Allosteric regulation of the carbohydrate-binding ability of a novel conger eel galectin by D-mannoside

Abstract

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of D-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that D-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.

FIGURE 1.

Nucleotide sequences and deduced amino acid sequences of Con-P and congerins. Con-P, congerin P; Con I, congerin I; Con II, congerin II. Dashes indicate gaps introduced to maximum sequence identity, and dots and asterisks represent the positions identical to Con-P in the nucleotide and amino acid sequences, respectively. The nucleotide sequences of Con I and Con II were from GenBank^TM database with accession codes AB010276 and AB010277, respectively.

FIGURE 2.

Amino acid sequence alignment of galectins from fish and human galectin-1. Con-P, congerin P; Con I, congerin I; Con II, congerin II; AJL1, Japanese eel galectin; Eele, electric eel galectin; hGal-1, human galectin-1. Asterisks show conserved amino acid residues in the CRD. The arrow indicates the Trp-117 residue of Con-P.

FIGURE 3.

Recombinant expression of Con II-tagged Con-P. Schematic representation of the expression plasmid for Con II-tagged Con-P (A) and time course expression levels of recombinant protein in E. coli in the absence (GroEL(−)) or presence (GroEL(+)) of chaperones (B) are shown. SDS-PAGE analysis with Coomassie Brilliant Blue staining (left) and Western blot analysis using anti-Con II antibody (right) are shown. Monomeric Con II-tagged Con-P appears at ∼28 kDa. The asterisk indicates native Con II.

FIGURE 4.

Restriction protease digestion of Con II-tagged Con-P fusion protein. Restriction digestion of Con II-tagged Con-P with α-thrombin and MLP at 37 °C (Α) and time course digestion of Con II-tagged Con-P with MLP at 15 °C (B) are shown. nCon II, native Con II. Following MLP digestion, cleaved Con II tag was detected by the presence of a 15-kDa band by using anti-Con II antibody.

FIGURE 5.

Purification of recombinant Con-P. SDS-PAGE analysis (A) and Western blot analysis of Con-P by using anti-Con II-tag antibody (B) or anti-Con-P antibody (C) for each purification step are shown. Restriction digestion of recombinant Con II-tagged Con-P with MLP was at 15 °C. Following MLP digestion, rCon-P was purified by mannose-immobilized TOYOPEARL AF-650 M column. The following are shown: E. coli lysate (lane i); soluble fraction containing Con II-tagged Con-P (lane ii); Con II-tagged Con-P purified by affinity purification on an HCl-treated Sepharose 4B column (lane iii); MLP-digested products of Con II-tagged Con-P (lane iv); purified rCon-P by mannose column (lane v), and native Con II (lane vi).

FIGURE 6.

Mannose-induced hemagglutination activity of Con-P. Relative hemagglutination activities of congerins at 250 μg/ml (A) and hemagglutination activity of MLP-digested Con II-tagged Con-P (B) are shown. The asterisk indicates no hemagglutinating activity. Con-P showed 30-fold greater hemagglutinating activity than Con I in the presence of 25 mm mannose but not absence of mannose. MLP-digested Con II-tagged Con-P contained equal amounts of Con-P and Con II. Hemagglutination activity of Con-P was assessed in the absence and presence of 25 mm mannose, respectively, and was completely inhibited by 20 mm lactose.

FIGURE 7.

Carbohydrate specificity of activated Con-P. Affinity constants of Con-P, Con I, and Con II for 22 PA sugars were determined by frontal affinity chromatography analysis using buffer containing 20 mm mannose. No data are available for the binding of Con I and Con II to oligo-mannose-type carbohydrates.

FIGURE 8.

Mannose-binding ability of Con-P. Titration of mannose (A and B) with 250 μm Con-P solution and Scatchard plots (C and D) at concentrations ranging from 1 to 100 μm (A and C) and from 5 to 125 mm (B and D), respectively. Fluorescence intensities at 320 nm between the free Trp-117 of Con-P and mannose at a given concentration were measured. Tryptophan excitation wavelength was set to 280 nm, and emission spectrum was recorded from 300 to 400 nm. The association constants K_a of Con-P toward mannose were estimated from Scatchard plots displaying ΔF/F₀ versus ΔF/F₀/[S]. ΔF, which is the difference in fluorescence intensity at 320 nm between free Con-P and with mannose at a given concentration [S], was normalized by F₀.