(Received for publication, August 23, 1994; and in revised form, October 25, 1994)
From the
Previously, we characterized murine mast cell
procarboxypeptidase A (MC-proCPA) as an inactive zymogen. To
investigate the mechanisms for this lack of enzymatic activity and the
processing of the zymogen to the active form, we now have performed
molecular modeling of the tertiary structure of murine MC-proCPA based
on the x-ray crystallographic structures of porcine pancreatic
procarboxypeptidases A and B. Our model predicts that MC-proCPA retains
a high degree of structural similarity to its pancreatic homologues.
The globular propeptide physically blocks access to the fully formed
active site of the catalytic domain and contains a salt bridge to the
substrate-binding region that precludes docking of even small
substrates. Based on consideration of the predicted tertiary structure
and charge field characteristics of the model, the activation site
(between Glu and Ile
) appears to be highly
exposed even after MC-proCPA binds to secretory granule proteoglycans.
Based on the steady-state levels of MC-proCPA versus MC-CPA,
cycloheximide inhibition of protein synthesis, and brefeldin A blockage
of protein sorting, we show that MC-proCPA is processed rapidly in
murine mast cell line KiSV-MC14 with a half-life of 26 ± 5 min
(mean ± S.D., n = 3), and the processing occurs
within the secretory granules. The enzyme responsible for this
processing may be a thiol protease since treatment of the KiSV-MC14
with 200 µM E-64d, a selective thiol-protease inhibitor,
increases MC-proCPA by 2.7 ± 0.2-fold (mean ± S.D., n = 3) within 6 h of application.
The secretory granules of mast cells contain various proteases
that possess maximal activities at extracellular pH, including
carboxypeptidase A
(MC-CPA)()(1, 2, 3, 4) ,
tryptases(5, 6, 7, 8) ,
chymases(5, 7, 9, 10, 11) ,
and an aminopeptidase(12) . Although tryptase has been
implicated in bronchospasm(13) , chymase in mucous secretion (14) and vasoconstriction (15) and MC-CPA in
vasodilation(16) , the actual physiologic functions and peptide
targets of the mast cell secretory proteases are unknown. These
proteases are similar to a broader set of immune/inflammatory cell
secretory proteases in that they are produced as inactive zymogens but
stored within the secretory granules in their fully active forms. These
immune/inflammatory proteases are activated at unusual processing sites (17, 18) . For example, mast cell tryptases are
activated by cleavage of their propeptides at a Gly residue; MC-proCPA,
mast cell chymases, neutrophil elastase and human cathepsin G, and most
lymphocyte granzymes are activated by cleavage at an acidic residue. We
have demonstrated that activation of the prochymases, protryptases, and
MC-proCPA requires three distinct mechanisms(18) . McGuire et al.(19) have shown that a lysosomal thiol
protease, dipeptidyl peptidase I, is involved in the activation of
proelastase and procathepsin G in neutrophils and granzymes in
cytotoxic T lymphocytes. We have shown that dipeptidyl peptidase-I is
involved in the processing of mast cell prochymases(18) . The
enzymes responsible for activation of protryptases and MC-proCPA remain
unknown. In this study, we have examined the properties of MC-proCPA
activation by molecular modeling and biochemical means.
Figure 1:
Comparisons of the mast cell and
pancreatic procarboxypeptidases. A, the amino acid sequence
alignments used as a basis for the molecular modeling are shown with
porcine pancreatic procarboxypeptidase B (PPB)(53) ,
murine mast cell procarboxypeptidase A (MMC)(26) ,
human mast cell procarboxypeptidase A (HMC)(2) , and
porcine pancreatic procarboxypeptidase A (PPA)(39) . B, percent amino acid sequence identities between the mast
cell and porcine pancreatic carboxypeptidases. C, RMS
deviations (in Å) were calculated comparing the C traces for
the indicated carboxypeptidases. Reported x-ray crystallographic data
were used for the porcine pancreatic
carboxypeptidases(39, 53) ; the coordinates from our
molecular models were used for the mast cell
carboxypeptidases.
A space-filling model
of murine MC-proCPA is shown in Fig. 2. The salient features of
the model are the globular propeptide domain that physically blocks
access to the substrate-binding pocket and the highly exposed loop
connecting the globular propeptide with the mature enzyme. This
predicted loop contains the activation site between Glu (Glu
of the activation domain) and Ile
(the first residue of the mature enzyme). Our models of murine
and human MC-proCPAs possess a high degree of structural similarity to
each other and to their pancreatic homologues (Fig. 1C and Fig. 2).
Figure 2:
Upper, the propeptide with activation loop (gray) and the catalytic domain (black) are indicated
in a space-filling model. This and subsequent models were created using
Biosym Insight II. Lower, the ribbon
structures for mouse MC-proCPA (black) and porcine pancreatic
proCPB (gray) are superimposed. The orientation is the same
for both upper and lower models.
Similar to the pancreatic
procarboxypeptidases, the models predict that the active sites of the
murine and human MC-proCPAs are fully formed in the zymogens, but
substrate binding is blocked or impaired by the propeptide. Detailed
examination of the murine MC-proCPA model revealed that a loop
comprised of residues A39-A44 of the propeptide projects into the
active site of the mature form, and the side chain carboxyl group of
Asp within this propeptide loop forms a salt bridge with
the guanidyl group of Arg
in the catalytic domain.
Formation of this salt bridge also causes the side chain of Arg
to assume a more extended conformation relative to its
conformation in the active enzyme. The predicted salt bridge between
the propeptide and the substrate-binding region of murine MC-proCPA is
found in porcine pancreatic proCPB and accounts for its complete lack
of enzymatic activity(20) . Interestingly, an alternative salt
bridge is possible in murine MC-proCPA between the side chain carboxyl
group of Asp
and Arg
. When this alternative
salt bridge was initially forced by rotation of the side chains of
Asp
and Arg
, it was found to be stable
during further energy minimization. In contrast, human MC-proCPA
contains no analogous acidic residue in the propeptide in proximity to
Arg
of the catalytic domain. The result is that the
propeptide loop of residues A39-A44 does not project as far into
the active site, and the Arg
side chain assumes a
position closer to that observed in the active enzyme. This lack of a
salt bridge between the propeptide and the substrate-binding region of
human MC-proCPA is similar to porcine pancreatic proCPA (21) and suggests that human MC-proCPA may possess some
activity toward small peptide substrates.
Electrostatic field
calculations were performed on the murine and human MC-proCPA and
MC-CPA models at the secretory granule pH of 5.5. The results for the
murine models are shown in Fig. 3and were substantially the
same for the human models. Positive charge fields nearly surrounded the
zymogen (270° of the circumference, Fig. 3A).
The only substantial area lacking a positive charge field was that of
the activation loop. With the propeptide removed, the electrostatic
fields were recalculated for the mature enzyme model. A positive charge
field again surrounded
270° of the circumference of MC-CPA,
including a significant positive charge field uncovered by the removal
of the propeptide and projecting from the substrate-binding site (Fig. 3B). No projections of negative charge were
observed in our MC-proCPA or MC-CPA models at pH 5.5, with the
exception of a small negative charge field emanating from the
activation loop of MC-proCPA. However, an intense negative charge field
was observed projecting from the MC-CPA propeptide, including the
activation loop, encompassing the entire side that had faced the
catalytic domain (Fig. 3C).
Figure 3: Electrostatic fields surrounding murine MC-proCPA, murine MC-CPA, and murine MC-CPA propeptide at secretory granule pH. The colored gradients depict the positive charge (blue) and negative charge (red) electrostatic fields surrounding MC-proCPA (A) and MC-CPA (B) and free MC-CPA propeptide (C). Also depicted is a proposed scheme for the binding of MC-proCPA and MC-CPA to negatively charged proteoglycans. The site of proteolytic processing (activation loop) and substrate-binding site is indicated. The orientations are the same as for Fig. 2. Calculations were performed based on models minimized at pH 5.5 using the Insight II program DelPhi with the following parameters: solute dielectric, 2.0; solvent dielectric, 80; full Coulombic boundary; ionic strength, 0.145; probe radius, 1.4 Å; and exclusion radius, 2.0 Å.
Figure 4: Processing of MC-proCPA in KiSV-MC14 in the absence of new protein synthesis. A, a representative immunoblot of KiSV-MC14 treated with cycloheximide for the indicated times. The blot was probed with anti-MC-CPA antibodies; detection was by chemiluminescence. B, two additional cycloheximide inhibition experiments were performed, and each of the three immunoblots was scanned with a laser densitometer to quantify the MC-proCPA. The scan results of all three experiments are plotted, and a best-fit curve has been calculated.
BMMC were pretreated with cycloheximide to deplete their stores of MC-proCPA and, after removing the cycloheximide, cultured in the presence or absence of brefeldin A to inhibit transport beyond the endoplasmic reticulum. Continued culture in the absence of brefeldin A resulted in no significant change in the amount of MC-proCPA in the BMMC (Fig. 5). In contrast, culture in the presence of brefeldin A caused a rapid and progressive accumulation of MC-proCPA (Fig. 5) with a 5.2-fold increase at 4 h.
Figure 5: Buildup of MC-proCPA in BMMC in the absence of protein transport out of the endoplasmic reticulum. A, BMMC were treated with cycloheximide (cyclohex) or not treated (initial) for 5 h. The cycloheximide was removed, and the cells were cultured for an additional 0-4 h in the presence or the absence of brefeldin A. Following SDS-polyacrylamide gel electrophoresis and transblotting, detection was by anti-MC-CPA antibodies and chemiluminescence. One of two experiments is shown. B, MC-proCPA levels in BMMC treated with brefeldin A (solidsymbols) or untreated (opensymbols) as detected by immunoblotting.
Figure 6: E-64d inhibits processing of MC-proCPA. KiSV-MC14 were cultured in the presence of 200 µM E-64d (solidsymbols) or in the absence of inhibitor (opensymbols). Levels of MC-proCPA were determined by immunoblotting. Three experiments (indicated by the threesymbolshapes) were performed separately, and all of the results are depicted along with linear regressions for the cumulative data. Linear regressions were also calculated separately for each experiment, allowing us to calculate the degree of increase at 6 h for each experiment. The mean increase in MC-proCPA at 6 h was 2.7 ± 0.2-fold (± S.D., n = 3) for E-64d-treated cells.
The principal requirement for pancreatic procarboxypeptidase
activation is removal of the propeptide from the active site.
Nevertheless, the proCPA and proCPB pancreatic zymogens possess
different properties that can be explained by structural differences in
their propeptides(21, 31) . In pancreatic proCPA, the
propeptide acts as a competitive inhibitor of CPA even after it is
disconnected from the catalytic domain(37) . This mechanism of
inhibition results in a zymogen that is active against small peptides
and even retains significant activity against protein substrates such
as casein(38) . The limiting event in pancreatic proCPA
activation is degradation of the propeptide, which has a rather stable
structure even when free from the enzyme(37) . Unlike
pancreatic CPA, CPB is not inhibited by its propeptide once cleavage
occurs at the activation site(39, 40) . Thus,
degradation of the propeptide following cleavage is not a limiting
factor. Furthermore, pancreatic proCPB is entirely devoid of
carboxypeptidase activity even against small substrates(39) .
This appears to be due to the formation of a salt bridge between
Asp of the propeptide and Arg
of the
catalytic domain (corresponding to Asp
and Arg
in MC-proCPA, see Fig. 1) that prevents binding of the
substrate carboxyl terminus(20) .
Our model of murine
MC-proCPA indicates that the propeptide not only serves to generally
block the substrate-binding pocket of the mature form (Fig. 2),
it also is linked through a salt bridge to the critical
substrate-binding residue Arg. This particular Arg
residue is conserved in all mammalian metallocarboxypeptidases,
including CPA, CPB, carboxypeptidase H, carboxypeptidase M, and plasma
CPB. The basic side chain of this Arg serves to form a salt bridge with
the acidic carboxyl terminus of substrates, thereby positioning the
peptide bond of the carboxyl-terminal amino acid in the reactive center
where it is cleaved. Since the propeptide of murine MC-CPA appears to
form a salt bridge with Arg
, the mechanism of inhibition
of murine MC-proCPA by its propeptide is likely the same as that for
pancreatic proCPB (20, 31) and, therefore, predicts
that MC-proCPA exists as a completely inactive zymogen. This view is
consistent with our prior experimental data regarding the lack of
activity of MC-proCPA even toward small substrates (18) and our
observation that purified murine MC-CPA is not inhibited by purified
MC-CPA propeptide expressed in bacteria. (
)In contrast to
murine MC-proCPA, human MC-proCPA cannot contain an analogous salt
bridge between the propeptide and Arg
, suggesting that
human MC-proCPA may exhibit significant reactivity to small substrates.
In addition to maintaining the zymogen in an inactive state, the
propeptide may serve other functions. We previously showed that
recombinant MC-CPA (i.e. lacking the propeptide) expressed in
mammalian cells (COS and U937) or bacterial cells (Escherichia
coli) is completely inactive. This suggests that the propeptide of
MC-proCPA assists in the proper folding of the catalytic domain. This
type of propeptide function has been described in subtilisin (41) and -lytic protease(42) . In these proteases,
the propeptides serve to lower the energy barrier between an
incompletely folded intermediate and the native conformation, resulting
in a substantial increase in the rate of a crucial folding
step(43, 44) .
We have shown that the steady-state
level of MC-proCPA is low relative to that of the active form in
KiSV-MC14 and BMMC ( Fig. 4and Fig. 5)(18) ,
indicating rapid processing of the zymogen. Because both mast cell
types have a doubling time of 48 h(45) , they are making
2% of their MC-CPA/h. Since we see
1% of the total MC-CPA in
the zymogen form, the MC-proCPA must be processed with a half-life of
<1 h. Using cycloheximide treatment (Fig. 4), we have
confirmed that MC-proCPA is rapidly turned over, with half-lives of 26
± 5 min in KiSV-MC14 and 28 ± 4 min in BMMC. The loss of
MC-proCPA did not result from secretion as evidenced by the complete
retention of the mature MC-CPA (Fig. 4A), which is
contained in the same secretory granules(18) , and the absence
of MC-proCPA in the culture medium (not shown). The processing event
appeared to be specific since no smearing or intermediate bands were
observed between MC-proCPA and MC-CPA.
The intracellular location for the activation of MC-proCPA was not previously determined. By confocal immunofluorescence microscopy and cellular activation studies, we showed that the majority of MC-proCPA is located in the secretory granules, and we speculated that this is where the processing occurred(18) . Since the cycloheximide treatment of KiSV-MC14 and BMMC indicated that the large majority of the MC-proCPA is quickly processed and the majority of the MC-proCPA is located in the secretory granules, we conclude that this is where the processing occurs. In support of this, blocking transport from the endoplasmic reticulum with brefeldin A caused a buildup of unprocessed MC-proCPA (Fig. 5), indicating that processing occurs in a post-endoplasmic reticulum compartment.
The mechanism of activation of MC-proCPA can be
considered in view of our molecular models. The segment connecting the
propeptide and the catalytic domains contains the highest degree of
non-conserved structure between pancreatic proCPB and pancreatic proCPA
and contains the longest single stretch of non-conserved sequence
between the pancreatic and mast cell forms (Fig. 1). Thus, it is
the most speculative structural element in the MC-proCPA models. The
residues specifying the primary site of proteolytic activation have
been determined to be among the most highly exposed residues in both of
the porcine pancreatic zymogens(20, 21) . The overall
motif of the connecting segment is a helix followed by a loop. The
helix and loop are much shorter in porcine pancreatic proCPB and in the
MC-proCPAs than in porcine pancreatic proCPA, due to the relative
deletion of 6 amino acids in pancreatic proCPB and the MC-proCPAs (Fig. 1). The activation segments of our modeled MC-proCPAs, and
particularly the loop portions, are exceptionally highly exposed (Fig. 2). This is enhanced by the high content of charged amino
acids (8 of 14) in the segments leading up to the activation site. By
comparison, porcine pancreatic proCPB and proCPA have only 4 and 6
charged amino acids, respectively, within their activation loops. Thus,
our model predicts that the activation site in the connecting segment
of the MC-proCPAs (between Glu and Ile
in
murine MC-proCPA) is highly susceptible to proteolytic cleavage.
Of the 8 charged amino acids in the 14-amino acid activation loop of murine and human MC-proCPA, 6 are negatively charged. This 3:1 ratio of negatively to positively charged amino acids is in marked contrast compared with MC-proCPA as a whole. Murine MC-proCPA has an excess of 23 positively charged amino acids compared with negatively charged amino acids at pH 5.5, including His residues. Human MC-proCPA has an excess of 20 positively charged amino acids. Thus, the predominance of negatively charged amino acids in the activation loop markedly alters the overall strong positive charge field characteristics of the MC-proCPA molecule (Fig. 3A). The predominance of negatively charged amino acids in the activation loop merits consideration in light of the fact that processing of MC-proCPA appears to occur in the secretory granules where highly negatively charged proteoglycans reside. We propose that the configuration of the overall positive charge field orients MC-proCPA such that binding to negatively charged proteoglycans leaves the activation loop exposed for cleavage by an activating protease (Fig. 3A) but provides protection from degradative proteases that are already activated within the secretory granule. Similarly, after removal of the propeptide, the cradle of positive charge surrounding MC-proCPA (Fig. 3B) serves to sequester it within proteoglycan complexes (1, 12) in an orientation that leaves the active site accessible to substrates but prevents degradative attack on the MC-CPA molecule itself.
In contrast to MC-CPA, the predominant field surrounding the propeptide is a negatively charged field emanating from the inner (active site) face (Fig. 3C). This negatively charged field may contribute substantial stability to the association of the propeptide domain with the catalytic domain while the zymogen is intact. The overall negative charge on the propeptide (19 acidic versus 17 basic residues at pH 5.5) may also serve another function, that of precluding attachment to the proteoglycan-protease complexes (1, 12, 46) once the propeptide has been severed from the zymogen. We have shown that the propeptide is rapidly degraded once it is cleaved from the zymogen, since no free propeptide is detectable within mast cells or in their culture medium despite our ability to detect it at levels below 0.5% that of the mature enzyme(18) . The overall negative charge of the propeptide and its presumed resultant inability to become incorporated into the protease-proteoglycan complexes may facilitate its rapid degradation.
Given a secretory granule pH of
5.5(47, 48, 49) , MC-proCPA appears to
be processed in an acidic environment. Because thiol proteases
typically have pH optima near pH 5.5, we considered a thiol protease to
be a likely candidate for the MC-proCPA processing enzyme.
Additionally, a thiol protease, dipeptidyl peptidase-I, is involved in
the processing of prochymases(18) , as well as neutrophil
proelastase and procathepsin G and cytotoxic T-lymphocyte
progranzymes(19) . Dipeptidyl peptidase-I does not participate
in MC-proCPA processing (18) .
E-64d is an epoxide-based,
irreversible inhibitor that is selective for thiol proteases, enters
the lysosomes of intact cells, and exhibits minimal cellular
toxicity(27, 28) . When intact KiSV-MC14 were treated
with E-64d, progressive accumulation of MC-proCPA occurred, consistent
with the inhibition of its processing to the active form (Fig. 6). The fact that dipeptidyl peptidase-I activity was
inhibited by 96% after 6 h of treatment indicates that the E-64d had
effectively entered the secretory granules, and the fact that chymase
and tryptase activities were not substantially decreased after 6 h of
treatment indicates that E-64d was acting to specifically inhibit thiol
proteases. In contrast, the protease inhibitors TLCK, TPCK, leupeptin,
and t-Boc-Gly-Phe-CHN did not inhibit MC-proCPA
processing in intact KiSV-MC14. However, TLCK and TPCK also did not
inhibit the activities of dipeptidyl peptidase-I, chymase, or tryptase
in the cells, indicating that they likely did not gain access to the
secretory granules. We conclude that a thiol protease within the
secretory granule, other than dipeptidyl peptidase-I, is involved in
the processing of MC-proCPA.
We found a pool of 10-20% of
MC-proCPA, equivalent to 0.05-0.1% of the total MC-CPA, that does
not appear to be susceptible to processing (note the non-zero asymptote
in Fig. 4B). This portion of the MC-proCPA may reside
in the secretory granules but be sequestered in a way that prevents its
activation. For example, the charge characteristics of murine MC-proCPA
(predicted to be +23 at pH 5.5) suggest that it can bind ionically
to acidic proteoglycans (Fig. 3A), as has been shown
for the active form(1) . MC-CPA has been shown to exist in both
lower molecular weight (M 2
10
) and higher molecular weight (M
> 1
10
) protease-proteoglycan
complexes(1) , where the higher molecular weight complexes are
formed from aggregates of the lower molecular weight
complexes(46) . The majority of MC-CPA is associated with the
higher molecular weight complexes. Due to the substantial positive
charge on the MC-proCPA, even at pH 7.4, it likely binds to
proteoglycan as soon as the two come in proximity. This may occur early
in the intracellular trafficking pathway, for example, within the
transport vesicles between the endoplasmic reticulum and Golgi, and
almost certainly as soon as MC-proCPA reaches the secretory granule.
Our model of MC-proCPA-proteoglycan interaction (Fig. 3A) predicts that MC-proCPA remains accessible to
processing even when bound in the lower molecular weight
protease-proteoglycan complexes. We speculate that processing of
MC-proCPA must occur while it is involved in the lower molecular weight
complexes and that a small percentage of MC-proCPA (0.05-0.1% of
the total MC-CPA) initially escapes processing and is then sequestered
in the large protease-proteoglycan aggregates where it is inaccessible
to the processing enzyme. Relevant to this is the fact that rat mast
cell protease-I (RMCP-I), while bound in large complexes with heparin
proteoglycans, is entirely stable even when incubated for 2 months at 4
°C. Once removed from the proteoglycans, RMCP-I becomes accessible
to cleavage and rapidly self-catabolizes even at 4 °C(50) .
Furthermore, RMCP-I bound to heparin in large complexes cannot digest
substrates larger in size than 17 kDa; unbound RMCP-I shows no size
discrimination for substrates(51) . Since rat MC-CPA is bound
in the same large protease-proteoglycan complexes as
RMCP-I(52) , access to it is likely severely limited once it is
fully incorporated. Our hypotheses concerning the affects of
proteoglycan binding and macromolecular complex formation on MC-proCPA
processing remain to be experimentally determined, but preliminary data
suggest that the processing-resistant MC-proCPA does reside in the
macromolecular complexes. (
)
Heparin has been identified
as a required cofactor in the activation of human prochymase. ()The negatively charged Glu residue immediately adjacent to
the activation site of prochymase likely repels the similarly charged
heparin in such a way that leaves the cleavage site freely available
for proteolytic attack by dipeptidyl peptidase-I. Since mouse and rat
MC-proCPAs have a Glu residue and human MC-proCPA has an Asp residue in
exactly the same position as the Glu residue in the propeptide of
prochymase, we speculate that a similar mechanism of heparin-mediated
activation may exist in the MC-proCPAs.