title: Translation and granule localization of mouse mast cell protease-5. Immunodetection with specific antipeptide Ig.
abstract: An antibody to mouse mast cell protease-5 (MMCP-5) was obtained by immunizing a rabbit with a 17-residue synthetic peptide corresponding to the unique amino acid sequence at residues 146 to 162 in this serine protease. After affinity purification, anti-MMCP-5(146-162) Ig reacted in SDS-PAGE immunoblots to recombinant MMCP-5 and to the native MMCP-5 protein present in the lysates of mouse serosal mast cells and the MC5 line of Kirsten sarcoma virus-immortalized mouse mast cells. Immunocytochemical staining localized MMCP-5 to the cytoplasmic granules of serosal mast cells and Kirsten sarcoma virus-immortalized mouse mast cells. Because mouse bone marrow-derived mast cells express abundant amounts of MMCP-5 mRNA, anti-MMCP-5(146-162) Ig was used to study the translation and granule accumulation of this protease when progenitor cells differentiate into these immature mouse mast cells. Maximal expression of MMCP-5 mRNA occurred after bone marrow cells had been cultured for 2 wk in IL-3-rich WEHI-3 cell conditioned medium, and MMCP-5 protein was detected in these cells. However, electron-microscopic analysis with gold-labeled antibody revealed that the amount of MMCP-5 in the individual granules of bone marrow-derived mast cells varied. The highest concentration of MMCP-5 was found in the most electron-dense secretory granules of the cells. These studies demonstrate the ultrastructural localization of the earliest transcribed mouse mast cell chymase, MMCP-5, and its granule accumulation during the differentiation of mouse bone marrow progenitor cells into immature mouse mast cells.
authors.affiliation: Department of Medicine, Harvard Medical School, Boston, MA 02115.
authors.forename: H P
authors.initials: HP
authors.lastname: McNeil
authors.forename: D P
authors.initials: DP
authors.lastname: Frenkel
authors.forename: K F
authors.initials: KF
authors.lastname: Austen
authors.forename: D S
authors.initials: DS
authors.lastname: Friend
authors.forename: R L
authors.initials: RL
authors.lastname: Stevens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053818:Chymases; D003594:Cytoplasmic Granules / Q000201:enzymology*; D015151:Immunoblotting; D007136:Immunoglobulins / Q000276:immunology*; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012697:Serine Endopeptidases / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
chemical_list: D007136:Immunoglobulins; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D053818:Chymases