Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins

Abstract

The carbohydrate binding specificity of the daffodil (Narcissus pseudonarcissus; NPA) and amaryllis (Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with [alpha]--glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal [alpha]--galactosyl groups, a synthetic linear [alpha]-1,6-mannan, and an [alpha]-1,3-mannan (DP = 30). Treatment of the linear [alpha]-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal [alpha]--mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for [alpha]--mannosyl units whereas -Glc and -GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was [alpha]-1,6-linked mannotriose, which was twice as good an inhibitor as Man[alpha] 1,6Man[alpha]-O-Me and 10 times better than methyl [alpha]--mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than -Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked [alpha]-mannosyl units. Various glycosylasparagine glycopeptides which contain [alpha]-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either [alpha]-1,3- or [alpha]-1,6-mannosyl residues were retarded on the immobilized HHA column; Man5-GlcNAc2-Asn [containing two Man[alpha] 1,3 (Man[alpha] 1,6) units] bound to the HHA column. On the contrary, glycopeptides with hybrid type glycan chains were not retarded on either column. These two new lectins which differ in their fine sugar binding specificity from each other, and also from the snowdrop lectin, should prove to be useful probes for the detection and preliminary characterization of glycoconjugates on cell surfaces and in solution.