title: Mast cell and neutrophil expression of dog mast cell protease-3. A novel tryptase-related serine protease.
abstract: In previous work the primary structure of a previously unknown protease was deduced from the sequence of a dog mastocytoma cDNA. The predicted preproprotein shares some features with mast cell tryptases but is no more than 49% identical in sequence to known trypsin-like enzymes, including dog tryptase. This study explores the expression of this protein, termed dog mast cell protease-3 (dMCP-3). A polyclonal Ab was raised to a peptide corresponding to residues 166-181 of the deduced sequence. Anti-dMCP-3(166-181) Ig recognizes dMCP-3 expressed as a CheY fusion protein in Escherichia coli and binds to a approximately 36-kDa protein in extracts of dog mastocytomas. The Ab does not recognize dog tryptase, dMCP-3s closest known relative in mastocytoma cells. When used with fluorescein-conjugated and alkaline phosphatase-conjugated secondary Abs, anti-dMCP-3(166-181) Ig yields punctate cytoplasmic staining in mastocytoma cells, suggesting localization to intracellular granules. Staining is greatly reduced by preincubation with synthetic dMCP-3 peptide, supporting the specificity of the Ab. Immunohistochemical staining of normal dog tissues reveals scattered dMCP-3 reactive cells in skin, intestine, trachea, and lung parenchyma. Double staining with Ab and methylene blue shows that anti-dMCP-3(166-188) Ig recognizes extravascular mononuclear tissue cells with metachromatic granules. In addition, cytoplasmic staining is seen in polymorphonuclear leukocytes within vessels in tissue sections and in leukocytes harvested from blood. Hybridization of dMCP-3 cDNA to dog skin RNA provides further evidence of dMCP-3 gene transcription in normal tissue. Thus, this study provides immunochemical evidence of dMCP-3 expression in dog mast cell tumors, normal tissue mast cells, and neutrophils.
authors.affiliation: Cardiovascular Research Institute, University of California, San Francisco 94143.
authors.forename: M J
authors.initials: MJ
authors.lastname: Yezzi
authors.forename: I E
authors.initials: IE
authors.lastname: Hsieh
authors.forename: G H
authors.initials: GH
authors.lastname: Caughey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004285:Dogs / Q000378:metabolism*; D007150:Immunohistochemistry; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D012697:Serine Endopeptidases; C092818:mast cell protease 3