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. 2023 Jul 11;14:1211295. doi: 10.3389/fimmu.2023.1211295

Figure 2.

Figure 2

 A phylogenetic tree of gzm A/K locus genes. A phylogenetic tree was constructed based on the relationships among gzm A/K locus genes of a number of mammals using both MrBase analysis program and Maximum-likelihood algorithm using the manual standard protocol (27). The phylogenetic trees were drawn in FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtre​e/). In (A) we show the phylogenetic tree of a large number of hematopoietic serine protease genes originating from the five major loci. As can be seen from the figure the genes from the different loci cluster together. One branch for chymase locus encoded gene, marked in red, one branch for met-ase locus genes, marked in green, one branch for the major fish serine proteases marked in brown, a separate small branch for a locus found in amphibians, reptiles, and birds but not in mammals marked in orange and finally a branch marked in dark purple with all the gzm A/K genes. In (B) we show an enlarged picture of the genes of the gzm A/K locus with the enzymes of major interest for this study and that have been analyzed by phage display are marked by red arrows. In (C, D) we show the nomenclature of the amino acids surrounding the cleaved peptide bond and the amino acids forming the active site pocket. (C) shows the amino acids N-terminal from the cleaved bond are termed P1 (where cleavage occurs, depicted by scissors), P2, P3, etc. Amino acids C-terminal of the cleaved bond are termed P1’ (adjacent to P1), P2’, P3’ etc. The corresponding interacting sub-sites in the enzyme are denoted with S. (D) shows the S1 pocket, which is important in determining the primary specificity of the chymotrypsin family. The important residues are shown, which determine chymotrypsin-, trypsin- or elastase-like specificity. Three residues corresponding to positions 189, 216, and 226 in bovine pancreatic chymotrypsinogen have been found to be the amino acids forming the major part of this pocket and thereby giving the primary specificity of the enzyme (20). These three amino acids are marked, in one letter code, after each protease in (B) Almost all of these enzymes have the DGG triplet, indicating tryptase specificity.