Lectinochemical studies on the affinity of Anguilla anguilla agglutinin for mammalian glycotopes

Abstract

Anguilla anguilla agglutinin (AAA) is a fucose-specific lectin found in the serum of the fresh water eel. It is suggested to be associated with innate immunity by recognizing disease-associated cell surface glycans, and has been widely used as a reagent in hematology and glycobiology. In order to gain a better understanding of AAA for further applications, it is necessary to elucidate its binding profile with mammalian glycotopes. We, therefore, analyzed the detailed carbohydrate specificity of AAA by enzyme-linked lectinosorbent assay (ELLSA) with our extended glycan/ligand collection and lectin-glycan inhibition assay. Among the glycans tested, AAA reacted well with nearly all human blood group Ah (GalNAcalpha1-->3[LFucalpha1-->2]Gal), Bh (Galalpha1-->3[LFucalpha1-->2]Gal), H LFucalpha1-->2Gal) and Leb (Fucalpha1-->2Galbeta1-->3[Fucalpha1-->4]GlcNAc) active glycoproteins (gps), but not with blood group Lea (Galbeta1-->3[Fucalpha1-->4]GlcNAc) substances, suggesting that residues and optimal density of alpha1-2 linked LFuc to Gal at the non-reducing end of glycoprotein ligands are essential for lectin-carbohydrate interactions. Blood group precursors, Galbeta1-3GalNAc (T), GalNAcalpha1-Ser/Thr (Tn) containing glycoproteins and N-linked plasma gps, gave only negligible affinity. Among the mammalian glycotopes tested, Ah, Bh and H determinants were the best, being about 5 to 6.7 times more active than LFuc, but were weaker than p-nitrophenylalphaFuc indicating that hydrophobic environment surrounding the LFuc moiety enhance the reactivity. The hierarchy of potency of oligo- and monosaccharides can be ranked as follows: p-nitrophenyl-alphaFuc > Ah, Bh and H > LFuc > LFucalpha1-->2Galbeta1-->4Glc (2'-FL) and Galbeta1-->4[LFucalpha1-->3]Glc (3'-FL), while LNDFH I (Leb hexa-), Lea, Lex (Galbeta1-->4[Fucalpha1-->3]GlcNAc), and LDFT (gluco-analogue of Ley) were inactive. From the present observations, it can be concluded that the combining site of AAA should be a small cavity-type capable of recognizing mainly H/crypto H and of binding to specific polyvalent ABH and Leb glycotopes.