Abstract
The cDNA and gene for mouse mast cell protease-6 (MMCP-6) have been sequenced and show MMCP-6 to be translated as a prepro-enzyme with a 21-amino acid hydrophobic leader peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The mature form of the enzyme has 73% amino acid sequence identity with human and dog mast cell tryptases. The MMCP-6 gene includes 6 exons, with a total span of 1.8 kilobases. A 208-base pair intron was defined which separates the 5'-untranslated sequence of MMCP-6 from the translation initiation codon, thereby presenting a gene organization which distinguishes tryptic serine proteases from chymotryptic serine proteases of the mast cell secretory granule. By RNA blot analysis with a gene-specific probe, MMCP-6 has a unique subclass distribution in being transcribed in mouse connective tissue mast cells but undetectable in mucosal mast cells. MMCP-6 is the first serine protease of any class to be shown to be significantly transcribed in progenitor, bone marrow-derived mast cells, which can reconstitute both mucosal mast cell and connective tissue mast cell populations in mast cell-deficient mice.
Highlights
From the Department of Medicine, Haruard Medical Schooland the Department of Rheumatologyand Immunology, Brigham and Women’s Hospital, Boston, Massachusetts 02115
The cDNA and gene for mouse mast cell protease-6 (MMCP-6)have been sequenced and show mouse mast cell proteases (MMCP)-6 to be translated as a prepro-enzymewith a 21-amino acid hydrophobicleader peptide, a 10-aminoacid activation peptide, and a 245-amino acid mature enzyme
MMCP-6 is the first serine protease of any class to be shown to be significantly transcribed in progenitor, bone marrow-derived mast cells, which can reconstitute both mucosal mast cell and connective tissue mast cell populationsin mast cell-deficient mice
Summary
Isolation and Characterization of cDNAs That Encode MMCP-6Total cellular RNA [19] was isolated from a mixture of 6 X 10’ cells of the DBA/2 strain mouse KiSV-MC lines MC1,MC5, and MC6 [20], and poly(A+)RNA was selected [21]. Was synthesized, gel-purified, and end-labeled [22] with [y3’P]ATP (-6000 Ci/mmol, Du Pont-New England Nuclear). This probe was used to screen approximately 20,000 recombinants from the KiSVMC cDNA library under hybridization and wash conditions previously described [23]. Transcripts barely detectable above backfragment of cDNA T2 was isolated by restriction digestion of the ground after prolonged exposure were scored as purified cDNA, agarose gel elec~rophoresis,and band interception
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