Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation.
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.
Vanderslice
P
P
Department of Medicine, University of California, San Francisco 94143.
Craik
C S
CS
Nadel
J A
JA
Caughey
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004285:Dogs; D004789:Enzyme Activation; D008407:Mast Cells / Q000201:enzymology*; D012515:Mast-Cell Sarcoma / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism
D004247:DNA; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D012697:Serine Endopeptidases