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

Abstract

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 Gal.beta.(1,4)GlcNAc or Gal.beta.(1,3)GlcNAc: .**GRAPHIC**. The asialotriantennary glycopeptides containing all .beta.(1,4)-lactosamine as the branches were designated Gal.beta.(1,4)GlcNAc-TRI while triantennary glycopeptides containing .beta.(1,3)-lactosamine as branch III were termed Gal.beta.(1,3)GlcNAc-TRI. The Gal.beta.(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 Gal.beta.(1,3) instead of Gal.beta.(1,4) substitution. Revised assignments are proposed for the H-1''s of Gal residues 6 (.delta. 4.464) and 8 (.delta. 4.471) [Vliegenthart, J.F.G., Dorland, L., and van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-373] in a Gal.beta.(1,4)GlcNAc-TRI. The proportion of Gal.beta.(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% Gal.beta.(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Gal.beta.(1,3)GlcNAc, the lower the affinity of the mixture. The Kd for Gal.beta.(1,4)GlcNAc-TRI was foundto 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., and 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 Kd of Gal.beta.(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.