Reconstruction of a probable ancestral form of conger eel galectins revealed their rapid adaptive evolution process for specific carbohydrate recognition

Recently, many cases of rapid adaptive evolution, which is characterized by the higher substitution rates of nonsynonymous substitutions to synonymous ones, have been identified in the various genes of venomous and biodefense proteins, including the conger eel galectins, congerins I and II (ConI and ConII). To understand the evolutionary process of congerins, we prepared a probable ancestral form, Con-anc, corresponding to the putative amino acid sequence at the divergence of ConI and ConII in phylogenetic tree with 76% and 61% sequence identities to the current proteins, respectively. Con-anc and ConII had comparable thermostability and similar carbohydrate specificities in general, whereas ConI was more thermostable and showed different carbohydrate specificities. Con-anc showed decreased specificity to oligosaccharides with alpha 2,3-sialyl galactose moieties. These suggest that ConI and ConII have evolved via accelerated evolution under significant selective pressure to increase the thermostability and to acquire the activity to bind to α2,3-sialyl galactose present in pathogenic bacteria, respectively. Furthermore, comparative mutagenesis analyses of Con-anc and congerins revealed the structural basis for specific recognition of ConII to α2,3-sialyl galactose moiety, and strong binding ability of ConI to oligosaccharides including lacto-N-biosyl (Galβ1-3GlcNAc) or lacto-N-neobiosyl (Galβ1-4GlcNAc) residues, respectively. Thus, protein engineering using a probable ancestral form presented here is a powerful approach not only to determine the evolutionary process but also to investigate the structure-activity relationships of proteins.