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Mast cell procarboxypeptidase A. Molecular modeling and biochemical characterization of its processing within
E B Springman1, M M Dikov, W E Serafin
1Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0111.
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 GluA94 and Ile1) 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 microM 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.
Insights
Murine mast cell procarboxypeptidase A (MC-proCPA) is an inactive zymogen. Molecular modeling and experiments reveal its rapid processing to active MC-CPA within secretory granules, likely by a thiol protease.
Area of Science:
- Biochemistry
- Cell Biology
- Structural Biology
Background:
- Murine mast cell procarboxypeptidase A (MC-proCPA) is characterized as an inactive zymogen.
- Understanding MC-proCPA's inactivity and activation mechanisms is crucial for mast cell function research.
Purpose of the Study:
- Investigate the structural basis for MC-proCPA's zymogenic inactivity.
- Determine the cellular mechanisms and kinetics of MC-proCPA processing to its active form.
Main Methods:
- Molecular modeling of MC-proCPA tertiary structure based on porcine procarboxypeptidases.
- Analysis of steady-state levels of MC-proCPA and MC-CPA.
- Inhibition studies using cycloheximide and brefeldin A.
- Treatment with a thiol-protease inhibitor (E-64d).
Main Results:
- Molecular model predicts structural similarity to pancreatic homologues, with the propeptide blocking the active site.
- MC-proCPA is rapidly processed in KiSV-MC14 cells with a half-life of 26 ± 5 min within secretory granules.
- Thiol protease inhibition significantly increases MC-proCPA levels, suggesting its involvement in processing.
Conclusions:
- The propeptide of MC-proCPA physically inhibits enzymatic activity by blocking the active site.
- MC-proCPA undergoes rapid, granule-localized processing, likely mediated by a thiol protease.
- These findings elucidate the activation pathway of a key mast cell protease.