We recently collated the full-length coding sequences of the entire mouse Psg gene family . In the present study we aimed to identify evolutionary signals embedded in Psg gene and PSG protein sequences to determine whether PSG protein function has diverged between the rodent and primate lineages, and to attempt to understand the reasons for the independent expansions of rodent and primate PSG gene families.
Mouse and human PSG protein amino-terminal N domains exhibit different patterns of evolution. McLenachan et al.  analysed the evolution of a subset of human PSGs using split decomposition analysis and found, in individual comparisons of N, A1, B2 and C domain exons, strong contradictions in alignments, which they suggested was due to gene conversion and/or positive selection. Our similar analysis of an expanded set of human PSG sequences revealed a detectable, but less marked, degree of homogenisation. Analysis of mouse N and A domain exons showed that, in general, there is less evidence of purifying selection compared to the human, although there are examples of gene conversions as described previously for the closely related Psg21 and Psg23 genes . Detailed analysis of alignments using plots of Dayhoff scores confirmed the difference between mouse and human N domain evolution.
s analysis for interspecies comparisons, we found that the PSG protein amino-terminal N and N1 domains are relatively conserved, consistent with conservation of function in rodents and primates. However, inspection of mouse PSG N1 domain alignments, and scrutiny of corresponding Dayhoff scores, revealed regions of apparently poor conservation. These regions correspond to the CFG face within the N1 domain of CEACAM1. In the CEACAM family, the CFG face interacts with pathogens and mammalian proteins. Comparisons of d
s values obtained from the CFG and ABED faces of mouse N1, N2 and N3 domains confirmed that the CFG face has evolved more rapidly than the ABED face in all three domains. The greatest effect was observed in the N1 domain exon with a doubling of the d
s ratio in the CFG face compared with the ABED face. The d
s ratio of 1.1 suggests weak positive selection on the CFG face of the N1 domain. The increase in the d
s ratio appears to be mainly due to an increase in the d
n value, indicative of diversification. The high dn/ds values for the CFG face in the N2 and N3 domains, which are not known to interact with ligands, could be due to a low contribution of these sequences to the structural integrity of the IgV-like domain.
Interestingly, the rat N1 domain CFG face does not appear to have evolved as rapidly as the mouse N1 domain, with a d
s ratio of 0.9. This observation, combined with the relatively smaller number of PSG genes identified in the rat (eight to date, compared to seventeen in the mouse) and the higher level of gene homogenisation implied by split decomposition analysis suggests that the rat PSG gene family has not expanded or diversified as extensively as the mouse. However, we cannot exclude the possibility that further rat PSG genes may yet be identified because there may be under-representation in the WGS database . Notwithstanding this possibility, there has clearly been ongoing turnover of the PSG gene family in all of the lineages analysed, as there are no known human orthologues of rat and mouse PSG s, and only four potential orthologous relationships between known rat and mouse PSG s.
These findings suggest partial conservation of PSG N domain function across rodent and primate lineages. However, the relaxed constraint on the CFG face of mouse PSGs suggests diversification of binding partners or modification of existing ligand-binding kinetics, analogous to the CEACAMs. This observation receives experimental support from the recent observation that treatment of mouse macrophages in vitro with recombinant mouse PSG17N, or human PSG1 or PSG11, induces cytokine expression; however, only in the case of mouse PSG17N does this depend on CD9 receptor expression . Divergence of PSG function is also suggested by differences in the level and developmental timing of expression of different mouse PSGs [7, 12], expansion of N domain number in PSG24, PSG30 and PSG31 , and loss of secretory signals in PSG32 and in the brain-specific splice variant of PSG16.
As noted above, the only PSG receptor identified to date is the integrin-associated tetraspanin, CD9, which binds the N1 domain of mouse PSG17 but not, apparently, to human PSGs . However, a peptide containing the RGD motif from the human PSG9 N domain binds to a receptor on a promonocytic cell line suggesting that some human PSGs may effect their functions through an integrin-type receptor . In this context, the high frequency of the RGD motif on an exposed loop in primate PSG N domains (seven of ten in human and five of fifteen in baboon) may be significant. Rodent PSG N1 domains do not have an RGD motif, but have a high frequency of the RGD-like motifs RGE, HGE and HAE on the CFG face. Under the null hypothesis that these motifs are unlikely to underpin structural integrity of the N1 domain and are therefore free of constraint, our analysis reveals evidence of unexpected conservation of RGD-like motifs in the N1 domain, which have been lost in the N2 and N3 domains. Given the high transition and transversion rates in the N1 domain and the fact that the mouse N1, N2 and N3 domains share a common ancestor after the divergence of the rodent/primate lineages, the conservation of RGD-like motifs exclusively in the N1 domain may have functional significance. We note that the RGE motif in the context of the POEM protein induced apoptosis of MC3T3-E1 cells in vitro . We speculate that certain RGE or RGE-like motifs may elicit weak cell attachment, followed by apoptosis – a combination of properties, reminiscent of snake venom disintegrins [30, 31], that could have important functional implications in the context of the extensive tissue remodelling that occurs during placentation .
In summary, our data are consistent with experimental evidence indicating functional convergence of rodent and primate PSGs, in spite of the independent expansions of the gene families in the two lineages. In the context of parent-offspring conflict, the homogenisation of human PSG sequences is consistent with the theory that placental hormones encoded by multigene families are monofunctional and selected for high expression, possibly due to coevolution with physiologically conflicting maternal mechanisms . However, the evidence for positive selection on the CFG face of the N1 domain implies divergent evolution of rodent PSGs. Allied to the evidence for functionality of putative integrin-interacting RGD-like motifs in rodents, a scenario can be envisaged whereby the different RGD-like motifs observed in human and baboon PSGs also suggest some degree of functional divergence in these species.