PURIFICATION AND CHARACTERIZATION OF DOG MAST-CELL PROTEASE-3, AN OLIGOMERIC RELATIVE OF TRYPTASES

The existence of a protein similar to 48% identical with mast cell try ptases was predicted previously from a dog mastocytoma cDNA Antibodies raised against a peptide based on the deduced sequence suggested that the protein (dog mast cell protease-3, dMCP-3) is expressed in mast c ells. In this report, characterization of the protein purified from ma stocytomas reveals an N-glycosylated, high molecular weight, tryptic s erine protease, which appears to be a tetramer of catalytic subunits, approximately half of which are Linked by disulfide bonds. The oligome ric complex yields a single NH2-terminal sequence, which is identical with that predicted by dMCP-3 cDNA. This finding, and the lack of clos ely related genes on blots of genomic DNA, predict that each subunit i s the product of one gene. Although dLMCP-3 binds to heparin, it is ac tive and stable at low ionic strength in heparin's absence. It resists inactivation by inhibitors in plasma but is sensitive to small inhibi tors, e.g. leupeptin and bis(5-amidino-2-benzimidazolyl)methane (BABIM ). dMCP-3 hydrolyzes extended peptidyl p-nitroanilides ending in basic residues, with P1 arginine preferred to lysine; it hydrolyzes the Arg (18)-Ser(19) bond of calcitonin gene-related peptide but cleaves neith er vasoactive intestinal peptide nor casein. These data suggest that d MCP-3 is a unique serine protease whose stability, formation of inters ubunit disulfide bonds, inhibitor susceptibilities and substrate prefe rences differ from those of its closest relatives, the mast cell trypt ases.