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Biochemical and immunological characterization of multiple glycoforms of mouse mast cell protease 1: comparison with an isolated murine serosal mast cell protease (MMCP-4).

ABSTRACT: Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinella spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double diffusion using a polyclonal, cross-absorbed, sheep antibody raised against mouse mast cell protease-1 (MMCP-1) and on the basis of N-terminal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five variants (MMCP-1 A-E) had similar characteristics, although highly significant (P = 0.025 to P < 0.0001) variations in Km and kcat, were detected. Against human alpha 1-proteinase inhibitor the Ki for MMCP-1C (45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-linked carbohydrate produced a polypeptide of approx. 28 kDa in each case which was, like the native enzyme, immunoreactive on Western blotting. A much less soluble 28 kDa enzyme was isolated from serosal mast cells and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was antigenically distinct from MMCP-1 and, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. This last technique established that the MMCP-1 variants were uniquely present in enteric mast cells, thereby providing a highly selective means of distinguishing the mucosal and connective tissue mast cell subsets in the mouse.

SUBMITTER: Newlands GF

PROVIDER: S-EPMC1134575 | biostudies-other | 1993 Aug

REPOSITORIES: biostudies-other

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Project description:Background:Thrombin could elicit degranulation of mast cells involved in numerous physiologic and pathologic processes; however, the detailed scrutiny of this procedure and further research of possible cell signaling pathways are lacking. Methods:P815 mouse mast cells were exposed to various concentrations of thrombin for 16?h. Expression of protease-activated receptor (PAR)1, PAR2, PAR3, and PAR4 mRNA in P815 was analyzed by quantitative real-time PCR (qRT-PCR) and the fittest concentration of thrombin was decided. Then, secretions of mediators from P815 stimulated by thrombin 0.2?U/ml were determined using enzyme-linked immunosorbent assay (ELISA) and Luminex liquichip; the possible cell signaling pathways were measured by immunoblotting. Furthermore, inhibition of thrombin inhibitor (hirudin), PAR1 inhibitor (SCH79797), and MAPK inhibitors (SB203580, PD98059, and SP600125) on the mediator section was evaluated by ELISA and Luminex liquichip. Results:Thrombin 0.2?U/ml induced the elevated expression of PAR1, PAR2, PAR3, and PAR4, as well as the increasing level of phospho-I?B?, phospho-SAPK/JNK MAPK, phospho-P38 MAPK (Thr180/Tyr182), and phospho-ERK1/2 MAPK (p44/42) in P815. Secretion of vascular endothelial growth factor (VEGF), tumor necrosis factor-? (TNF-?), interleukin- (IL-) 2, IL-6, chemokine ligand- (CCL-) 2, chemokine (C-X-C motif) ligand- (CXCL-) 1, and CXCL-5 from P815 increased apparently; this effect could be diminished by hirudin, whereas SCH79797 and MAPK inhibitors (SB203580, PD98059, and SP600125) diminish the secretions with weaker effect. Conclusion:We found the expression of PAR mRNA in P815, activation of signaling pathways of nuclear factor-kappaB (NF-?B), and mitogen-activated protein kinases (MAPKs) including C-Jun NH2-terminal kinase (JNK), P38, and extracellular signal-regulated kinase 1/2 (ERK1/2), and the release of multiple inflammatory mediators stimulated by thrombin, as well as the inhibition of the inflammatory releases by hirudin, SCH79797, and MAPK inhibitors including SB203580, PD98059, and SP600125.

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Biochemical and immunological characterization of multiple glycoforms of mouse mast cell protease 1: comparison with an isolated murine serosal mast cell protease (MMCP-4).

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