Partial identification of carbohydrate-binding sites of a Galα1,3Galβ1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

The Galα1,3Galβ1,4GlcNAc-specific lectin from the mushroom Marasmius oreades (MOA) contains a ricin B chain-like (QXW)3 domain at its N-terminus that is composed of three identical subdomains (α,β, and γ) and a C-terminal domain of unknown function. Here, we investigate the structure–function relationship of MOA to define the number and location of its carbohydrate-binding sites. Based on the sequence alignment of MOA to the ricin B-chain lactose-binding sites, we systematically constructed mutants by site-directed mutagenesis. We have used precipitation and hemagglutination assay for the primary analyses, and surface plasmon resonance for the kinetic analysis. Among amino acid residues at the putative carbohydrate-binding sites, Gln46 in the α subdomain and Trp138 in the γ subdomain have been identified to be important amino acid residues directly or indirectly involved in carbohydrate recognition. By surface plasmon resonance, Q46A and W138A were 2.4- and 4.3-fold less active than that of the wild-type MOA (Ka=2×107), respectively. A double-site mutant (Q46A/W138A) had activity similar to W138A. The C-terminal deletion mutant MOAΔC showed hemagglutination and precipitation activity, although its binding constant was 12.5-fold less active (Ka=1.6×106) than that of the wild-type MOA. A C-terminal deletion mutant with mutations at both Gln46 and Trp138 (MOAΔC-Q46A/W138A) was 12,500-fold less active (Ka=1.6×103) than that of the wild-type MOA. On the basis of this observation, we conclude that both α and γ subdomains are most probably involved in carbohydrate binding, but the β subdomain appears to be inactive.