Structural Basis for Elastolytic Substrate Specificity in Rodent α-Chymases

Abstract

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent ␣-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-Å resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val form an easily identifiable triplet in all known rodent ␣-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent ␣-chymases.Chymases (EC 3.4.21.39), serine proteases with a chymotrypsin-fold, are stored within the secretory granules of mast cells along with histamine, tryptases, and other inflammation mediators. When released during mast cell degranulation in various tissues, chymases participate in a variety of biological functions including regulation of vasoactive peptide processing, modulation of inflammatory response, stimulation of submucosal gland secretion, and degradation of extracellular matrix (1). Human chymase has been linked to various pathologic conditions such as allergic inflammatory reactions that can contribute to asthma (2), Crohn disease (3), inflammatory kidney disease (4), and cardiovascular disorders (5, 6).Based on the catalytic triad consisting of His, Asp, and Ser (in that order in the sequence), chymases belong to the S1A family of serine proteases that include, for example, trypsin, chymotrypsin, elastase, and cathepsin G (7, 8). Mammalian chymases divide into two phylogenetic groups, termed ␣-and ␤-chymases (9 -11). The genomes of primates, dogs, ruminants, and rodents apparently contain only one functional ␣-chymase, whereas rodents typically have several ␤-chymases. Until recently, all chymases were thought to possess chymotryptictype substrate specificity, preferring Tyr and Phe (as well as Trp and Leu, to a lesser extent) at the P1 substrate position (Schechter and Berger nomenclature (12)). However, two recent studies have clearly established elastolytic specificity (i.e. preference for small aliphatic residues Ala, Val, and Ile at the P1 position) for mouse chymase-5 (mouse mast cell protease-5; mMCP5) 4 and rat chymase-5 (rMCP5) (13,14). Neither enzyme hydrolyzed a typical chymase substrate with Phe at P1. Site-directed mutagenesis and computer modeling studies suggested Val 216 as a major contributor to this unusual substrate specificity (14).Hamster ch...

Guinea Pig Chymase Is Leucine-specific

“…Residues with side chains of intermediate size, such as Val 216 in murine and hamster ␣-chymases and Ser 216 in granzyme M, are associated with elastase and Met-ase activity, respectively, but have little if any ability to hydrolyze peptides after aromatic amino acids. The "gatekeeping" role of residue 216 also is supported by the structural and modeling studies (19,20,23,31), including those in the present work, which suggests that the 216 side chain influences the volume, shape, and hydrophobicity of the binding pocket accommodating the P1 residue of the substrate (see Fig. 6).…”

“…In the case of elastolytic chymases, notably the mouse chymase MCP-5 and its rat ortholog, the switch from chymotryptic to elastolytic specificity is attributable to natural mutation of a single amino acid (19,20). Similar elastolytic properties have recently been described for hamster ␣ chymase (23). Thus, small changes in structure, including alteration of just one amino acid in the vicinity of the substrate-binding site, can cause large changes in function.…”

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).…”

Mast cell chymase limits the cardiac efficacy of Ang I–converting enzyme inhibitor therapy in rodents

“…The hamster has 2 known chymases. Hamster chymase-1, which converts Ang I to Ang II (35), is a homolog of MMCP4 (27), and hamster chymase-2, which does not, is a homolog of MMCP5 (36). We tested and found that 4-[1-(naphthalen-1-yl methyl)-1H-benzimidazol-2-yl sulfanyl]-butyric acid (CI-B) inhibits purified hamster chymase-1 with a K I of 30.6 ± 3.75 nM (n = 4).…”