Dipeptide processing activates recombinant human prochymase.
Human chymase (h-chymase) is a serine protease that efficiently converts angiotensin I to II. Its structure and homology to other serine proteases suggest that it is synthesized as a zymogen, and is processed to the active form by cleavage of a 19-residue signal peptide and of a dipeptide pro-segment. To evaluate maturational processing of this enzyme, the proteins encoded by three h-chymase cDNA constructs (wild-type, lacking the pro- or lacking the prepro-segment) were characterized after expression in COS-1 cells. These recombinant proteins were not catalytically active. Purification and NH2-terminal sequence analysis of the protein expressed from the wild-type construct revealed processing to the proenzyme. Prochymase activation was achieved by incubation with a B-cell lymphoma homogenate, which apparently contains a heterologous processing enzyme sensitive to thiol protease inhibitors. NH2-terminal sequence analysis of the activated h-chymase revealed cleavage of the pro-segment, and its biochemical characteristics were identical to those of native h-chymase purified from the myocardium. These findings indicate that processing of the dipeptide pro-segment is necessary and sufficient for activation of human chymase. Such processing is probably also required for the activation of related serine proteases, e.g., cathepsin G, which have homologous dipeptide pro-segments.
Urata
H
H
Department of Cardiovascular Biology, Cleveland Clinic Foundation, Ohio 44195-5069.
Karnik
S S
SS
Graham
R M
RM
Husain
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D053818:Chymases; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004151:Dipeptides / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D016393:Lymphoma, B-Cell / Q000201:enzymology; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
D018076:DNA, Complementary; D004151:Dipeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053818:Chymases