Abstract
BackgroundConger eel galectins, congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution. We recently reconstructed a probable ancestral form of congerins, Con-anc. It showed properties similar to those of ConII in terms of thermostability and carbohydrate recognition specificity, although it shares a higher sequence similarity with ConI than ConII.ResultsIn this study, we have focused on the different amino acid residues between Con-anc and ConI, and have performed the protein engineering of Con-anc through site-directed mutagenesis, followed by the molecular evolution analysis of the mutants. This approach revealed the functional importance of loop structures of congerins: (1) N- and C-terminal and loop 5 regions that are involved in conferring a high thermostability to ConI; (2) loops 3, 5, and 6 that are responsible for stronger binding of ConI to most sugars; and (3) loops 5 and 6, and Thr38 residue in loop 3 contribute the specificity of ConI toward lacto-N-fucopentaose-containing sugars.ConclusionsThus, this methodology, with tracing of the molecular evolution using ancestral mutants, is a powerful tool for the analysis of not only the molecular evolutionary process, but also the structural elements of a protein responsible for its various functions.
Highlights
Conger eel galectins, congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution
The following 3 Con-anc mutants were prepared: (1) Con-anc-N/C, in which the N- and C-termini were substituted with the corresponding residues of ConI; (2) Con-anc-L5, in which the L5 region was substituted with the corresponding sequence of ConI; and (3) Con-anc-N/C/L5, which had mutations of both Con-anc-N/C and Con-anc-L5 (Figure 1B)
The Con-anc mutants, in which the L6 and L3 regions were substituted with the corresponding sequences of ConI, i.e., Con-anc-N/C/L5/L6, Con-ancN/C/L5/L3, and Con-anc-N/C/L5/L6/L3 (Figure 1B), were prepared, and their carbohydrate-binding activities were determined by frontal affinity chromatography (FAC)
Summary
Congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution. Molecular evolution refers to the evolutionary process at the macromolecular level, such as at the DNA, RNA, and protein levels It encompasses the reconstruction of the evolutionary history of organisms and macromolecules (i.e., molecular phylogeny) on the basis of the sequence data of nucleic acids and proteins. The primary event in molecular evolution is a mutational change in genes that may be caused by the substitution or insertion/deletion of a nucleotide, recombination, etc.; otherwise, in general, DNA sequences are copied exactly during the process of chromosome replication. They spread in a population by genetic drift and/or natural selection, and eventually get established in a species [1,2]. On the basis of their structures, galectins are classified into three types: proto-, chimera-, and tandem repeat-type galectins [11]
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