Binding of [1‐¹³C]galactose‐enriched hen ovalbumin to Erythrina cristagalli agglutinin as studied by ¹³C‐NMR spectroscopy

Abstract

A study of the equilibrium binding of glycoproteins to lectins was undertaken using the titled compounds as a model system for such interactions. The binding of hen ovalbumin, enriched in galactose specifically 13C-labelled at C1, to Erythrina cristagalli agglutinin was studied by 13C-NMR spectroscopy. The lectin was shown to be bivalent for ovalbumin with an apparent estimated association constant, at infinite dilution, of about lo4 M-'. The observed association is similar to that found for the corresponding disaccharide, Gal(j1-4)GlcNAc.The results strongly suggest that only the N-acetyllactosamine moiety in the carbohydrate side chain of galactoseenriched ovalbumin participates in the binding to the lectin with little, if any, interactions from other parts of either the side chain or the polypeptide backbone of ovalbumin.Lectins are proteins which agglutinate cells and bind carbohydrates specifically [l]. Binding of carbohydrates to lectins has been examined by a variety of techniques [2]. NMR spectroscopy was successfully employed in previous studies by utilising signals arising from a specifically labelled part of the carbohydrate molecule, and the magnetic isotopes used were I3C, 2H and I9F [3-81. In other more favourable cases, natural-abundance NMR spectroscopy was employed [9 -3 11. To study the interactions between two large molecules, it is advantageous to use specific isotope labelling in that it isolates and labels desired NMR signals and reduces signal overlap [12-141. To date, no direct binding study of glycoproteins to lectins has been reported. The major difficulty in such studies is, no doubt, related to the fact that the known glycoproteins which carry oligosaccharide side chains of either complex or hybrid types exhibit structural microheterogeneity with respect to these sugar side chains. Therefore, these glycoproteins, by definition constitute poorly defined ligands for such binding studies.Erythrina cristagalli agglutinin (ECA) is a well characterised glycoprotein which shows a pronounced preference for binding N-acetyllactosamine [15, 161. ECA is composed of two subunits and, according to studies with monosaccharides, it has two sugar-binding sites [15-181. It binds oligosaccharides containing multiple N-acetyllactosamine branches with greater afinity than the corresponding unbranched oligosaccharides [18].

Neoglycoconjugates

“…These reconstructed cell surface glycoconjugates containing only a single specific type of sialyl linkages enabled them to probe specificities of viral binding. In another example of reconstruction of glycoconjugates, Berman et al (Berman et al, 1985;Berman et al, 1986;Berman and Lis, 1987), used a galactosyltransferase to incorporate 13C-labeled Gal onto GlcNAc for binding studies by CMR. Chemical synthesis of such 13C-labeled oligosaccharide chains would be extremely laborious.…”