title: 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.
authors.affiliation: Cardiovascular Research Institute, University of California, San Francisco, California 94143-0911, USA. kfang@itsa.ucsf.edu
authors.forename: K C
authors.initials: KC
authors.lastname: Fang
authors.forename: W W
authors.initials: WW
authors.lastname: Raymond
authors.forename: J L
authors.initials: JL
authors.lastname: Blount
authors.forename: G H
authors.initials: GH
authors.lastname: Caughey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D053818:Chymases; D017364:Collagenases / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D004285:Dogs; D015536:Down-Regulation; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D008407:Mast Cells / Q000201:enzymology*; D012515:Mast-Cell Sarcoma / Q000201:enzymology; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D012333:RNA, Messenger; D005973:Glutamine; D010649:Phenylalanine; D012697:Serine Endopeptidases; D053818:Chymases; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; C022440:procollagenase; C069860:progelatinase