Shuh-Chyung Song

A Guide to the Carbohydrate Specificities of Applied Lectins-2

The lectins, that can be used as tools to study glycobiological systems are defined as applied lectins (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).They are easily obtained, stable and have their own binding specificity extending beyond the monosaccharide(1, 2, 3, 4,15, 16, 17, 18, 19).Their biochemical application has been reviewed extensively by Goldstein and Poretz (19), Sharon and Lis (8) and Goldsteinet al. (11) Hundreds of lectins are used as applied lectins. Thus, organizing and grouping binding properties of these lectins should facilitate the selection of lectins as structural probes for studying glycans as well as the interpretation, distribution and properties of the carbohydrate chains on the cell surface. From the information provided by inhibition assays and binding properties, the carbohydrate specificities of applied lectins are classified into six groups according to their specificities to monosaccharides. The subgroups are based on lectin affinities to GalNAca1-0 to Ser(Thr) of the peptide chain, disaccharides, trisaccharides and, the number and the location of LFucal-’linked to oligosaccharides; all of these structures are found mainly in soluble glycoproteins and as cell surface glycoconjugates in mammals (1, 2, 3, 4,18). Reviews concerning coding and classification of DGa1, GaINAc and Galf31-3/4G1cNAc specificities of applied lectins are given in Secion 1–4 of this proceeding (18) together with references 1, 2, 3, 4. A scheme of the classification is shown as follows.

STUDIES ON THE BINDING OF WHEAT GERM AGGLUTININ (TRITICUM VULGARIS) TO O-GLYCANS

The binding profile of Triticum vulgaris (WGA, wheat germ) agglutinin to 23 O‐glycans (GalNAcα1→Ser/Thr containing glycoproteins, GPs) was quantitated by the precipitin assay and its specific interactions with O‐glycans were confirmed by the precipitin inhibition assay. Of the 28 glycoforms tested, six complex O‐glycans (hog gastric mucins, one human blood group A active and two precursor cyst GPs) reacted strongly with WGA and completely precipitated the lectin added. All of the other human blood group A active O‐glycans and human blood group precursor GPs also reacted well with the lectin and precipitated over two‐thirds of the agglutinin used. They reacted 4–50 times stronger than N‐glycans (asialo‐fetuin and asialo‐human α1 acid GP). The binding of WGA to O‐glycans was inhibited by either p‐NO2‐phenyl α,βGlcNAc or GalNAc. From these results, it is highly possible that cluster (multivalent) effects through the high density of weak inhibitory determinants on glycans, such as GalNAcα1→Ser/Thr (Tn), GalNAc at the non‐reducing terminal, GlcNAcβ1→ at the non‐reducing end and/or as an internal residue, play important roles in precipitation, while the GlcNAcβ1→4GlcNAc disaccharide may play a minor role in the precipitation of mammalian glycan‐WGA complexes.

Multi‐antennary Galβ1→4GlcNAc and Galβ1→3GalNAc clusters as important ligands for a lectin isolated from the sponge Geodia cydonium

Based on the present and previous results, it is proposed that tri‐antennary Galβ1→4GlcNAc and Galβ1→3GalNAc clusters, in addition to GalNAcα1→3GalNAc and GalNAcα1→3Gal, are also important ligands for binding; and sialic acid of glycoprotein does interfere with binding.

Interaction of native and asialo rat sublingual glycoproteins with lectins

The binding properties of the rat sublingual glycoprotein (RSL) and its asialo product with lectins were characterized by quantitative precipitin(QPA) and precipitin inhibition(QPIA) assays. Among twenty lectins tested for QPA, native RSL reacted well only with Artocarpus integrifolia (jacalin), but weakly or not at all with the other lectins. However, its asialo product (asialo-RSL) reacted strongly with many Gal and GalNAc specific lectins-it bound best to three of the GalNAc alpha 1-->Ser/Thr (Tn) and/or Gal beta 1-->4GlcNAc (II) active lectins [jacalin, Wistaria floribunda and Ricinus communis agglutinins] and completely precipitated each of these three lectins. Asialo-RSL also reacted well with Abrus precatorius, Glycine max, Bauhinia purpurea alba, and Maclura pomifera agglutinins, and abrin-a, but not with Arachis hypogeae and Dolichos biflorus agglutinins. The interaction between asialo-RSL and lectins were inhibited by either Gal beta 1-->4GlcNAc, p-NO2-phenyl alpha-GalNAc or both. The mapping of the precipitation and inhibition profiles leads to the conclusion that the asialo rat sublingual glycoprotein provides important ligands for II (Gal beta 1-->4GlcNAc beta 1-->) and Tn (GalNAc alpha 1-->Ser/Thr) active lectins.

Native and/or asialo‐Tamm‐Horsfall glycoproteins Sd(a+) are important receptors for Triticum vulgaris (wheat germ) agglutinin and for three toxic lectins (abrin‐a, ricin and mistletoe toxic lectin‐I)

The binding properties of human Tamm‐Horsfall Sd(a+) urinary glycoprotein (THGP) and asialo‐THGP with Triticum vulgaris agglutinin(WGA) and three toxic lectins (abrin‐a, ricin, and Mistletoe toxic lectin‐I) were investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins reacted strongly with abrin‐a, precipitating over 80% of the lectin nitrogen tested. THGP also bound well to mistletoe toxic lectin‐I and precipitated 86% of this lectin added, while the precipitability of its asialo product decreased by 28%. The native glycoprotein completely precipitated the WGA added, but its reactivity was reduced dramatically after desialylation. On the contrary, the poor reactivity of THGP with ricin increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein‐lectin interactions were inhibited by one or several of the following haptens, p‐NO2‐phenylαGalNAc, p‐NO2‐phenylβGalNAc, Galβ1→4GlcNAc, Galβ1→4Glc, GlcNAcβ1→4GlcNAc, and/or GlcNAc. From the above results, it is concluded that native and/or asialo Tamm‐Horsfall glycoproteins serve as important receptors for these three toxic lectins and for WGA.

A sheep hydatid cyst glycoprotein as receptors for three toxic lectins, as well as Abrus precatorius and Ricinus communis agglutinins

The binding properties of a glycoprotein with blood group P1 specificity isolated from sheep hydatid cyst fluid with Gal and GalNAc specific lectins was investigated by quantitative precipitin and precipitin inhibition assays. The glycoprotein completely precipitated Ricinus communis agglutinin (RCA1), Abrus precatorius agglutinin (APA) and Mistletoe toxic lectin-I (ML-I). Only 1.0 microgram of P1 glycoprotein was required to precipitate 50% of 5.1 micrograms ML-I nitrogen. It also reacted well with abrin-a and ricin, precipitating over 73% of the lectin nitrogen added, but poorly or weakly with Dolichos biflorus (DBL), Vicia villosa (VVL, a mixture of A4, A2B2 and B4), VVL-B4, Arachis hypogaea (PNA), Maclura pomifera (MPL), Bauchinia purpurea alba (BPL) and Wistaria floribunda (WFL) lectins. When an inhibition assay in the range of 5.1 micrograms N to 5.9 micrograms N of lectins (ML-I, abrin-a; ricin, RCA1, and APA, and 10 micrograms P1 active glycoprotein interaction was performed; from 76 to 100% of the precipitations were inhibited by 0.44 and 0.52 mumol of Gal alpha 1-->4Gal and Gal beta 1-->4GlcNAc, respectively, but not or insignificantly with 1.72 mumol of GlcNAc. The Gal alpha 1-->4Gal disaccharide found in this P1 active glycoprotein is a frequently occurring sequence of many glycosphingolipids located at the surface of mammalian cell membranes, especially human erythrocytes and intestinal cells for ligand binding and microbial toxin attachment. The present finding suggests that the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in this P1 active glycoprotein is one of the best glycoprotein receptors for three toxic lectins (ricin, abrin-a, and ML-I) as well as for APA, and RCA1, and the result of inhibition assay implies that these lectins are recognizing part or all of the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in the P1 active glycoprotein.

DIFFERENTIAL BINDING OF HUMAN BLOOD GROUP SD(A+) AND SD(A-) TAMM-HORSFALL GLYCOPROTEINS WITH DOLICHOS BIFLORUS AND VICIA VILLOSA-B4 AGGLUTININS

The binding patterns of human blood group Sd(a+) and Sd(a−) Tamm‐Horsfall glycoproteins (THGPs) with respect to four GalNAc specific agglutinins were studied by quantitative precipitin assay (QPA) and enzyme linked lectinosorbent assay (ELLSA). Of the native and asialo Sd(a+) and Sd(a−) THGP tested by QPA and ELLSA, only native and asialo Sd(a+) bound well with Dolichos biflorus (DBA) and Vicia villosa‐B4 (VVA‐B4), while Sd(a−) THGP reacted poorly with these two lectins. Neither Sd(a+) nor Sd(a−) THGPs reacted with two other GalNAc α‐anomer specific lectins: Codium fragile subspecies tomentosoides and Artocarpus integrifolia. Furthermore, the binding of asialo Sd(a+)THGP‐VVA‐B4 and native Sd(a+)THGP‐DBA through GalNAcβ→ was confirmed by inhibition assay. These results demonstrate that DBA and VVA‐B4 are useful reagents to differentiate between Sd(a+) and Sd(a−) THGP.

Fraction A of armadillo submandibular glycoprotein and its desialylated product as sialyl‐Tn and Tn receptors for lectins

Fraction A of the armadillo submandibular glycoprotein (ASG‐A) is one of the simplest glycoproteins among mammalian salivary mucins. The carbohydrate side chains of this mucous glycoprotein have one‐third of the NeuAcα2→6GalNAc (sialyl‐Tn) sequence and two thirds of Tn (GalNAcα→Ser/Thr) residues. Those of the desialylated product (ASG‐Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinant). When the binding properties of these glycoproteins were tested by a precipitin assay with Gal, GalNAc and GlcNAc specific lectins, it was found that ASG‐Tn reacted strongly with all of the Tn‐active lectins and completely precipitated Vicia villosa (VVL both B4 and mixture of A and B), Maclura pomifera (MPA), and Artocarpus integrifolia (jacalin) lectins. However, it precipitated poorly or negligibly with Ricinus communis (RCA1); Dolichos biflorus (DBA); Viscum album, ML‐1; Arachis hypogaea (PNA), and Triticum vulgaris (WGA). The reactivity of ASG‐A (sialyl‐Tn) was as active as that of ASG‐Tn with MPA and less or slightly less active than that of ASG‐Tn with VVL‐A+B, VVLB4, HPA, WFA, and jacalin, as one‐third of its Tn was sialylated. These findings indicate that ASG‐A and its desialylated product (ASG‐Tn) are highly useful reagents for the differentiation of Tn, T (Galβ1→3GalNAc), A (GalNAcα1→3Gal) or Gal specific lectins and monoclonal antibodies against such epitopes.

Interaction of a human blood group Sd(a−) Tamm‐Horsfall glycoprotein with applied lectins

Unlike the human blood group Sd(a+) Tamm‐Horsfall glycoprotein (THGP), the Sd(a−) one lacks terminal GalNAcβ1 → residues at the nonreducing ends. The binding properties of this glycoprotein and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition assays. Among 20 lectins tested by QPA, both native and asialo Sd(a−) THGP reacted best with Abrus precatorius and Ricinus communis and completely precipitated the lectin added. They also precipitated well Wistaria floribunda (WFA), Glycine max (SBA), Bauhinia purpurea alba, abrin‐a and ricin, all of which recognize the Galβ1 → 4GlcNacβ1 → sequence, although at different strength. The lectin‐glycan interactions were inhibited by Galβ1 → 4GlcNAc and Galβ1 → 4Glc. When the precipitability of Sd(a−) THGP was compared with that of the Sd(a+) phenotype, the native Sd(a−) THGP exhibited a 40% lesser affinity for WFA, SBA, WGA and mistletoe lectin‐I (ML‐I). Mapping the precipitation and inhibition profiles of the present study and the results of THGP Sd(a+), it is concluded that Sd(a−) THGP showed a strongly diminished affinity for GalNAcβ1 → active lectins (SBA and WFA) than the Sd(a+) phenotype.