Sequence Analysis of Four Acidic β-Crystallin Subunits of Amphibian Lenses: Phylogenetic Comparison between β- and γ-Crystallins

Abstract

β-Crystallins composed of the most heterogeneous group of subunit chains among the three major crystallin families of vertebrates, i.e. α-, β- and γ-Crystallins, are less well understood at the structural and functional levels than the other two. They comprise a multigene family with at least three basic (βB1–3) and four acidic (βA1–4) subunit polypeptides. In order to facilitate the determination of the primary sequences of all these ubiquitous crystallin subunits present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+mRNA isolated from bullfrog eye lenses. We report here a protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding β-crystallin acidic subunit polypeptides by polymerase chain reaction (PCR). Four complete full-length reading frames with two each of 597 and 648 base pairs, which cover four deduced protein sequences of 198 (βA1-1 and βA1-2) and 215 (βA3-1 and βA3-2) amino acids including the universal initiating methionine, were revealed by nucleotide sequencing. They show about 96–98% sequence similarity among themselves and 76–80%, 80–83% to the homologous βA1/A3 crystallins of bovine and human species respectively, revealing the close structural relationship among acidic subunits of all β-crystallins even from remotely related species. In this study a phylogenetic comparison based on amino-acid sequences of various βA1/A3 crystallins plus the major basic β-crystallin (βBp) and γ-crystallin from different vertebrate species is made using a combination of distance matrix and approximate parsimony methods, which correctly groups these βA crystallin chains together as one family distinct from basic β-crystallins and γ-crystallin and further corroborates the supposition that β- and γ-crystallins form a superfamily with a common ancestry.