Analysis of vertebrate genomes suggests a new model for clade B serpin evolution

BACKGROUND: The human genome contains 13 clade B serpin genes at two loci, 6p25 and 18q21. The three genes at 6p25 all conform to a 7-exon gene structure with conserved intron positioning and phasing, however, at 18q21 there are two 7-exon genes and eight genes with an additional exon yielding an 8-exon structure. Currently, it is not known how these two loci evolved, nor which gene structure arose first – did the 8-exon genes gain an exon, or did the 7-exon genes lose one? Here we use the genomes of diverse vertebrate species to plot the emergence of clade B serpin genes and to identify the point at which the two genomic structures arose. RESULTS: Analysis of the chicken genome indicated the presence of a single clade B serpin gene locus, containing orthologues of both human loci and both genomic structures. The frog genome and the genomes of three fish species presented progressively simpler loci, although only the 7-exon structure could be identified. The Serpinb12 gene contains seven exons in the frog genome, but eight exons in chickens and humans, indicating that the additional exon evolved in this gene. CONCLUSION: We propose a new model for clade B serpin evolution from a single 7-exon gene (either Serpinb1 or Serpinb6). An additional exon was gained in the Serpinb12 gene between the tetrapoda and amniota radiations to produce the 8-exon structure. Both structures were then duplicated at a single locus until a chromosomal breakage occurred at some point along the mammalian lineage resulting in the two modern loci

The major human and mouse granzymes are structurally and functionally divergent

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges

A Natural Genetic Variant of Granzyme B Confers Lethality to a Common Viral Infection

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining ‘Asp-ase ’ activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV

Identification of Serpinb6b as a species-specific mouse granzyme A inhibitor suggests functional divergence between human and mouse granzyme A

Background: There are conflicting reports on the ability of granzyme A (GzmA) to kill cells. Results: Substrate specificity mapping of human and mouse GzmA was used to identify Serpinb6b as a mouse-specific GzmA inhibitor. Conclusion: Mouse but not human GzmA is controlled by an intracellular inhibitor. Significance: The GzmA debate is partly explained by species-specific divergence in cytotoxicity. The granzyme family serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme (Gzm) A is controversial, with significant debate over its ability to induce death in target cells. Here, we investigate the natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse (m) and human (h) GzmA at the peptide and proteome-wide levels and we used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 +/- 0.8 x 10(5) m(-1) s(-1) and a stoichiometry of inhibition of 1.8. Mouse GzmA is over five times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, whereas transfection of target cells with a Serpinb6b cDNA increases the EC50 value of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution

Conservation of the extended substrate specificity profiles among homologous granzymes across species

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility

A Novel Serpin Regulatory Mechanism

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