Haruko Ueda

Interaction of a lectin from Psathyrella velutina mushroom with N‐acetylneuraminic acid

A lectin from the fruiting body of Psathyrella velutina has been used as a specific probe for non‐reducing terminal N‐acetylglucosamine residues. We reveal in this report that P. velutina lectin recognizes a non‐reducing terminal N‐acetylneuraminic acid residue in glycoproteins and oligosaccharides. Binding of biotinyl P. velutina lectin to N‐acetylneuraminic acid residues was prevented by desialylation of glycoconjugates and was distinguished from the binding to N‐acetylglucosamine. Sialooligosaccharides were retarded or bound and eluted with N‐acetylglucosamine on a P. velutina lectin column, being differentiated from each other and also from the oligosaccharides with non‐reducing terminal N‐acetylglucosamine which bound more strongly to the column.

Screening of a unique lectin from 16 cultivable mushrooms with hybrid glycoprotein and neoproteoglycan probes and purification of a novel N-acetylglucosamine-specific lectin from Oudemansiella platyphylla fruiting body

Hybrid glycoprotein and neoproteoglycan probes were prepared by coupling various glycoproteins or polysaccharides to peroxidase or biotinyl bovine serum albumin, respectively. Lectins recognizable by the neoglycoconjugate probes were extracted from 16 cultivable mushrooms. Dot-blot assay revealed five extracts to be reactive with only hybrid glycoprotein probes, but others also reacted with neoproteoglycan probes. According to the reactivity pattern with probe screening, the one lectin from Oudemansiella platyphylla extract (OPL) bound best with asialotransferrin-- and asialoagalactotransferrin--peroxidase probes and was isolated using an asialotransferrin column, but it did not bind with other hybrid glycoprotein or neoproteoglycan probes. OPL, consisting of two polypeptides with high homology in the N-terminal amino acid sequences, exhibited weak hemagglutinating activity. Purified OPL specifically bound the beta-GlcNAc probe among various biotinylated polymeric sugar probes, while it exhibited essentially the same binding specificity toward neoglycoconjugate probes as that of the crude extract, showing a preference for the asialobiantennary complex type of N-linked glycans. These results indicate that the neoglycoconjugate probes are valuable in lectin screening.

Solubility–insolubility interconversion of sophoragrin, a mannose/glucose-specific lectin in Sophora japonica (Japanese pagoda tree) bark, regulated by the sugar-specific interaction

Sophoragrin, a mannose/glucose-specific lectin in Sophora japonica (Japanese pagoda tree) bark, was the first lectin found to show self-aggregation that is dependent on the sugar concentration accompanying the interconversion between solubility and insolubility [Ueno, Ogawa, Matsumoto and Seno (1991) J. Biol. Chem. 266, 3146-3153]. The interconversion is regulated by the concentrations of Ca(2+) and specific sugars: mannose, glucose or sucrose. The specific glycotopes for sophoragrin were found in the sophoragrin subunit and an endogenous galactose-specific lectin, B-SJA-I (bark S. japonica agglutinin I), and the lectin subunit that binds to the glycotope was identified by photoaffinity glycan probes. Remarkably, the insoluble polymer of sophoragrin is dissociated by interaction with B-SJA-I into various soluble complexes. Based on these results, self-aggregation of sophoragrin was shown to be a unique homopolymerization due to the sugar-specific interaction. An immunostaining study indicated that sophoragrin localizes mainly in vacuoles of parenchymal cells coincidently with B-SJA-I. These results indicate that sophoragrin can sequester endogenous glycoprotein ligands via sugar-specific interactions, thus providing new insights into the occurrence and significance of the intravacuolar interaction shown by a legume lectin.

Psathyrella velutina Lectin as a Specific Probe for N-Acetylneuraminic acid in Glycoconjugates

Publisher Summary This chapter describes a high throughput purification protocol and methods to study the binding activity of psathyrella velutina lectin (PVL) toward monosaccharides, oligosaccharides, or glycoproteins and elucidates its specificity toward NeuAc-containing glycoconjugates by comparing it with other NeuAc-binding lectins. PVL has been used as an N -acetylglucosamine (GlcNAc)-specific reagent for the detection of glycoconjugates. Chemically-labeled PVL could detect sialyl residues and differentiate them from GlcNAc residues when used in combination with the glycosidase treatment of glycoconjugates. The binding activity of PVL for acidic polysaccharides, such as heparin and polygalacturonic acid, was blocked specifically by the biotinylation of PVL possessing GlcNAc/NeuAcbinding activity. PVL-Sepharose is also a useful tool for the separation of glycoconjugates containing NeuAc or terminal GlcNAc residues from others in terms of the number or linkage of these sugar residues.

Preparation and utility of neoproteoglycan probes in analyses of interaction with glycosaminoglycan-binding proteins.

Publisher Summary Proteoglycans have been attracting increasing biological interest because of their specific signaling roles or modulatory functions in important cellular events, for example, growth factor–receptor interaction and signaling, viral infection, extracellular matrix assembly, and neurite outgrowth and plasticity. Such functions are exhibited mostly by glycosaminoglycanbinding proteins (lectins or receptors) that recognize repeating disaccharide units with microheterogeneities in the sugar residues and the negative charges. There is a need for a detailed study in the specificity of lectins to glycoconjugates, because certain multispecificities may call into question the validity of the lectins when used as carbohydrate-specific probes. This chapter discusses two types of neoproteoglycan probes—one is used to immobilize glycosaminoglycans or polysaccharides effectively on solid substrates and the other is a biotin- or peroxidase-labeled neoproteoglycan probe that is used to detect binding with high sensitivity.