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Extended cleavage specificity of a Chinese alligator granzyme B homologue, a strict Glu-ase in contrast to the mammalian Asp-ases.
Granzyme B (GzmB) is primarily expressed by mammalian cytotoxic T cells and serves as one of the key components in the defense against viral infection by the induction of apoptosis in virus infected cells. By direct cell to cell contact and delivery into target cells by perforin, cytotoxic T cells activate apoptosis through the action of GzmB by both caspase-dependent and -independent pathways. In search for early ancestors of GzmB we have in the current study identified and characterized a GzmB homologue from a reptile, the Chinese alligator. This enzyme is encoded from the same locus as the mammalian counterparts, the chymase locus. Phage display analysis of the cleavage specificity of the recombinant alligator enzyme (named MCP1A-like) shows that it is a relatively strict Glu-ase, with strong preference for glutamic acid in the P1 position of a substrate. The majority of mammalian GzmB:s are, in marked contrast to the alligator enzyme, relatively strict Asp-ases. The alligator enzyme also showed strong preference for Ala, Pro and Gly in the P2 position and Val in the P3 position indicating that it has a narrow specificity, similar to the mammalian counterparts. Analysis of the three amino acids forming the substrate binding pocket (S1 pocket) in three amphibian homologues to MCP1A-like, from the frogs Xenopus laevis and Xenopus tropicalis, shows that these amphibian enzymes have similar substrate binding pocket as their mammalian counterparts. This finding, together with the apparent lack of GzmB homologs in fish, indicates that the ancestor of GzmB did appear with the amphibians at the base of tetrapod evolution. This study is a first step in a larger effort to understand the evolutionary processes involved in shaping anti-viral immunity in non-mammalian vertebrates.
Fig. 1. The chymase locus and a related locus of a panel of non-mammalian tetrapod species. An in-scale figure of a panel of non-mammalian tetrapods ranging from amphibians to birds. The genes are color coded. The human locus is present as a reference. In the human locus the granzymes are shown in dark blue, α-chymases in light blue, β-chymases as blue with a darker tint and cathepsin G in green. In the amphibians and reptiles the genes cannot be assigned to any of the major groups of mammalian enzymes and are therefore shown in a light-dark blue color. The genes for which we have produced recombinant proteins are marked by black stars. The protease genes within an additional locus present in birds, reptiles and amphibians but that has been lost in mammals are shown in orange. The protease of interest in this study Chinese alligator MCP1A-like is marked by a yellow arrow. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. A phylogenetic tree of selected chymase locus genes. A phylogenetic tree was constructed based on the relationships among chymase locus genes of a number of tetrapods using both MrBase analysis program and Maximum-likelihood algorithm. The enzyme of major interest for this study, the Chinese alligator MCP1A-like, is marked by a large orange arrow. Other enzymes of particular interest for this study are marked by smaller arrows in yellow. Panel A shows a highly compressed version of all the genes involved in the analysis just to show that the chymase locus encoded genes form a clearly defined separate branch in the tree. Panel B shows an enlargement of the chymase locus encoded genes from panel A. For details of the different genes and the accession numbers see an earlier publication on the evolution of the hematopoietic serine proteases involving enlarged figures of also the genes encoded from the met-ase locus, the granzyme A/K locus and the fish serine proteases (Akula et al., 2015). A larger review on the evolution of the chymase locus has also recently been published (Akula et al., 2021). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. SDS-PAGE gel of the Chinese alligator granzyme B-like enzyme used in this study. The Chinese alligator enzyme was produced as an inactive enzyme containing an N terminal His6-tag and an enterokinase site. The enzyme was produced in the human cell line HEK293-EBNA with the episomal vector pCEP-Pu2. Entrokinase (EK) was used to cleave of the N-terminal tail to activate the chymase. Panel A shows the inactive enzyme with the N-terminal purification tag and the active enzyme analyzed by separation on a 4–12% gradient SDS-PAGE gel and visualized with Coomassie Brilliant Blue staining. PAGE Ruler was used as marker. M: marker; +EK: with entrokinase; -EK: without entrokinase. The arrow indicates the position of the recombinant Chinese alligator enzyme. The more prominent band above the recombinant enzyme is bovine serum albumin originating from the cell medium. Panel B shows the amino acid sequence of Chinese alligator MCP1A-like with the catalytic triad marked in red, the amino acids of the S1 pocked in blue and the three potential N-glycosylation sites in green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Phage display analysis of Chinese alligator MCP1A-like after six rounds of selection. After the last round of selection, the released phages were collected for sequencing. The amino acid sequences were aligned into a P5–P4’ consensus. The cleavage occurs between positions P1 and P1’. The amino acids are color coded according to their side chain properties as shown in the bottom corner of the figure. The cleavage site is marked with red arrow heads. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. Analysis of the cleavage specificity of Chinese alligator MCP1A-like by the use of recombinant protein substrates. In panel A, the overall structure of the recombinant protein substrates used for the analysis of the cleavage by Chinese alligator MCP1A-like is depicted. The sequences analyzed were positioned between two thioredoxin molecules with a His6-tag attached to the C terminal of the second Trx molecule. Two unique restriction sites (BamHI and SalI) were used for the insertion of the target sequences between the two thioredoxin molecules. Panel B shows a schematic example of this type of analysis. In panels C, D and E the results from the cleavage of a set of different substrates are presented. The sequences and the time in minutes of cleavage are indicated above the lanes of the different samples. The bands of a size of approximately 26 kDa represent un-cleaved substrates and those between 10 kDa and 15 kDa represent cleaved substrates. The difference in size of the cleaved bands are caused by the presence of the His6-tag and the EK site in the C-terminal end of one of the Trx molecules, representing the upper band. The amino acid residues that differ from the consensus sequence are marked in red. The cleavage sites are marked with red arrow heads. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6. Alignment of the chymase locus encoded genes from reptiles, and amphibians. The amino acid sequences of the active protease, lacking signal sequence and activation peptide were aligned using the DNASTAR program to visualize the residues forming the S1 pocket of the enzyme. The sequences for human chymase, human granzyme B and H as well as granzyme B;s from mouse, opossum and platypus were included as reference sequences. The three amino acids forming the S1 pocket and that can give important information concerning the primary specificity of the enzyme is marked by red arrows and summarized as the resulting triplet in the end of the figure, marked with pale red. The three residues forming the S1 pocket are residue 189, 216 and 226 following the position numbering of bovine chymotrypsin (Schechter and Berger, 1967). The presence of Cys residues outside of the common Cys residues of members of this protease family encoded from the chymase locus are marked in light green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)