©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mast Cell Procarboxypeptidase A
MOLECULAR MODELING AND BIOCHEMICAL CHARACTERIZATION OF ITS PROCESSING WITHIN SECRETORY GRANULES (*)

(Received for publication, August 23, 1994; and in revised form, October 25, 1994)

Eric B. Springman (2)(§)(¶) Michael M. Dikov(§) (1) William E. Serafin (2) (1)(**)

From the  (1)From theDepartment of Medicine, Allergy Division, and the (2)Department of Pharmacology, Clinical Pharmacology Division, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232-0111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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^1) 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.


INTRODUCTION

The secretory granules of mast cells contain various proteases that possess maximal activities at extracellular pH, including carboxypeptidase A (MC-CPA)(^1)(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.


MATERIALS AND METHODS

MC-proCPA Model Construction

Coordinates for the x-ray crystallographic structures of porcine pancreatic procarboxypeptidase B (proCPB) (20) and proCPA (21) were generously provided by Dr. Robert Huber (Max Planck Institute for Biochemistry, Martinsreid bei Munchen, Germany). Coordinate files for bovine pancreatic CPA (pdb3cpa.ent) and CPB (pdb1cpb.ent) were obtained from the Brookhaven Protein Data Bank. Modeling was performed using the Biosym (San Diego, CA) Insight II molecular graphics suite running on a Silicon Graphics (Mountain View, CA) IRIS Indigo workstation. The amino acid sequences for murine and human MC-proCPA were aligned with those of porcine proCPA and proCPB as shown in Fig. 1A, and the calculated amino acid sequence identities are shown in Fig. 1B. The spatial coordinates of structurally conserved regions in porcine proCPB and proCPA were applied directly to the corresponding amino acid sequences of murine and human MC-proCPA using the Insight II program Homology. Four short stretches, consisting of residues A90-4, 150-154, 213-216, and 286-287 of both murine and human MC-proCPA, could not be directly assigned and were assigned by searching sets of energetically favored loops. Energy minimization was performed using the Insight II program Discover. The only constraint used during the minimizations was to maintain the four zinc-ligand atoms His:N1, Glu:O1, Glu:O2, and His:N1 in their original coordinates to preserve the structure of the zinc-binding site, although the zinc ion itself was not included during the minimization process. The details of the energy minimization process for murine MC-proCPA are shown in Table 1. A similar minimization scheme was used for human MC-proCPA. As a control, porcine pancreatic proCPB was modeled in the same way starting with its amino acid sequence. The RMS deviation between the backbone coordinates of the modeled proCPB and the original crystallographic coordinates was 0.24 Å; the final energy of the model was -12,867 kcal/mol with a 0.0073 kcal/Å root mean square derivative. This indicates that our procedure for modeling of the MC-proCPAs likely did not introduce any unreasonable conformations or interactions.


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 Calpha 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.





Treatment of Mast Cells with Cycloheximide and Brefeldin A

Murine bone marrow-derived mast cells (BMMC) (22) and KiSV-MC14 (23) were cultured, harvested in log-phase growth, resuspended at a concentration of 3 times 10^5/ml in 10 ml of fresh culture medium containing cycloheximide (0.5 mM), and incubated at 37 °C, 5% CO(2)(24) . In a separate experiment, existing MC-proCPA was depleted by pretreatment of the cells with cycloheximide (0.5 mM) for 5 h as described above, the cycloheximide was removed from the culture with three successive washes, and the culture was continued in the absence of cycloheximide with or without the addition of brefeldin A (5 µg/ml) (25) . In each experiment, 3 times 10^5 cells were removed from the culture at timed intervals, and 1.5 times 10^5 cell equivalents were subjected to immunoblotting as previously described using anti-MC-CPA antiserum(18) . Using this antiserum, specific immunoreactivity (i.e. competable by recombinant MC-CPA) is found in mast cells but not in U937 human promonocytes(18) , murine 3T3 fibroblasts, or murine WEHI-3B myelomonocytic cells (not shown). To quantify MC-proCPA, laser densitometric scanning (Ultroscan 2202, LKB, Bromma, Sweden) was performed on the exposed films, and the MC-proCPA was expressed as area units (OD times mm). To ensure accuracy, three scans of each lane were performed, and the average of the three scans was used.

Treatment of Mast Cells with Protease Inhibitors

KiSV-MC14 were pelleted by centrifugation at 150 times g for 10 min, resuspended at a concentration of 3 times 10^5/ml in fresh complete medium, and cultured in the absence or presence of protease inhibitor in the medium. E-64d (Matreya, Inc., Pleasant Gap, PA) (27, 28, 29) was added at 200 µM, or N-p-tosyl-L-lysine chloromethyl ketone (TLCK, Sigma) was added at 100 µM; N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, Sigma) was added at 20 µM, or t-Boc-Gly-Phe diazomethyl ketone (synthesis as previously described(18, 30) ) was added at 10 µM. Timed samples containing 1.8 times 10^6 cells were withdrawn; 0.15 times 10^6 cell equivalents were subjected to immunoblotting, and the remaining cells were used for the determination of dipeptidyl peptidase-I, chymase, and tryptase activities as previously described (18) .


RESULTS

Molecular Modeling

Based upon areas of sequence identity and predicted secondary structural similarities, we aligned the sequences of mast cell and pancreatic procarboxypeptidases (Fig. 1A) and calculated the percent sequence identities for the proenzymes, propeptides, and mature enzymes (Fig. 1B). The proposed alignment lends credibility to the modeling of the MC-proCPAs using porcine pancreatic proCPB for the following four reasons. First, the propeptides of MC-proCPA and pancreatic proCPB share 40% sequence identity and 60% sequence similarity when conservative substitutions are considered; as a whole, MC-proCPA and porcine pancreatic proCPB share 65% sequence similarity when conservative substitutions are considered. Second, no sequence gaps were required for alignment of the propeptides, and only one gap of a single amino acid in porcine pancreatic proCPB was required for alignment of the mature forms. Third, the propeptides of MC-proCPA and porcine pancreatic proCPB share more identity with each other (40%) than do the propeptides of porcine pancreatic proCPB and porcine pancreatic proCPA (29%); yet, the two pancreatic propeptides have been separately determined by x-ray crystallography to have highly similar tertiary structures(20, 21, 31) . Fourth, the overall amino acid sequence identities between all four procarboxypeptidases are 40-50% (Fig. 1B). Comparisons of homology-based molecular models where crystallographic coordinates were subsequently determined indicate that a sequence identity of 30% or greater is often sufficient for homology-based modeling of functionally similar proteins (32, 33) . For example, modeling of HIV-protease based on the crystal structure of Rous sarcoma virus-protease (34) and subsequent crystallography of HIV-protease (35) have demonstrated that homology-based molecular models can be remarkably accurate, even for proteins that share only 30% sequence identity(36) .

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^1 (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.



Cellular Location and Rate of MC-proCPA Processing

To determine whether the low amount of MC-proCPA present in mast cells is actively processed or represents a small percentage of MC-proCPA that has escaped appropriate processing, we treated KiSV-MC14 with cycloheximide and studied the fate of the MC-proCPA in the absence of new protein synthesis. Cycloheximide treatment resulted in a rapid decrease in the MC-proCPA within the cells while the levels of MC-CPA remained constant. A representative experiment is shown in Fig. 4A. Quantitative analyses revealed exponential decreases in the level of the zymogen, with a half-life of 26 ± 5 min (mean ± S.D., n = 3) in KiSV-MC14 (Fig. 4B). This analysis also suggested that 10% of the MC-proCPA in KiSV-MC14 was inaccessible to the processing machinery (note the asymptote in Fig. 4B). Similar results were obtained when BMMC were treated with cycloheximide, yielding a half-life of 28 ± 4 min (mean ± S.D., n = 2) for MC-proCPA and 17% MC-proCPA inaccessible to processing (not shown).


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.



Effects of Protease Inhibitors on MC-proCPA Processing

When KiSV-MC14 were cultured in the presence of the 200 µM E-64d for 7 h, a progressive increase in the level of MC-proCPA was observed (Fig. 6), but in the absence of inhibitor the levels of MC-proCPA remained constant. In the presence of leupeptin, TLCK, TPCK, or t-Boc-Gly-Phe-CHN(2), the levels of MC-proCPA were unaffected (not shown). Protease activities were examined in KiSV-MC14 after 6 h of incubation with E-64d, as well as in untreated KiSV-MC14 cultured in parallel. After incubation of KiSV-MC14 with 200 µM E-64d for 6 h, we found that dipeptidyl peptidase-I activity was inhibited by 96 ± 1% (mean ± S.D., n = 2). In the same samples we found that chymase activity was reduced by 13 ± 1% (mean ± S.D., n = 2), while tryptase activity was unaffected. In contrast, treatment of KiSV-MC14 with the protease inhibitors TLCK and TPCK did not significantly affect the activities of dipeptidyl peptidase-I, chymase, or tryptase in the secretory granules.


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.




DISCUSSION

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. (^2)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 alpha-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 leq1% 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^1 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(2) 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(r) 2 times 10^5) and higher molecular weight (M(r) > 1 times 10^7) 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. (^3)

Heparin has been identified as a required cofactor in the activation of human prochymase. (^4)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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI31273 and GM15431, a starter grant from the Burroughs Wellcome Fund, and the Immunology Development Fund, Vanderbilt University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work.

Supported by National Institutes of Health Training Grant GM-07569 and subsequently by a postdoctoral fellowship from the American Lung Association.

**
To whom correspondence should be addressed: 847 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0111; Fax: 615-322-7194. The coordinates for the murine and human MC-proCPA and MC-CPA models are available by E-mail by request to SERAFIN{at}CTRVAX.VANDERBILT.EDU.

(^1)
The abbreviations used are: MC-CPA, mast cell carboxypeptidase A; BMMC, bone marrow-derived mast cells; KiSV-MC, Kirsten sarcoma virus-immortalized mast cells; MC-proCPA, mast cell procarboxypeptidase A; RMCP-I, rat mast cell protease-I; proCPB, procarboxypeptidase B; HIV, human immunodeficiency virus; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

(^2)
M. M. Dikov and W. E. Serafin, unpublished data.

(^3)
E. B. Springman and W. E. Serafin, unpublished data.

(^4)
M. Murakami, S. S. Karnik, and A. Husain, personal communication.


ACKNOWLEDGEMENTS

Dr. Ahsan Husain (The Cleveland Clinic Foundation) made his manuscript concerning the processing of mast cell chymases available prior to its submission for publication. Mrs. Daphne Mitchell expertly cultured the BMMC. We thank Dr. Marcia Newcomer for her assistance with the modeling software and Dr. John Murray for use of the high performance liquid chromatography system and autoinjector. The Vanderbilt Cancer Center allowed us access to the Biosym software. Dr. Carsten Kaerlein (Max Planck Institute for Biochemistry, Martinsreid bei Munchen, Germany) arranged transfer of the molecular coordinate data for the pancreatic procarboxypeptidases. Dr. Suresh Yeola and Sam Saleh synthesized the t-Boc-Gly-Phe-CHN(2).


REFERENCES

  1. Serafin, W. E., Dayton, E. T., Gravallese, P. M., Austen, K. F., and Stevens, R. L. (1987) J. Immunol. 139, 3771-3776 [Abstract/Free Full Text]
  2. Reynolds, D. S., Gurley, D. S., Stevens, R. L., Sugarbaker, D. J., Austen, K. F., and Serafin, W. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9480-9484 [Abstract]
  3. Everitt, M. T., and Neurath, H. (1980) FEBS Lett. 110, 292-296 [CrossRef][Medline] [Order article via Infotrieve]
  4. Goldstein, S. M., Kaempfer, C. E., Proud, D., Schwartz, L. B., Irani, A. M., and Wintroub, B. U. (1987) J. Immunol. 139, 2724-2729 [Abstract/Free Full Text]
  5. Reynolds, D. S., Stevens, R. L., Lane, W. S., Carr, M. H., Austen, K. F., and Serafin, W. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3230-3234 [Abstract]
  6. Reynolds, D. S., Gurley, D. S., Austen, K. F., and Serafin, W. E. (1991) J. Biol. Chem. 266, 3847-3853 [Abstract/Free Full Text]
  7. Vensel, W. H., Komender, J., and Barnard, E. A. (1971) Biochim. Biophys. Acta 250, 395-407 [Medline] [Order article via Infotrieve]
  8. Schwartz, L. B., Lewis, R. A., and Austen, K. F. (1981) J. Biol. Chem. 256, 11939-11943 [Abstract/Free Full Text]
  9. Serafin, W. E., Reynolds, D. S., Rogelj, S., Lane, W. S., Conder, G. A., Johnson, S. S., Austen, K. F., and Stevens, R. L. (1990) J. Biol. Chem. 265, 423-429 [Abstract/Free Full Text]
  10. Serafin, W. E., Sullivan, T. P., Conder, G. A., Ebrahimi, E., Marcham, P., Johnson, S. S., Austen, K. F., and Reynolds, D. S. (1991) J. Biol. Chem. 266, 1934-1941 [Abstract/Free Full Text]
  11. Schechter, N. M., Choi, J. K., Slavin, D. A., Deresienski, D. T., Sayama, S., Dong, G., Lavker, R. M., Proud, D., and Lazarus, G. S. (1986) J. Immunol. 137, 962-970 [Abstract/Free Full Text]
  12. Serafin, W. E., Guidry, U. A., Dayton, E. T., Kamada, M. M., Stevens, R. L., and Austen, K. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5984-5988 [Abstract]
  13. Sekizawa, K., Caughey, G. H., Lazarus, S. C., Gold, W. M., and Nadel, J. A. (1989) J. Clin. Invest. 83, 175-179 [Medline] [Order article via Infotrieve]
  14. Sommerhoff, C. P., Caughey, G. H., Finkbeiner, W. E., Lazarus, S. C., Basbaum, C. B., and Nadel, J. A. (1989) J. Immunol. 142, 2450-2456 [Abstract/Free Full Text]
  15. Urata, H., Kinoshita, A., Misono, K. S., Bumpus, F. M., and Husain, A. (1990) J. Biol. Chem. 265, 22348-22357 [Abstract/Free Full Text]
  16. Serafin, W. E., Burch, W., Quershi, H., and Murray, J. J. (1993) J. Allergy Clin. Immunol. 91, 255
  17. Springman, E. B., and Serafin, W. E. (1994) in The Biology of Mast Cell Proteases (Caughey, G. H., ed) pp. 169-201, Marcel Dekker, New York
  18. Dikov, M. M., Springman, E. B., Yeola, S., and Serafin, W. E. (1994) J. Biol. Chem. 269, 25897-25904 [Abstract/Free Full Text]
  19. McGuire, M. J., Lipsky, P. E., and Thiele, D. L. (1993) J. Biol. Chem. 268, 2458-2467 [Abstract/Free Full Text]
  20. Coll, M., Guasch, A., Avilés, F. X., and Huber, R. (1991) EMBO J. 10, 1-9 [Abstract]
  21. Guasch, A., Coll, M., Avilés, F. X., and Huber, R. (1992) J. Mol. Biol. 224, 141-157 [Medline] [Order article via Infotrieve]
  22. Razin, E., Ihle, J. N., Seldin, D., Mencia-Huerta, J. M., Katz, H. R., LeBlanc, P. A., Hein, A., Caulfield, J. P., Austen, K. F., and Stevens, R. L. (1984) J. Immunol. 132, 1479-1486 [Abstract/Free Full Text]
  23. Reynolds, D. S., Serafin, W. E., Faller, D. V., Wall, D. A., Abbas, A. A., Dvorak, A. M., Austen, K. F., and Stevens, R. L. (1988) J. Biol. Chem. 263, 12783-12791 [Abstract/Free Full Text]
  24. Hayes, N., Biswas, C., Strout, H. V., and Berger, J. (1993) Biochem. Biophys. Res. Commun. 190, 881-887 [CrossRef][Medline] [Order article via Infotrieve]
  25. Sugumaran, G., Katsman, M., and Silbert, J. E. (1992) Biochem. Biophys. Res. Commun. 183, 357-361 [Medline] [Order article via Infotrieve]
  26. Reynolds, D. S., Stevens, R. L., Gurley, D. S., Lane, W. S., Austen, K. F., and Serafin, W. E. (1989) J. Biol. Chem. 264, 20094-20099 [Abstract/Free Full Text]
  27. Varughese, K., Su, Y., Cromwell, D., Hasnain, S., and Xuong, N. (1992) Biochemistry 31, 5172-5176 [Medline] [Order article via Infotrieve]
  28. Hanada, K., Tamai, M., Adachi, T., Oguma, K., Kashiwagi, K., Ohmura, S., Kominami, E., Towatari, T., and Katanuma, N. (1983) in Protease Inhibitors: Medical and Biological Aspects (Katunuma, N., Umezawa, H., and Holzer, H., eds) pp. 25-36, Springer-Verlag, New York
  29. Yamamoto, D., Ishida, T., and Inoue, M. (1990) Biochem. Biophys. Res. Commun. 171, 711-716 [Medline] [Order article via Infotrieve]
  30. Green, G. D. J., and Shaw, E. (1981) J. Biol. Chem. 256, 1923-1928 [Free Full Text]
  31. Avilés, F. X., Vendrell, J., Guasch, A., Coll, M., and Huber, R. (1993) Eur. J. Biochem. 211, 381-389 [Abstract]
  32. Greer, J. (1980) J. Mol. Biol. 153, 1027-1042
  33. Blundell, T. L., Sibanda, B. L., Sternberg, M. J. E., and Thornton, J. M. (1987) Nature 326, 347-352 [CrossRef][Medline] [Order article via Infotrieve]
  34. Weber, I. T., Miller, M., Jaskolski, M., Leis, J., Skalka, A. M., and Wlodawer, A. (1989) Science 243, 928-931 [Medline] [Order article via Infotrieve]
  35. Navia, M. A., Fitzgerald, P. M. D., McKeever, B. M., Leu, C.-T., Heimbach, J. C., Herber, W. K., Sigal, I. S., Darke, P. L., and Springer, J. P. (1989) Nature 337, 615-620 [CrossRef][Medline] [Order article via Infotrieve]
  36. Weber, I. T. (1990) Proteins Struct. Funct. Genet. 7, 172-184 [Medline] [Order article via Infotrieve]
  37. Vendrell, J., Cuchillo, C. M., and Avilés, F. X. (1990) J. Biol. Chem. 265, 6949-6953 [Abstract/Free Full Text]
  38. Uren, J. R., and Neurath, H. (1974) Biochemistry 13, 3512-3520 [Medline] [Order article via Infotrieve]
  39. Burgos, F. J., Salvà, M., Villegas, V., Soriano, F., Mendez, E., and Avilés, F. X. (1991) Biochemistry 30, 4082-4089 [Medline] [Order article via Infotrieve]
  40. Vendrell, J., Guasch, A., Coll, M., Villegas, V., Billeter, M., Wider, G., Huber, R., Wüthrich, K., and Avilés, F. X. (1992) Biol. Chem. Hoppe-Seyler 373, 387-392 [Medline] [Order article via Infotrieve]
  41. Zhu, X., Ohta, Y., Jordan, F., and Inouye, M. (1989) Nature 339, 483-484 [CrossRef][Medline] [Order article via Infotrieve]
  42. Silen, J. L., and Agard, D. A. (1989) Nature 341, 462-464 [CrossRef][Medline] [Order article via Infotrieve]
  43. Eder, J., Rheinnecker, M., and Fersht, A. R. (1993) Biochemistry 32, 18-26 [Medline] [Order article via Infotrieve]
  44. Baker, D., Sohl, J., and Agard, D. A. (1992) Nature 356, 263-265 [CrossRef][Medline] [Order article via Infotrieve]
  45. Tchekneva, E., and Serafin, W. E. (1994) J. Immunol. 152, 5912-5921 [Abstract/Free Full Text]
  46. Serafin, W. E., Katz, H. R., Austen, K. F., and Stevens, R. L. (1986) J. Biol. Chem. 261, 15017-15021 [Abstract/Free Full Text]
  47. Deyoung, M. B., Nemeth, E. F., and Scarpa, A. (1987) Arch. Biochem. Biophys. 254, 222-233 [Medline] [Order article via Infotrieve]
  48. Lagunoff, D., and Rickard, A. (1983) Exp. Cell Res. 144, 353-360 [Medline] [Order article via Infotrieve]
  49. Johnson, R. G., Carty, S. E., Fingerhood, B. J., and Scarpa, A. (1980) FEBS Lett. 120, 75-79 [CrossRef][Medline] [Order article via Infotrieve]
  50. Woodbury, R. G., Everitt, M., and Neurath, H. (1981) Methods Enzymol. 80, 588-609 [Medline] [Order article via Infotrieve]
  51. Le Trong, H., Neurath, H., and Woodbury, R. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2266-2270
  52. Schwartz, L. B., Riedel, C., Schratz, J. J., and Austen, K. F. (1982) J. Immunol. 128, 1128-1133 [Free Full Text]
  53. Vendrell, J., Avilés, F. X., Genescà, E., San Segundo, B., Soriano, F., and Médez, E. (1986) Biochem. Biophys. Res. Commun. 141, 517-523 [Medline] [Order article via Infotrieve]

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