Engineering of the glycan-binding specificity of Agrocybe cylindracea galectin towards α(2,3)-linked sialic acid by saturation mutagenesis

Abstract

Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.

Conformational change of a unique sequence in a fungal galectin from Agrocybe cylindracea controls glycan ligand‐binding specificity

“…Recent site-directed mutagenesis studies on galectins have revealed that only slight modification of ligand-binding sites can dramatically change the specificity of lectins [3,24,25]. In this study, we revealed the structural basis by which ACG specifically recognizes GalNAca1-3Galb-containing glycans.…”

A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin

“…Human podoplanins, purified from LN229/hPDPN, LN319, and CHO/hPDPN cells, were applied to a lectin microarray19. Figure 1b shows that podoplanins from LN319 and CHO/hPDPN react with sialic acid ± core1 binders (Jacalin, Agaricus bisporus agglutinin (ABA), Amaranthus caudatus agglutinin (ACA)), sialo-mucin binders ( Maackia amurensis hemagglutinin (MAH), wheat germ agglutinin (WGA)), and an α2-3 sialic acid binder ( Agrocybe cylindricea galectin (ACG), which shows a high affinity for α2-3 sialyl lactose and α2-3 sialyl LacNAc, as well as LacNAc, α2-3 core1, and core120). Although podoplanin from CHO/hPDPN reacts with Maclura pomifera agglutinin (MPA), podoplanin from LN319 did not.…”

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Homodimer 2 Gal-β1,4-GlcNAc na nd (Ban et al 2005;Imamura et al 2011;Kuwabara et al 2013;Liu et al 2008;Yagi et al 2001;Yagi et al 1997) Neu5Ac-α2,3-Gal-β1,4-…”