Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers

Abstract

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…By the end of the last millennium, only two structures, representing CBM families 13 and 18, had been solved in complex with ligands by X-ray crystallography. These were the well-known plant-derived lectins ricin toxin B-chain [19] and WGA (wheat germ agglutinin) [20]. The structure of a family 20 starch-binding module in complex with β-cyclodextrin was determined by NMR spectroscopy [9].…”

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

“…Recently, Schwientek et al (2007) have utilized serial lectin affinity chromatography in combination with two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-offlight (MALDI-ToF) mass spectrometry in an innovative glycoproteomic approach, showing the usefulness of lectin binding properties as analytical tools in highthroughput glycobiological studies. Here, we have used one of the most commonly employed lectin in glycobiology, wheat germ agglutinin (WGA), which selectively recognizes sialic acid and N-acetylglucosaminyl sugar residues (Bhavanandan and Katlic, 1979;Bains et al, 1992;Muraki et al, 2002).…”

Differences in hydropathic properties of ligand binding at four independent sites in wheat germ agglutinin‐oligosaccharide crystal complexes

“…gested the prcscnce of two cquivalent sites/monomer (four per dimer) (Privat et al, 1974b;Kronis & Carver, 1985;Baines et al, 1992). In contrast, combined evidence from crystal structures of several types of oligosaccharide compleses revealed that all four unique sites are functional (Wright, 1980(Wright, , 1984(Wright, , 1990(Wright, , 1992.…”

“…to a single domain of (Lotan & Sharon, 1973;Nagata & Burger, 1974;Privat et al, 1974a;Baines et al, 1992). Clearly, high-affinity binding requires auxiliary contacts from across the dimer interface, particularly in the case of N-acetylated sialosides (Wright, 1992).…”