Cooperation between Mast Cell Carboxypeptidase A and the Chymase Mouse Mast Cell Protease 4 in the Formation and Degradation of Angiotensin II

Abstract

The octapeptide angiotensin II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by angiotensin-converting enzyme, but there is also evidence for non-angiotensin-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4 ؊/؊ cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-10) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(1-9) and Ang-(1-7) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4 ؊/؊ cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.

Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan, HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation

“…The recently developed protease-specific knock-out mouse strains have become important tools in identifying such potential in vivo substrates, and examples of substrates identified using these knock-out mice are fibronectin, endothelin, and vasointestinal peptide. Previous work also showed that chymase and CPA3 could cooperate in the degradation of potential substrates (4,6,7,34). Our results also suggest that chymase may be required for the initiation of the degradation process.…”

“…The following mouse strains were used for in vivo and in vitro studies: wild type (WT); the serglycin-deficient mouse strain (SG Ϫ/Ϫ ) (32,33); and the mouse mast cell protease 4-deficient mouse strain (Mcpt4 Ϫ/Ϫ ) (6,34,35). The following mouse strains were used for in vitro studies only: the mast cell-deficient W sash mice; the heparin-deficient mouse strain (NDST2 Ϫ/Ϫ ) (14); the two carboxypeptidase A-deficient mouse strains, Cpa3 Ϫ/Ϫ (which also lack MCPT5) and Cpa3 inact (which carry an inactivating mutation of the catalytic site and maintain MCPT5 expression) (36,37); and the mouse mast cell protease 6-deficient mouse strain (Mcpt6 Ϫ/Ϫ ) (10).…”

Characterization of Carboxypeptidase A6, an Extracellular Matrix Peptidase

“…In the case of CPA1 His cleavage has been exploited as a method to remove His 6 tags from the C terminus of recombinant proteins (29). CPA3 has been shown to be able to cleave His 9 of [des-Leu 10 ]angiotensin I in a similar manner as that observed in this study for CPA6 (30). The cleavage of the C-terminal His tag is not an intramolecular reaction, based on the finding that co-transfection of catalytically inactive CPA6 together with the active enzyme resulted in cleavage of the His 6 tag from the inactive enzyme.…”

Advanced Glycation End Products Activate a Chymase-Dependent Angiotensin II–Generating Pathway in Diabetic Complications

“…This was measured by ELISA, adapting the method of Lundequist et al 26 Briefly, VSMCs were distributed onto 24-well plates at a concentration of 0.5ϫ0 6 cells per well and grown to confluence. After being rendered quiescent as described above, they were stimulated with AGEs (50 g/mL) or control BSA (50 g/mL) for 24 hours.…”