Albumin Is a Substrate of Human Chymase

Abstract

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 bestknown 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 Cu 2؉ or Zn 2؉ . A synthetic peptidic inhibitor, diphenyl N ␣ -benzoxycarbonyl-L-Arg-Glu-Thr-Phe P -phosphonate, was designed from the preferred P4 -P1 substrate sequence. This inhibitor is highly potent (IC 50 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.

An evolving story of angiotensin-II-forming pathways in rodents and humans

“…Incubation of plasma membranes from these atrial myocytes with 125 I-Ang-(1-12) resulted in the rapid production of Ang II that was prevented in the presence of chymostatin and not inhibited by lisinopril (Figure 3). As discussed in our study [68], while chymase cleaving activity in rodents occurs at amino acid residues containing the sequence of Tyr 4 -Ile 5 [70], human chymase hydrolytic activity is specific for the Phe 8 -His 9 sequence. The latter sequence is present within the amino acid composition of human Ang-(1-12) [H-Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 - Phe 8 -His 9 -Leu 10 -Val 11 -Ile 12 -OH].…”

Mast Cell and Neutrophil Peptidases Attack an Inactivation Segment in Hepatocyte Growth Factor to Generate NK4-like Antagonists

“…These proteins and HGF evidently present Leu-containing segments in a more cleaveable conformation than in short peptides with little secondary structure. It bears noting that combinatorial screening suggests that the residues occupying the P4 -P2 positions (Ile-Val-Asn) of HGF adjacent to the site hydrolyzed by chymase are favorable when present in short peptides (30). This suggests that P1 side chain accessibility, combined with P4 -P2 side chains interacting favorably with the extended substrate-binding site, contribute to the ability of chymase to hydrolyze at Leu 480 .…”

“…Identification of Leu at the site of hydrolysis itself argues for an unusual degree of exposure, because Leu is not favored at the scissile bond in short peptides cleaved by chymotryptic peptidases generally and chymase particularly. A profiling of the substrate preferences of human chymase with a combinatorial peptide library found the cleavage of substrates with P1 Leu at the site of hydrolysis to be far less likely than when the P1 residue is Phe or Tyr (30). Profiling with selected 4-nitroanilide and angiotensin-like peptides similarly suggests preferences for P1 aromatic residues (31,32).…”

Mast Cell Proteases as Protective and Inflammatory Mediators

“…Indeed, in human airway, chymase-positive mast cells are a high fraction of mast cells lying within 20 μm of submucosal glands 152 . The proteolytic activity of human chymase extends to albumin 153 ; however, this cleavage would not increase oncotic pressure because the nicked fragments remain joined by disulfide linkages. Acting subacutely or chronically, chymase-like peptidases also may promote inflammatory angiogenesis, as suggested by sponge granulomas in hamsters 154 and by mast cell-dependent angiogenesis in a model of skin carcinogenesis 49 .…”