Guinea Pig Chymase Is Leucine-specific

Abstract

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the ␣ clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig ␣-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-ProLeu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe 8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by ␣ 1 -antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster ␣-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe-and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala 216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.

How Immune Peptidases Change Specificity: Cathepsin G Gained Tryptic Function but Lost Efficiency during Primate Evolution

“…Subsequent mutations led to acquisition of specialized activity and higher catalytic efficiency. More generally, these results reveal how small changes in structure in this family of immune peptidases can cause large changes in function, as also occurred in mouse, rat and hamster α-chymase (56, 57), which switched from chymotryptic to elastolytic activity, and in guinea pig α-chymase, which switched from chymotryptic to leu-ase activity (45). Although cathepsin G appears to be microbicidal in mice (6), in the context of chronic airway infection in humans with cystic fibrosis cathepsin G interferes with killing of certain bacteria (11).…”

“…The mouse triad (Ser 189 /Gly 216 /Ala 226 ) is typical in that it matches cathepsin G consensus sequence at all three positions. It also matches the human chymase triad, even though chymase is less closely related in overall sequence than human cathepsin G and belongs to a distinct clade distinct from mammalian cathepsin G (45). As shown in Fig.…”

“…Angiotensin-cleaving activity of wild type mouse and human cathepsin G was compared with that of recombinant human mast cell chymase (produced as described (43)) by incubating enzymes with 19 μM angiotensin I for 40 min at 37 °C in PBS containing 0.01% Triton X-100. Hydrolyzed fragments were detected by reverse phase-HPLC as in this laboratory’s published work (44, 45). General proteinase activity of human chymase and wild type and mutant cathepsin G was assessed by incubation of purified enzymes with bovine casein in PBS for 1 hour at 37 °C, followed by SDS-PAGE and staining with Coomassie Blue.…”

Mast cell proteases as pharmacological targets

“…This tendency is especially pronounced when preferences are profiled using small (peptidic) substrates (Andersson et al, 2009; Powers et al, 1985; Raymond et al, 2003). However, when confronting globular proteins, which tend to bury aromatic residues in hydrophobic interiors where they are inaccessible to hydrolytic attack, chymase can cleave after leucine (Caughey et al, 2008; Raymond et al, 2006). In vitro, human chymase is not highly selective, and is capable of cleaving a variety of peptide and protein targets, both endogenous and exogenous (as from pathogens) (Powers et al, 1985).…”

“…Although the biological targets of MCP-1 remain to be identified, studies in mice lacking MCP-1 suggest that this enzyme helps to defend against certain intestinal parasites (Knight et al, 2000) and may influence bronchial responses to allergic inflammation (Sugimoto et al, 2012). The mouse enzyme that is the phylogenetic equivalent of human chymase is MCP-5 (product of the Cma1 gene) (Caughey et al, 2008; Gallwitz et al, 2006; Gurish et al, 1993; Huang et al, 1991; McNeil et al, 1991) and is expressed in similar locations in mice and humans. However, mutations in the active site introduced during rodent evolution changed specificity so that it is no longer chymotryptic (see additional discussion below) (Kunori et al, 2002).…”