PhD (Doctor of Philosophy)
Date of Award
Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased 13- and 6-fold upon fibroblast-conditioned media and butyrate induction, respectively. However, tryptase antigen levels increased dramatically from 2.3 $\mu$g/10$\sp6$ uninduced cells to 125 (54-fold) and 75 (33-fold) $\mu$g/10$\sp6$ cells induced with fibroblast-conditioned media or butyrate, respectively. A cDNA library was constructed in $\lambda$gt10 from ABFTL-6 cell poly(A)$\sp+$ RNA, and screened with dog mast cell tryptase and rat mast cell chymase cDNAs. Clones encoding two distinct tryptases (mouse tryptases I and II), a chymase (mouse chymase I) and a novel carboxyl terminal chymase (mouse chymase II) were isolated and sequenced. Mouse tryptases I and II have 75% and 70% sequence identity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequence for the mature active enzyme for each mouse tryptase contains 245 residues and all the characteristics of a serine protease. Asp is found in the substrate binding pockets, consistent with a trypsin-like specificity for Arg-X and Lys-X bonds. It is predicted that tryptases are synthesized with prepropeptides, requiring signal peptidase processing and removal of a three amino acid propeptide for activation. Mouse chymase I consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. An Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine protease.
Dissertation - Open Access
Chu, Wei, "Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization" (1991). Electronic Theses and Dissertations. Paper 2656. http://dc.etsu.edu/etd/2656