Cloning and characterization of a second complementary DNA for human tryptase

• Published in: The Journal of clinical investigation 1990-09, Vol.86 (3), p.864-870
• Main Authors: MILLER, J. S, MOXLEY, G, SCHWARTZ, L. B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Blotting, Southern; chromosome 16; Chromosomes, Human, Pair 16; Cloning, Molecular; DNA - genetics; Genes; Humans; Mast Cells - enzymology; Medical genetics; Medical sciences; Molecular Sequence Data; Oligonucleotide Probes; Peptide Hydrolases - genetics; Polymerase Chain Reaction; Restriction Mapping
• ISSN: 0021-9738; 1558-8238
• DOI: 10.1172/jci114786

A second cDNA for human tryptase, called beta-tryptase, was cloned from a mast cell cDNA library in lambda ZAP. Its nucleotide sequence and corresponding amino acid sequence were determined and compared with those of a previously cloned tryptase cDNA, now called alpha-tryptase. The 1,142-base sequence of beta-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 beta-tryptase is 90% identical with that of alpha-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 alpha-tryptase and beta-tryptase cDNA molecules. Modest differences between the nucleic acid sequences of alpha- and beta-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, beta-tryptase has one carbohydrate binding site compared to two in alpha-tryptase, and one additional amino acid in the catalytic sequence. Regions of the substrate binding pocket in beta-tryptase (DSCQ, residues 218-221; SWG, residues 243-245) differ slightly from those in alpha-tryptase (DSCK, residues 217-220; SWD, residues 242-244). The presence of both alpha- and beta-tryptase sequences in each haploid genome was indicated by finding alpha- and beta-tryptase specific fragments after amplification by PCR of genomic DNA in 10 unrelated individuals. Localization of both alpha- and beta-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.