Binding of n-linked bovine fetuin glycopeptides to isolated rabbit hepatocytes: Gal/GalNAc hepatic lectin discrimination between Galβ(1,4)GlcNAc and Galβ(1,3)GlcNAc in a triantennary structure

Glycopeptides were isolated from bovine fetuin after digestion with Pronase, aminopeptidase M, and carboxypeptidase Y. The glycopeptides were derivatized with tert-butyloxycarbonyltyrosine and separated on the basis of peptide by using reverse-phase high-performance liquid chromatography. Using 400-MHz ¹H NMR, the asialotriantennary oligosaccharides at each of the three N-linked glycosylation sites were found to be combinations of the following two structures in which the third branch is either Galβ(1,4)GlcNAc or Galβ(1,3)GlcNAc: (formula omitted) The asialotriantennary glycopeptides containing all β(1,4)-lactosamine as the branches were designated Galβ(1,4)GlcNAc-TRI while triantennary glycopeptides containing β(1,3)-lactosamine as branch III were termed Galβ(1,3)GlcNAc-TRI. The Galβ(1,3)GlcNAc unit was localized predominantly to the branch III arm on the basis of a downfield shift (-0.027 ppm) in the H-1 and upfield shift (0.01 ppm) in the NAc methyl signals from the branch III GlcNAc resulting from Galβ(1,3) instead of Galβ(1,4) substitution. Revised assignments are proposed for the H-l's of Gal residues 6 (δ 4.464) and 8 (δ 4.471) [Vliegenthart, J. F. G., Dorland, L., & van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209–373] in a Galβ(l,4)GlcNAc-TRI. The proportion of Galβ(1,3)GlcNAc-TRI glycopeptides from the Asn-Asp, Asn-Gly, and Asn-Cys sites was found to be 40%, 60%, and 20%, respectively. Analysis of the binding of these glycopeptides, containing from 20% to 60% Galβ(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Galβ(1,3)GlcNAc, the lower the affinity of the mixture. The K_d for Galβ(1,4)GlcNAc-TRI was found to be between 3.6 and 5.4 nM (P = 0.10) with a mean of 4.4 nM from binding data analyzed by using the Ligand program [Munson, P. J., & Rodbard, D. (1980) Anal. Biochem. 107, 220–239] and computer simulations of the binding of two ligands as a mixture to one receptor site. The K_d of Galβ(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.