Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution

Abstract

The acidic granules of natural killer (NK) cells, T cells, mast cells, and neutrophils store large amounts of serine proteases. Functionally, these proteases are involved, e.g., in the induction of apoptosis, the recruitment of inflammatory cells, and the remodeling of extra-cellular matrix. Among the granule proteases are the phylogenetically related mast cell chymases, neutrophil cathepsin G, and T-cell granzymes (Gzm B to H and Gzm N), which share the characteristic absence of a Cys(191)-Cys(220) bridge. The genes of these proteases are clustered in one locus, the mast cell chymase locus, in all previously investigated mammals. In this paper, we present a detailed analysis of the chymase locus in cattle (Bos taurus) and opossum (Monodelphis domestica). The gained information delineates the evolution of the chymase locus over more than 200 million years. Surprisingly, the cattle chymase locus contains two alpha-chymase and two cathepsin G genes where all other studied chymase loci have single genes. Moreover, the cattle locus holds at least four genes for duodenases, which are not found in other chymase loci. Interestingly, duodenases seem to have digestive rather than immune functions. In opossum, on the other hand, only two chymase locus-related genes have been identified. These two genes are not arranged in one locus, but appear to have been separated by a marsupial-specific chromosomal rearrangement. Phylogenetic analyses place one of the opossum genes firmly with mast cell alpha-chymases, which indicates that the alpha-chymase had already evolved as a separate, clearly identifiable gene before the separation of marsupials and placental mammals. In contrast, the second gene in opossum is positioned phylogenetically between granzymes, cathepsin G, and the duodenases. These genes, therefore, probably evolved as separate subfamilies after the separation of placental mammals from marsupials. In platypus, only one chymase locus-like sequence could be identified. This previously published "granzyme" does not cluster clearly with any of the chymase locus gene families, but shares the absence of the Cys(191)-Cys(220) bridge with the other chymase locus proteases. These findings indicate that all chymase locus genes are derived from a single ancestor that was present more than 200 million years ago.

Structural Basis for Elastolytic Substrate Specificity in Rodent α-Chymases

“…The results strongly suggest that, during evolutionary diversification, the rodent ␣-chymases have separated relatively recently from other chymases, whereas neutrophil and pancreatic elastases have evolved independently of chymases. Thus, despite the elastolytic specificity, comparison of substrate-binding sequence regions of chymases and other serine proteases agree with an earlier hypothesis that rodent ␣-chymases are more closely related to chymase-like enzymes than to elastases (39,41,43).…”

“…An extensive comparison of crystal structures within the trypsin family of serine proteases has revealed that specificity-conferring residues are most often located at positions 189, 216, and 226 (40,41,44 diminish the space available for a P1 substrate side chain. In trypsin, the negatively charged Asp 189 , which forms a direct ionic interaction with the basic P1 residues Lys or Arg, at the bottom of the S1 pocket is the main determinant for trypsinlike specificity (40 , and Val 216 in all known rodent ␣-chymases form an easily identifiable triplet that can be used in the prediction of elastolytic specificity for any novel chymase-like sequence.…”

“…The amino acid at position 216, which is located on the wall of the S1 pocket and controls access to the pocket, is generally known to have a profound effect on the specificity of proteases with a chymotrypsin fold (40,41,44) and thus can be referred to as a "gatekeeper." This residue is typically Gly in enzymes exhibiting tryptic or chymotryptic specificity, allowing access of large substrate side chains to the base of the pocket.…”

“…6). The alignment, together with earlier alignments (14,41,42), shows that rodent ␣-chymases have Asn, Val, and Val at positions 189, 190, and 216, respectively, whereas human and several other chymases have almost invariably Ser/Thr, Ala/Ser, and Gly, respectively, at these positions. Structural analysis of HAM2 presented here revealed that these residues are the major determinants of the conversion of the substrate specificity from chymotryptic to elastolytic.…”

Optimization of Volumetric Computed Tomography for Skeletal Analysis of Model Genetic Organisms

“…Monodelphis domestica, the South American gray short-tailed opossum, is the first metatherian mammal to have its genome sequenced (Mikkelsen et al, 2007). Evolutionary (Gallwitz et al, 2006;Belov et al, 2007;Goodstadt et al, 2007) and functional (Ek et al, 2006;Freyer et al, 2007) studies have been extensively conducted, but morphological studies are sparse (Sanchez-Villagra and Maier, 2003;Kitchener et al, 2006). Because of its size similarities to mouse and rat, Monodelphis domestica is also emerging as a useful tool for preclinical studies (Chan et al, 2002).…”