Carbohydrate specificity of an insecticidal lectin isolated from the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates

Abstract

Preliminary studies indicated that the potent insecticidal lectin, Gleheda, from the leaves of Glechoma hederacea (ground ivy) preferentially agglutinates human erythrocytes carrying the Tn (GalNAcalpha1-Ser/Thr) antigen. However, no details have been reported yet with respect to the fine specificity of the lectin. To corroborate the molecular basis of the insecticidal activity and physiological function of Gleheda, it is necessary to identify the recognition factors that are involved in the Gleheda-glycotope interaction. In the present study, the requirement of high-density multivalent carbohydrate structural units for Gleheda binding and a fine-affinity profile were evaluated using ELLSA (enzyme-linked lectinosorbent assay) with our extended glycan/ligand collections, a glycan array and molecular modelling. From the results, we concluded that a high-density of exposed multivalent Tn-containing glycoproteins (natural armadillo and asialo ovine salivary glycoproteins) were the most potent factors for Gleheda binding. They were, on a nanogram basis, 6.5x10(5), 1.5x10(4) and 3.1x10(3) times more active than univalent Gal (galactose), GalNAc (N-acetylgalactosamine) and Tn respectively. Among mono- and oligo-saccharides examined, simple clustered Tn (molecular mass <3000 Da) from ovine salivary glycoprotein was the best, being 37.5 and 1.7x10(3) times better than GalNAc and Gal respectively. GalNAc glycosides were significantly more active than Gal glycosides, indicating that the N-acetamido group at C-2 plays an important role in Gleheda binding. The results of glycan array support the conclusions drawn with respect to the specificity of Gleheda based on the ELLSA assays. These findings combined with the results of the molecular modelling and docking indicate the occurrence of a primary GalNAcalpha1-binding site in the Gleheda monomer. However, the extraordinary binding feature of Gleheda for glycoproteins demonstrates the importance of affinity enhancement by high-density multivalent glycotopes in the ligand-lectin interactions in biological processes.

Figure 1. Binding of Gleheda to microtitre plates coated with serially diluted human blood group A, B, H, Le^a and Le^b active gps, sialo- and asialo-gps

The amount of lectin used was 10 ng/well. The total volume of the assay was 50 μl. A₄₀₅ was recorded after 2 h of incubation.

Figure 2. Carbohydrate structural units in salivary gps

(i) Native ASG-Tn and asialo HSM [45,46]. (ii) Asialo OSM (over 75% Tn) [47].

Figure 3. Inhibition of Gleheda binding to asialo PSM coated on ELLSA plates by various gps

The quantity of gp in the coating solution was 2 ng/well. The quantity of lectin used for inhibition assay was 10 ng/well. The final Gleheda content was 5 ng/well. Total volume was 50 μl. A₄₀₅ was recorded after 2 h of incubation. The amount (ng) of gp required to induce 50% inhibition was determined.

Figure 4. Inhibition of Gleheda binding to asialo PSM coated on ELLSA plates by various saccharides

The amount of gp in the coating solution was 2 ng/well. The lectin (10 ng/well) was pre-incubated with an equal volume of serially diluted inhibitor. The final Gleheda content was 5 ng/well. Total volume was 50 μl. A₄₀₅ was recorded after 2 h of incubation.

Figure 5. Docking of Tn and T antigen in the carbohydrate-binding site of Gleheda

(A) Docking of Tn antigen in the carbohydrate-binding site. The network of hydrogen bonds (dark broken lines) connecting the sugars to residues Asp⁷³, Gly⁹³, Asn¹²², Trp¹¹⁷, Thr²⁰¹ and Asn²⁰² (Corey–Pauling–Koltun code) forming the monosaccharide-binding site of Gleheda. Residue His¹¹⁵ which stacks against the pyranose ring of the GalNAc is not shown. (B) Docking of GalNAcα1-Ser into the carbohydrate-binding cavity of Gleheda. (C) Docking of the T antigen into the carbohydrate-binding cavity of Gleheda showing the clash of the Gal residues with residues Asp⁷³, His¹¹⁵ and Asn¹¹⁷. Both the carbohydrate-binding site and Tn or T antigen are similarly oriented in (A), (B) and (C). The Figure was drawn using PyMOL.