Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin

Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a mod el to investigate the structural basis of selective recognition of protein- bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin s hows little binding to the isolated RNaseB-Man(8) glycoform, and no binding to Man(5-6) glycoforms, In contrast, when the protein moiety is reduced an d denatured we observe that conglutinin binds strongly to the isolated RNas eB-Man(8) glycoform and weakly to the Man(5-6) glycoforms, These results ar e in accord with observations on the binding to the N-glycans in the absenc e of carrier protein. NMR analyses of native RNaseB-Man, and -Man(5-6) glyc oforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (BNaseA), Thus the re are no perceptible differences between the RNase protein forms that coul d account for differential availability of the N-glycan for conglutinin-bin ding, After reduction and denaturation, the NMR spectrum became typical of a nonstructured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man(8) olig osaccharide retained the average conformational behavior of the free oligos accharide irrespective of the carrier protein fold. This conformational fre edom is clearly not translated into full availability of the oligosaccharid e for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in rel ation to the protein surface within the glycan:carrier protein ensemble.