Shunji Sugii

Coding and classification of d-galactose, N-acetyl-d-galactosamine, and β-d-Galp-[1→3(4)]-β-d-GlcpNAc, specificities of applied lectins☆☆☆

Abstract Grouping of lectin-binding properties, based on determinant structure rather than monosaccharide-inhibition pattern, should facilitate the selection of lectins as structural probes for glycans, as well as for the interpretation of the distribution and the properties of the carbohydrate chains on the cell surface. Based on the binding specificities studied with glycan by precipitin-inhibition, competitive-binding, and hemagglutinin-inhibition assays, twenty d -galactose-or N -acetyl- d -galactosamine-(or both)-specific lectins have been divided into six classes according to their specificity for a disaccharide unit, as all or part of the determinants, and the α- d -Gal p NAc-(1→3)-Ser(Thr) unit of the glycopeptide chain. A scheme of classification is shown as follows: (a) F-specific lectins [α- d Gal p NAc-(1→3)- d -GalNAc, Forssman specific disaccharide]: Dolichos biflorus (DBL), Helix pomatia (HPL), hog peanut (ABL, Amphicarpaea bracteata ), and Wistaria floribunda (WFL) lectins. (b) A-specific lectins [α- d -Gal p NAc-(1→3)- d -Gal blood group A-specific disaccharide]: Griffonia (Bondeiraea) simplicifolia-A 4 (GSI-A 4 ), lima bean (LBL), soy bean(SBL), Vicia villosa (VVL), Wistaria floribunda (WFL), Dolichos biflorus (DBL), and Helix pomatia (HPL) lectins. (c) Tn-specific lectins [α- d -Gal p NAc-(1→3)-Ser(Thr) of the protein core]: Vicia villosa B 4 (VVL-B 4 ), Salvia sclarea (SSL), Machura pomifera (MPL), Bauhinia purpurea alba (BPL), HPL, and WFL, lectins. (d) T-specific lectins [β- d -Gal p -(1→3)- d -GalNAc, the mucin-type sugar sequences on human erythrocyte membrane and T antigen, or the terminal, nonreducing disaccharide end-groups of the gangliosides]: Peanut (PNA), Bauhinia purpurea alba (BPL), Machura pomifera (MPL), Sophora japonica (SJL), Artocarpus integrifolia (Jacalin, AIL), and Artocarpus lakoocha (Artocarpin) lectins. (e) Type I and II specific lectins [β- d -Gal p -(1→3 or 4)- d -GlCNAc, the disaccharide residues at the nonreducing end of the carbohydrate chains derived from either N - or O -glycans]: Ricinus communis agglutinin (RCAl), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL), lectins. (f) B-specific lectin [α- d -Gal p -(1→3)-β- d -Gal p , human blood group B-specific disaccharide]: Griffonia (Bandeiraea) simplicifolia B 4 (GSI-B 4 ) lectin. Many other GalNAc- or Gal-(or both)-specific lectins that can be used as tools are also described.

Differential Binding Properties of GalNAc and/or Gal Specific Lectins

Grouping of lectin binding properties, based on determinant structure rather than monosaccharide inhibition pattern, should facilitate the selection of lectins as structural probes for glycans as well as for the interpretation of the distribution and the properties of the carbohydrate chains on the cell surface. Based on the binding specificities studied with glycan by precipitin-inhibition, competitive-binding and hemagglutinin-inhibition assays, twenty Ga1 and/or Ga1NAc specific lectins have been divided into six classes according to their specificity for the disaccharide as all or part of the determinants and Ga1NAc alpha 1----Ser(Thr) of the peptide chain. The differential affinities of these lectins were characterized by quantitative precipitin assay. Abbreviation of the following six lectin determinants can also be used to classify these lectins. (1) F determinant (GalNAc alpha 1----3GalNAc, Forssman specific disaccharide). (2) A (Af) determinant (GalNAc alpha 1----3Gal, Human blood group A specific disaccharide; Af, fucosylated A, (GalNAc alpha 1----3 [LFuc alpha 1----2]Gal). (3) Tn determinant (GalNAc alpha 1----0 to Ser (Thr) of the protein core, Tn antigen). (4) T determinant (T antigen, Gal beta 1----3GalNAc alpha 1----0 to Ser (Thr) of the protein core, the mucin type sugar sequence on the human erythrocyte membrane or Gal beta 1----3GalNAc beta 1---- at the nonreducing end of ganglioside). (5) I and II determinants (human blood group type I and II carbohydrate sequences). Most of the lectins reactive to Gal beta 1----4GlcNAc (II) are also reactive to Gal beta 1----3GlcNAc (I). Lectin I (II) determinants (i.e. Gal beta 1----3 (4) GlcNAc residues) can be found at the nonreducing end of the carbohydrate chains derived from either N-glycosidic or O-glycosidic linkages. (6) B determinant (Gal alpha 1----3Gal, Human blood group B specific disaccharide). Their carbohydrate specificities are classified as following: (Table see text). The differential binding properties of lectins can be defined from comparisons of their carbohydrate specificities listed above.

A guide for carbohydrate specificities of lectins.

Lectins are carbohydrate-binding proteins of nonimmune origin, which have been widely used in the fields of cell biology, biochemistry and histochemistry to isolate and/or to characterize cell surface carbohydrates(1–3). Lectins require configurational and structural complementarity of sugars for interaction to occur. All lectin molecules have two or more carbohydrate binding sites, a property essential to their ability to agglutinate cells or to precipitate complex carbohydrates(1, 2, 4). Until the early seventies, the carbohydrate specificities of lectins were mainly determined by the abilities of monosaccharides or their glycosides to inhibit lectin-induced haemagglutination(1, 4). Makela(5), in the late fifties, divided lectin-reactive monosaccharides into four classes, based on their configuration at C-3 and C-4 of the pyranose form, as shown in Fig. 1. Lectins that bind to Makela’s group II sugars are Gal-specific whereas those reacting with sugars of group III are Man and/or Glc-specific lectins. Fucose-binding lectins are specific for group I sugars. Lectins binding sugars belonging to group IV have not yet been reported.

Hemagglutinating and binding properties of botulinum C2 toxin

To characterize the binding substance(s) for botulinum C2 toxin, the hemagglutinating activity of component II of botulinum C2 toxin (C2II) was studied by hemagglutination and hemagglutination inhibition. Human and animal erythrocytes were agglutinated by trypsinized C2II much more strongly than by untreated C2II. Trypsinized C2II agglutinated neuraminidase-treated erythrocytes more strongly than intact, trypsin- and pronase-treated ones. On the other hand, trypsin- and pronase-treated erythrocytes were more weakly hemolyzed by trypsinized C2II than intact and neuraminidase-treated ones, and trypsinized C2II showed both hemagglutinating and hemolytic activities to these erythrocytes. Hemagglutination of trypsin-treated human type B erythrocytes was inhibited by galactose, N-acetylgalactosamine, N-acetylglucosamine, L-fucose and mannose. Thyroglobulin and bovine salivary mucin were much stronger inhibitors. From these findings, the binding substance(s) for botulinum C2 toxin on erythrocytes is(are) suggested to be glycoprotein(s).

Studies on the binding substances on human erythrocytes for the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

The binding substance for the heat-labile enterotoxin (LTp) isolated from porcine enterotoxigenic Escherichia coli was studied by competitive binding assays. The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1 among inhibitors used. Mono-, di- and polysaccharides, glycoproteins and lectins were over 10(4)-times less potent inhibitors. Similar results were also obtained in competitive binding assays with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B. On the other hand, hemagglutination of neuraminidase-treated human type A erythrocytes by LTp was inhibited by methyl alpha-D-galactopyranoside, galactose, melibiose and some glycoproteins, but not effectively inhibited by ganglioside GM1 at the highest concentration used. Preincubation of LTp with an appropriate amount of ganglioside GM1 resulted in much higher hemagglutination than LTp alone. Although these findings show that there may be fundamental differences between interactions with ganglioside GM1 in hemagglutination compared to interactions with ganglioside GM1 in binding, the predominant binding substance for LTp on neuraminidase-treated human type A erythrocytes is suggested to be ganglioside GM1.

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enteroxigenic Escherichia coli

The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.