Abstract
We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.
Highlights
Inhumans, chymase-like proteinases havebeenisolated regions of the ratchymase I1 gene
Primary structure was determined by direct amino acid sequence analysis of these peptides, and in all cases the amino acid sequences were found to be encoded in an open reading
Trypsin Cleavage and Amino Acid Sequencing-Human heart chy- frame after isolation and nucleotide sequencing of the human mase (52 pg) was purifiedas described previously (Urata et al, 199Ob) heart chymase cDNA and gene (Figs. 2 and 3)
Summary
Potential regof chymases show extensive homology to a group of chymotrypsin-like serine proteinasesincluding neutrophil cathepsin G and granzymes (Salveson et al, 1987; Jenne et al, 1989) and cytotoxic cell proteases (Lobe et al, 1986; Meier et al, 1990) Their catalytic and physicochemical properties differ markedly with respect to substrate specificity, catalytic efficiency, netcharge,and solubility(Woodbury et al, 1978; Yoshida et al, 1980; Powers et al, 1985; Le Trong et al, 1987; Caughey et al, 1988b; Urata et al, 1990b). ' The abbreviations used are: RMCP 11, rat mast cell protease 11; Ang I, angiotensin I; Ang 11, angiotensin 11; HPLC, high performance liquid chromatography;RMCP I, ratmast cell protease I; SDS, sodium dodecyl sulfate; PCR,polymerase chain reaction; kbk, ilobase; bp, base pair The determinantsof its specific functional characteristics.We report here the cloning of the gene and cDNA for human
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