title: Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by alpha-chymase.
abstract: We previously reported that mast cell alpha-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. Alpha-chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1's alpha-chymase-mediated cleavage products reveals hydrolysis at Phe(12)-Cys(13) and Phe(23)-Val(24) in loop 1 and Phe(101)-Val(102) and Trp(105)-Asn(106) in loop 3 of the NH(2)-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to alpha-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, alpha-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with alpha-chymase increases gel B activity 2- to 5-fold, suggesting that alpha-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of alpha-chymase blocks degranulation-induced TIMP-1 processing (absent in alpha-chymase-deficient HMC-1 cells). Purified alpha-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell alpha-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.
authors.affiliation: Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, CA 94143, USA.
authors.forename: B T
authors.initials: BT
authors.lastname: Frank
authors.forename: J C
authors.initials: JC
authors.lastname: Rossall
authors.forename: G H
authors.initials: GH
authors.lastname: Caughey
authors.forename: K C
authors.initials: KC
authors.lastname: Fang
mesh_terms: D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D015550:Cell Degranulation; D002478:Cells, Cultured; D053818:Chymases; D004285:Dogs; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology* / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D019272:Leukocyte Elastase / Q000378:metabolism; D000071068:Lipocalin-2; D054834:Lipocalins; D046911:Macromolecular Substances; D008407:Mast Cells / Q000201:enzymology* / Q000378:metabolism; D012515:Mast-Cell Sarcoma / Q000201:enzymology / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D015513:Oncogene Proteins; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins; D012697:Serine Endopeptidases / Q000378:metabolism*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D004792:Enzyme Precursors; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D046911:Macromolecular Substances; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053818:Chymases; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase