Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe88-Gln89 and Phe91-Glu92 bonds of the catalytic domain

Abstract

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell alpha-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by alpha-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2. 3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog alpha-chymase yielded an approximately 84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89-98 and 92-101 of the cDNA predicted sequence, respectively. Thus, alpha-chymase cleaves the catalytic domain of gelatinase B at the Phe88-Gln89 and Phe91-Glu92 bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by alpha-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its proform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a approximately 15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by approximately 5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell alpha-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.