Molecular Evolution of the bg Lens Crystallin Superfamily: Evidence for a Retained Ancestral Function in gN Crystallins?

Within the vertebrate eye, bc crystallins are extremely stable lens proteins that are uniquely adapted to increase refractory power while maintaining transparency. Unlike a crystallins, which are well-characterized, multifunctional proteins that have important functions both in and out of the lens, bc lens crystallins are a diverse group of proteins with no clear ancestral or contemporary nonlens role. We carried out phylogenetic and molecular evolutionary analyses of the bccrystallin superfamily in order to study the evolutionary history of the cN crystallins, a recently discovered, biochemically atypical family suggested to possess a divergent or ancestral function. By including nonlens, bc-motifcontaining sequences in our analysis as outgroups, we confirmed the phylogenetic position of the cN family as sister to other c crystallins. Using maximum likelihood codon models to estimate lineage-specific nonsynonymous-tosynonymous rate ratios revealed strong positive selection in all of the early lineages within the bc family, with the striking exception of the lineage leading to the cN crystallins which was characterized by strong purifying selection. Branch-site analysis, used to identify candidate sites involved in functional divergence between cN crystallins and its sister clade containing all other c crystallins, identified several positively selected changes at sites of known functional importance in the bc crystallin protein structure. Further analyses of a fish-specific cN crystallin gene duplication revealed a more recent episode of positive selection in only one of the two descendant lineages (cN2). Finally, from the guppy, Poecilia reticulata, we isolated complete cN1 and cN2 coding sequence data from cDNA and partial coding sequence data from genomic DNA in order to confirm the presence of a novel cN2 intron, discovered through data mining of two pufferfish genomes. We conclude that the function of the cN family likely resembles the ancestral vertebrate bc crystallin more than other bc families. Furthermore, owing to the presence of an additional intron in some fish cN2 crystallins, and the inferred action of positive selection following the fish-specific cN duplication, we suggest that further study of fish cN crystallins will be critical in further elucidating possible ancestral functions of cN crystallins and any nonstructural role they may have.