of pro-PC2 processing. PC2
mRNA was translated in the egg extract for 2 h. Aliquots were then
taken and adjusted to the pH shown by addition of 0.1 vol of 1 M sodium acetate (pH 4.0 and 4.5), 1 M MES (pH 5.5, 6.0,
and 6.5) or 1 M TES (pH 7.0). The reactions were then
incubated at 21 °C for 6 h. Membranes were prepared as described
under ``Materials and Methods,'' and samples were analyzed by
SDS-PAGE and fluorography. Track M denotes C-labeled protein markers, with the molecular mass in kDa. Arrowheads on the right point to the 75- and 68-kDa
forms of PC2. These bands were quantified by laser scanning
densitometry and plotted to show the percentage processing (ratio of
68- to 75-kDa bands) against pH of the chase
buffer.
Figure 8:
Effect of calcium on pro-PC2 processing.
PC2 mRNA was translated in the egg extract for 2 h. Aliquots were then
adjusted to the calcium concentrations shown by addition of a 10-fold
stock of CaCl. The reactions were then chased for 20 h
before membranes were prepared as described under ``Materials and
Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Arrowheads on the left indicate the positions of
C-labeled protein markers, with the molecular mass in kDa.
The arrowheads on the right indicate the 75- and
68-kDa forms of PC2. These bands were quantified by scanning
densitometry and plotted as percentage processing (ratio of 68- to
75-kDa bands) against concentration of added
calcium.
Figure 9:
Time course of pro-PC2 processing. PC2
mRNA was translated in the egg extract for 2 h. The translation
reaction mix was then adjusted to pH 5.5 and 10 mM CaCl, and the reaction mix was incubated at 21 °C.
Aliquots were removed at the indicated times, and membranes were
prepared as described under ``Materials and Methods.''
Samples were analyzed by SDS-PAGE and fluorography. Track M denotes
C-labeled protein markers, with the molecular
mass in kDa. The arrowheads on the right indicate the
75- and 68-kDa forms of PC2. The bands were quantified by scanning
laser densitometry and plotted to show the percentage processing (ratio
of 68- to 75-kDa bands) with time.
We
have previously shown that the maturation of PC2 involves an
autocatalytic reaction (Matthews et al., 1994). We now wished
to determine whether the same mechanism occurred under optimal
conditions of pH and Ca. This was achieved by using
two mutant PC2 molecules: PC2M4, which contained a change of the
catalytically important Asp
to Asn, and PC2M7, which
contains a deletion of 214 amino acids from the COOH-terminal end of
the molecule. This part of the protein has been shown to be important
for furin activity (Creemers et al., 1993). When translated in
the Xenopus egg extract, PC2M7 remained unprocessed after an
18-h chase period (Fig. 10, tracks 1 and 2).
Wild-type pro-PC2 was completely processed to the 68-kDa polypeptide (Fig. 10, tracks 3 and 4). PC2M4 underwent a
small degree of processing, principally to a 71-kDa intermediate (Fig. 10, track 6). This is likely to be due to the
activity of an endogenous egg extract protease during the chase
conditions. When PC2M7 and PC2M4 were translated in the same reaction,
there was no change in the pattern of processing (Fig. 10, tracks 7 and 8), confirming that both PC2M7 and PC2M4
are catalytically inactive. However, PC2M7 was completely processed in trans by cotranslating PC2M7 mRNA with wild-type PC2 mRNA (Fig. 10, tracks 9 and 10). As shown
previously (Matthews et al., 1994), the mutant PC2M4 pro-PC2
polypeptide migrates slightly faster than the wild-type pro-PC2
polypeptide. Co-incubation of pro-PC2M4 with wild type pro-PC2 resulted
in the appearance of two processed molecules indicating that pro-PC2M4
had also undergone processing in trans by the wild-type
pro-PC2 or PC2 molecule(s) (Fig. 10, tracks 11 and 12).
Figure 10:
Processing of PC2 is autocatalytic and
intermolecular. PC2 (W/T), PC2M7, and PC2M4 were translated
separately (tracks 1-6) and in the combinations shown (tracks 7-12) for 2 h. The pH was adjusted to pH 5.5 by
the addition of 1 M MES, pH 5.5, the calcium concentration was
adjusted to 10 mM by the addition of CaCl, and the
reactions were continued for 18 h. Membranes were prepared after the
2-h pulse and 18-h chase periods as described under ``Materials
and Methods,'' and were analyzed by SDS-PAGE and
fluorography.
These results confirm that cleavage of pro-PC2 can
occur via an intermolecular reaction. The question remained as to
whether the pro-PC2 itself could cleave other pro-PC2 molecules or
whether following an initial activation step further cleavage involved
the newly formed mature enzyme. To investigate whether the pro-PC2
could cleave in an intermolecular reaction we used the mutant PC2M3 (Fig. 1), which contains a deletion of the tetrabasic processing
site RKKR. This mutation blocks processing of the 75-kDa
pro-PC2 polypeptide to the mature 68-kDa form. Translation of PC2M3
mRNA in the Xenopus egg extract resulted in accumulation of
the unprocessed 75-kDa propolypeptide and the 71-kDa intermediate (Fig. 11, tracks 1 and 2). The 71-kDa
intermediate is generated through cleavage of the 75-kDa PC2M3
polypeptide at the sequence KRRR
. When PC2M3 mRNA was
cotranslated in the egg extract with PC2M7 mRNA, the 75- and 71-kDa
pro-PC2M3 polypeptides had no effect on the processing of pro-PC2M7 (Fig. 11, tracks 5 and 6). Pro-PC2M7 was,
however, completely processed when cotranslated with wild-type PC2 mRNA (Fig. 11, tracks 7 and 8). These results
indicated that the pro-PC2 molecules were not involved in the
processing of other pro-PC2 molecules in trans.
Figure 11:
The 75- and 71-kDa molecular forms of
pro-PC2 are not catalytically active. PC2M3 (tracks 1 and 2), PC2M7 (tracks 3 and 4), PC2M3 plus PC2M7 (tracks 5 and 6), and PC2WT plus PC2M7 (tracks 7 and 8) were translated for 2 h. The pH was adjusted to pH
5.5 by the addition of 1 M MES, pH 5.5, the calcium
concentration was adjusted to 10 mM by the addition of
CaCl, and the reactions were chased for 18 h. Membranes
were prepared after the pulse and the chase period as described under
``Materials and Methods,'' and were analyzed by SDS-PAGE and
fluorography. Track M denotes
C-labeled protein
markers, with the molecular mass in kDa.
To determine the class of protease responsible for the maturation of pro-PC2 and pro-PC3, chase reactions were performed in the presence of various protease inhibitors. Maturation of pro-PC2 and pro-PC3 was inhibited by high (1 mM) but not low (1 µM) concentrations of leupeptin, unaffected by pepstatin A or phenylmethylsulfonyl fluoride, and inhibited by high (10 mM) but not low (2 mM) concentrations of EDTA (data not shown). In these respects the inhibitor profile of maturation was similar to that of PC2 and PC3 activity against peptide substrates (Shennan et al., 1991a; Bailyes et al., 1992).
We next looked for
structural differences between pro-PC2 and pro-PC3 that might explain
the different rate of maturation of the two molecules. Most members of
the subtilisin family of proteases have a conserved asparagine that
stabilizes the oxyanion transition state. PC2 differs from other
members of the subtilisin-like family in that it contains an aspartate
in place of asparagine in this position. We therefore changed
Asp to Asn in PC2M5. PC2M5 was translated in the Xenopus egg extract for 2 h and then chased for up to 18 h in
buffer at pH 7.0 in the absence of added Ca
, i.e. the conditions that favored rapid processing of pro-PC3. Under
these conditions pro-PC2M5 was processed slightly more efficiently than
pro-PC2, but the rate of processing was nonetheless more like PC2 than
PC3 (Fig. 12).
Figure 12:
Effect of D285N mutation on time course
of processing of pro-PC2 at neutral pH. PC2 and PC2M5 mRNA were
translated in the egg extract for 1 h, and then RNase was added. Chase
reactions were performed at pH 7.0, and aliquots were removed at
various times. Membranes were prepared as described under
``Materials and Methods,'' and were analyzed by SDS-PAGE and
fluorography. Track M denotes C-labeled protein
markers, with the molecular mass in kDa.
Pro-PC2 and pro-PC3 are glycosylated at three and two sites, respectively. To determine whether the glycosylation state would affect maturation of either protease, translations were carried out in the presence of a tripeptide (Asn-Tyr-Thr) that acts as a substrate for glycosylation, competing for glycosylation of newly synthesized proteins (Lau et al., 1983). While the tripeptide inhibited glycosylation of both pro-PC2 (Fig. 13A, tracks 1 and 3) and pro-PC3 (Fig. 13B), the nonglycosylated pro-PC2 and pro-PC3 were processed to their mature forms with similar efficiencies as glycosylated pro-PC2 and pro-PC3.
Figure 13:
Effect of glycosylation on pro-PC2 and
pro-PC3 maturation. A, PC2 mRNA was translated in the egg
extract for 2 h in the presence or absence of 1.67 mM Asn-Tyr-Thr. The extract was then adjusted to pH 5.5 and 10 mM calcium and the reaction chased for 18 h at 21 °C. Membranes
were then prepared and analyzed by SDS-PAGE and fluorography. Track
M denotes C-labeled protein markers, with the
molecular mass in kDa. B, PC3 mRNA was translated in the egg
extract for 2 h in the presence or absence of 1.67 mM Asn-Tyr-Thr. Membranes were then prepared and analyzed by SDS-PAGE
and fluorography. Track M denotes
C-labeled
protein markers, with the molecular mass in
kDa.
We show here that the processing of pro-PC3 and pro-PC2
exhibits different pH optima and calcium requirements. Pro-PC3 (88-kDa
form) was rapidly processed (t < 10 min) to the active
80-kDa form of the enzyme in an autocatalytic reaction that occurred at
pH 7.0-8.0, and which was not dependent on additional
Ca. The exact Ca
concentration in
the Xenopus egg extract was not measured, but it is likely to
be within the micromolar range. The enzymic properties of autocatalytic
activation are, therefore, very different from those of the mature
enzyme, which has been shown to be optimally active in the pH range
5.5-6.5 and to exhibit a requirement for Ca
in
the millimolar range (K
= 2 mM)
(Jean et al., 1993; Rufaut et al., 1993; Zhou and
Lindberg, 1993; Vindrola and Lindberg, 1993; Bailyes et al.,
1992). We have not determined whether cleavage involves an intra- or
intermolecular reaction. If it is intramolecular, then our results
would suggest that conformational changes occur on removal of the
propeptide, which may result in different pH and calcium requirements
of the mature protease. Differences have previously been noted between
autoprocessing of furin and processing of substrates by furin (Creemers et al., 1993) and similarly between autoprocessing of Kex2 and
processing of substrates by Kex2 (Brenner et al., 1993).
The time course of pro-PC3 processing in the Xenopus egg
extract is similar to that observed when PC3 was expressed in GH4 cells
(Benjannet et al., 1992; Zhou and Lindberg, 1993) and in Cos-7
cells. ()Endogenous pro-PC3 in AtT20 cells is also rapidly
processed (Lindberg, 1994), suggesting that these events occur in an
early secretory compartment, most likely the endoplasmic reticulum.
This may be important for PC3 involvement in early processing events of
prohormone maturation. For example, PC3 is implicated in some of the
earliest events in pro-opiomelanocortin processing (Bloomquist et
al., 1991), and in proinsulin processing, PC3 is thought to
perform the initial processing step at the B-chain/C-peptide junction
before PC2 can act at the C-peptide/A-chain junction (Rhodes et
al., 1992). The observation that pro-PC3 processing occurs
efficiently at pH 7.0 and is not dependent on the addition of
Ca
is therefore compatible with current views on the
internal ionic environment of the endoplasmic reticulum (Anderson and
Orci, 1988). However, simple removal of the propeptide may not be
sufficient for enzyme activation. A furin mutant containing an
endoplasmic reticulum retention signal was efficiently processed to the
mature form in the endoplasmic reticulum, but this was enzymatically
inactive (Molloy et al., 1994) suggesting that movement
through the secretory pathway may confer further modifications leading
to activation. In the case of PC3, although it may be processed in the
endoplasmic reticulum, because of the low pH and high calcium
requirements of the mature protease, it is unlikely to be active until
later in the secretory pathway.
In GH4, AtT20, or mouse L cells, the
80-kDa form of PC3 undergoes further cleavage to generate 75- and
66-kDa polypeptides that are secreted. These molecules are
enzymatically active and are generated by COOH-terminal cleavage of the
80-kDa polypeptide, possibly by an autocatalytic mechanism that takes
place in the secretory vesicles of the regulated pathway (Zhou and
Lindberg, 1994). These smaller PC3-related peptides were not generated
in the Xenopus egg extract under various conditions of pH and
Ca concentration. In COS-7 cells
and
microinjected Xenopus oocytes (Bailyes et al., 1992)
the major secreted form of PC3 exhibits a molecular size of 80 kDa with
no further cleavage observed.
Pro-PC2 (75 kDa) was processed only slowly (t = 8 h) to the active (68 kDa) form in a reaction that required acidic pH and millimolar concentrations of calcium. The enzymic properties of pro-PC2 maturation were therefore identical to those of the mature enzyme when assayed both against small peptide fluorogenic substrates (Shennan et al., 1991a) and potential in vivo substrates (Rhodes et al., 1993). The conditions required for pro-PC2 maturation (acidic pH and high calcium) would suggest that pro-PC2 is not activated until late in the secretory pathway, i.e. in the trans-Golgi network or possibly not until the secretory granules. This interpretation, however, is at variance with the results of Guest et al.(1992) who showed that mature PC2 was present in subcellular fractions enriched in Golgi and endoplasmic reticulum elements.
We have confirmed our previous results (Matthews et al., 1994) that pro-PC2 maturation occurs by an autocatalytic reaction. Our present results show that mature PC2 can activate catalytically inactive pro-PC2 mutants by an intermolecular reaction. This is in contrast to Kex2 (Germain et al., 1992) and furin (Leduc et al., 1992; Creemers et al., 1993) where activation of the precursor molecules has been shown to be exclusively intramolecular. Our data would favor a two-step (intermolecular) reaction rather than a simple one-step (intramolecular) reaction for the maturation of pro-PC2. However, to define a process as intramolecular, the reaction must be shown to be independent of the enzyme concentration as, for example, in the maturation of pro-cathepsin B (Mach et al., 1994). A definitive answer to this question must await full kinetic analysis of pro-PC2 processing following overexpression and purification of pro-PC2 on a larger scale.
The catalytic importance of the middle/P-domain
of pro-PC2 has been shown by the inability of the deletion mutant PC2M7
either to process itself or another catalytically inactive mutant in trans. It is unlikely that this was simply due to
inappropriate folding as wild-type PC2 was able to process PC2M7 in trans. The P-domain of both Kex2 (Gluschankof and Fuller,
1994) and furin (Creemers et al., 1993) has previously been
shown to be necessary for catalytic activity; in furin the essential
region has been mapped to a 21-amino acid stretch from Glu to Glu
.
Pro-PC2 differs from the other
subtilisin-like serine proteases in having an aspartate residue,
instead of an asparagine residue, in a position which is thought to
stabilize the oxyanion transition state formed during substrate
processing. It has been suggested that the aspartate in PC2 might act
as an oxyanion hole when protonated, restricting its activity to acidic
pH (Steiner, 1991); however, the fact that PC3 activity is also
restricted to acidic pH despite having an asparagine as the oxyanion
hole might discount this view. The major differences we have found in
pH requirements for activation of pro-PC2 and pro-PC3 suggested that
the aspartate of the oxyanion hole of PC2 might be important in
restricting the pH at which maturation occurs. The mutant PC2M5, in
which the oxyanion aspartate was altered to an asparagine, was more
efficiently processed to its mature form at neutral pH, but the time
course of processing was still slow compared with that of pro-PC3. The
reverse substitution in pro-Kex2, i.e. Asn to Asp, had no
effect either on the rate of maturation of pro-Kex2 or its pH
requirements, but it did affect the activity of the mature protease
against substrates (Brenner et al., 1993). It is possible that
pro-PC2 has an additional oxyanion binding site and, indeed, there are
in pro-PC2 further residues around the Asp site that are
different in pro-PC3. For example PC2 has an Asp residue at position
291 that is an Asn in PC3 and, conversely, PC2 has an Asn at position
293 whereas PC3 has an Asp at the equivalent position. The importance
of these substitutions in the maturation or substrate specificity of
PC2 and PC3 is currently being investigated.