Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation

Abstract

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.

Characterization of Human γ-Tryptases, Novel Members of the Chromosome 16p Mast Cell Tryptase and Prostasin Gene Families

“…Intron splice junctions were predicted by reference to dog mastin cDNA (27) and patterns of intron phase and placement in known tryptase genes (22,23) by examination of open reading frames and by application of "GT. .…”

“…This suggests that mMCP-6 and -7 are not the equivalents, respectively, of ␣-and ␤-tryptases and that ancestors of ␣/␤-tryptases diverged from each other after the point when known mouse and human tryptases shared a common ancestor (23,26). In dogs, our laboratory characterized a mast cell protease termed mastin, a relative of tryptase sufficiently different from known tryptases that it forms a separate branch of the tree (27)(28)(29). No expressed human or mouse homologue has been identified.…”

Mast Cell-restricted Tryptases: Structure and Function in Inflammation and Pathogen Defense

“…The fact that some of the initially isolated hTryptase cDNAs are nearly identical added to the confusion as to how many tryptase genes are present in the human genome. It tentatively has been concluded that two adjacent genes on chromosome 16p13.3 give rise to the hTryptases ␣, ␤1, ␤2, and ␤3 transcripts which encode tetramer-forming proteases that are 93-99% identical (17,(22)(23)(24).Sequence analysis of mouse chromosome 17A3.3 led to the identification of the Prss31 gene (18) …”

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

“…Tryptases are major constituents of the secretory granules of mast cells in the mouse (1)(2)(3)(4), rat (5-7), dog (8), gerbil (9), and human (10 -13). In the mouse, two tryptases, designated mouse mast cell protease (mMCP) 1 -6 and mMCP-7, have been identified whose overall amino acid sequences are 71% identical.…”