Open Access Research article

Protein engineering of conger eel galectins by tracing of molecular evolution using probable ancestral mutants

Ayumu Konno1, Shintarou Yonemaru1, Atsushi Kitagawa2, Koji Muramoto1, Tsuyoshi Shirai2 and Tomohisa Ogawa1*

Author Affiliations

1 Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Sendai 981-8555, Japan

2 Nagahama Institute of Bio-Science and Technology and Japan Science and Technology Agency, BioInfomatic Research Division, Nagahama, Shiga 526-0829, Japan

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BMC Evolutionary Biology 2010, 10:43  doi:10.1186/1471-2148-10-43

Published: 14 February 2010

Abstract

Background

Conger 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.

Results

In 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.

Conclusions

Thus, 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.