Cloning and characterization of a second complementary DNA for human tryptase

Abstract

A second cDNA for human tryptase, called β-tryptase, was cloned from a mast cell cDNA library in lambda ZAP. Its nucieotide sequence and corresponding amino acid sequence were determined and compared with those of a previously cloned tryptase cDNA, now called α-tryptase. The 1,142-base sequence of β-tryptase encodes a 30-amino acid leader sequence of 3,089 D and a 245-amino acid catalytic region of 27,458 D. The amino acid sequence of β-tryptase is 90% identical with that of α-tryptase, the first 20 amino acids of the catalytic portions being 100% identical. This identity, together with recognition of each recombinant protein by monoclonal antibodies directed against purified tryptase validate the tryptase identity of both α-tryptase and β-tryptase cDNA molecules. Modest differences between the nucleic acid sequences of α- and β-tryptase occurred throughout the cDNA molecules except in the 3′ noncoding regions, which were identical. Although most highly conserved regions of amino acid sequence in the trypsin superfamily are conserved in both tryptase molecules, β-tryptase has one carbohydrate binding site compared to two in α-tryptase, and one additional amino acid in the catalytic sequence. Regions of the substrate binding pocket in β-tryptase (DSCQ, residues 218-221; SWG, residues 243-245) differ slightly from those in α-tryptase (DSCK, residues 217-220; SWD, residues 242-244). The presence of both α- and β-tryptase sequences in each haploid genome was indicated by finding α- and β-tryptase specific fragments after amplification by PCR of genomic DNA in 10 unrelated individuals. Localization of both α- and β-tryptase sequences to human chromosome 16 was then performed by analysis of DNA preparations from 25 human/hamster somatic hybrids by PCR. It is now possible to assess the expression of each tryptase cDNA by mast cells and the relationship of each gene product to the active enzyme.