Cloning and characterization of complementary DNA for human tryptase.
The amino acid sequence of human mast cell tryptase was determined from corresponding cDNA cloned from a lambda ZAP library made with mRNA derived from a human mast cell preparation. Tryptase is the major neutral protease present in human mast cells and serves as a specific marker of mast cells by immunohistologic techniques and as a specific indicator of mast cell activation when detected in biologic fluids. Based on nucleic acid sequence, human tryptase consists of a 244-amino acid catalytic portion of 27,423 D with two putative N-linked carbohydrate binding sites and a 30-amino acid leader sequence of 3,048 D. A His74, Asp120, Ser223 catalytic triad and four cystine groups were identified by analogy to other serine proteases. Regions of amino acid sequence that are highly conserved in serine proteases, in general, were conserved in tryptase. The catalytic portion of human tryptase had an 84% amino acid sequence similarity with that of dog tryptase; their leader sequences had a 67% similarity. Asp217 in the substrate binding pocket of human tryptase is consistent with a specificity for Arg and Lys residues at the site of cleavage (P1), whereas Glu245 is consistent with the known preference of human tryptase for substrates with Arg or Lys also at P3, analogous residues also being present in dog tryptase. Asp244, which is substituted for the Gly found in dog tryptase and in most serine proteases, is present in the putative substrate binding pocket and may confer additional substrate specificity on human tryptase for basic residues. Further studies now can be designed to elucidate these structure-function relationships.
Miller
J S
JS
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Westin
E H
EH
Schwartz
L B
LB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004285:Dogs; D006801:Humans; D008407:Mast Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger; D004247:DNA; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase