Albumin is a substrate of human chymase. Prediction by combinatorial peptide screening and development of a selective inhibitor based on the albumin cleavage site.
Human chymase is a chymotryptic serine peptidase stored and secreted by mast cells. Compared with other chymotryptic enzymes, such as cathepsin G and chymotrypsin, it is much more slowly inhibited by serum serpins. Although chymase hydrolyzes several peptides and proteins in vitro, its target repertoire is limited compared with chymotrypsin because of selective interactions in an extended substrate-binding site. The best-known natural substrate, angiotensin I, is cleaved to generate vasoactive angiotensin II. Selectivity of angiotensin cleavage depends in major part on interactions involving substrate residues on the carboxyl-terminal (P1'-P2') side of the cleaved bond. To identify new targets based on interactions with residues on the aminoterminal (P4-P1) side of the site of hydrolysis, we profiled substrate preferences of recombinant human chymase using a combinatorial, fluorogenic peptide substrate library. Data base queries using the peptide (Arg-Glu-Thr-Tyr-X) generated from the most preferred amino acid at each subsite identify albumin as the sole, soluble, human extracellular protein containing this sequence. We validate the prediction that this site is chymase-susceptible by showing that chymase hydrolyzes albumin uniquely at the predicted location, with the resulting fragments remaining disulfide-linked. The site of hydrolysis is highly conserved in vertebrate albumins and is near predicted sites of metal cation binding, but nicking by chymase does not alter binding of Cu2+ or Zn2+. A synthetic peptidic inhibitor, diphenyl N alpha-benzoxycarbonyl-l-Arg-Glu-Thr-PheP-phosphonate, was designed from the preferred P4-P1 substrate sequence. This inhibitor is highly potent (IC50 3.8 nM) and 2,700- and 1,300-fold selective for chymase over cathepsin G and chymotrypsin, respectively. In summary, these findings reveal albumin to be a substrate for chymase and identify a potentially useful new chymase inhibitor.
Raymond
Wilfred W
WW
Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, San Francisco, California 94143-0911, USA.
Ruggles
Sandra Waugh
SW
Craik
Charles S
CS
Caughey
George H
GH
D000418:Albumins / Q000737:chemistry*; D000803:Angiotensin I / Q000737:chemistry; D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry; D002412:Cations; D053818:Chymases; D002918:Chymotrypsin / Q000737:chemistry / Q000494:pharmacology; D003300:Copper / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013053:Spectrophotometry; D015032:Zinc / Q000737:chemistry
D000418:Albumins; D002412:Cations; D004220:Disulfides; D004791:Enzyme Inhibitors; D008670:Metals; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D003300:Copper; D000803:Angiotensin I; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D053818:Chymases; D015032:Zinc