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

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.

Figure 1

Structure and mutants of Con-anc. (A) 3D structure of Con-anc with liganded lactose, which was predicted by homology modeling, based on congerin I. The amino acid residues in N- and C-termini (NT and CT, respectively) and loops 3, 5, and 6 (L3, L5, and L6, respectively) of Con-anc mutants are represented by the space-filling model. (B) Aligned amino acid sequences of ConI, ConII, Con-anc, and Con-anc mutants. The sequences were aligned using the ClustalW program. Residue numbers of ConI were used as reference for all the congerins and mutants in this study. Positions of the strands (S1--S6, F1--F5) and loops (L1--L10) are indicated by thick and thin horizontal lines, respectively. The mutated amino acid positions are boxed in color.

Figure 2

The molecular phylogenetic tree of galectins including Con-anc and mutants. The phylogenetic tree was inferred from the amino acid sequences.

Figure 3

Thermal stabilities of ConI, ConII, Con-anc, and Con-anc mutants. The residual hemagglutination activities were measured after incubation at various temperatures for 30 min.

Figure 4

The relative sugar-binding activities of Con II and Con-anc mutants when compared with that of Con I. Scale for the PA sugars, except for LNFP-II, LNFP-III, LNDFH, and A-heptasaccharide (#44, #45, #46, and #48, respectively), was expanded. The PA sugar numbers are provided in Additional File 3: Supplemental Figure S1.

Figure 5

The relative sugar-binding activities of Con I and Con-anc mutants when compared with that of Con II. Asterisk (*) indicates no binding activities against sugars #29, #30, and #33, and asterisks (**) indicate no activities against #40 in addition to #29, #30, and #33. The PA sugar numbers are provided in Additional File 3: Supplemental Figure S1.

Figure 6

Correlation of inter- and intra-loop hydrogen bond formation during MD simulation. Correlation coefficients of hydrogen bond formation rates are shown on the structure of ConI. L3, L4, L5, and L6 represent the intra-loop bonds. L2--L4, L2--L6, and L3--L5 indicate the inter-loop bonds. Lac-R28 and Lac-R47 are the bonds between the residues and lactose. The colors of the arrows connecting the labels indicate the degree of correlation from positive (red) to negative (blue), as shown in the color bar.

Figure 7

Cytotoxic effects of ConI, ConII, and Con-anc mutants on Jurkat cells. Solid square and continuous line represent ConI; solid circle and continuous line represent ConII; solid triangle and continuous line indicate Con-anc; hollow circle and dotted line indicate Con-anc-N/C/L5/L6; and hollow diamond and broken line represent Con-anc-N/C/L5/L6-T38 M. The calculated ED₅₀ values (50% effective dose) were as follows: ConI, 0.098; ConII, 0.27; Con-anc, 1.2; Con-anc-N/C/L5/L6, 0.12; and Con-anc-N/C/L5/L6/L3, 0.11.

Figure 8

Evolutionary pathway of ConI. Each step in the pathway represents one functional evolutionary process. *A subset of sugars includes LNFP-II, LNDFH, and A-heptasaccharide (#44, #46, and #48, respectively).