Partial characterization of the N‐linked oligosaccharides occurring on rat hepatocyte glycoproteins

Abstract

Primary monolayers of isolated rat hepatocytes were cultured in the presence of [2-3H]mannose and [ l -' 4 C ] f~~~~e to label metabolically the carbohydrate portion of glycoproteins. Subsequently, glycopeptides were prepared by extensive pronase digestion in order that the relative occurrence of N-linked oligosaccharides could be examined by lectin-agarose affinity chromatogra-The results indicate that isolated rat hepatocytes are able to synthesize many of the N-linked oligosaccharides known to be present in whole-rat-liver tissue and in rat-serum glycoproteins. Differences were noticed, however, in the relative occurrence of N-linked glycans and their degree of galactosylation. Biantennary complex-type glycans were predominant in isolated rat hepatocytes, whereas the majority of the N-linked complex-type glycans in rat-liver tissue glycoproteins has been reported to be of the tetraantennary type. Furthermore, the degree of P-galactosylation of the glycans of the hepatocytes appeared to be substantially lower than reported for the peripheral branches of liver tissue complex-type glycans. However, most of the P-linked Gal residues present appeared to be substituted by sialic acid.phy. Abbreviations ConA, ConcanavalinA; WGA, wheat germ agglutinin; PEA, Pisum sativum lectin; E-PHA, Phaseolus vulgaris erythroagglutinating phytohemagglutinin; L-PHA, Phaseolus vulgaris leukoagglutinating phytohemagglutinin;RCa, , Ricinus communis agglutinin I; mGlc, methyl-a-D--glucopyranoside; mMan, methyl-a-D-mannopyranoside; CO, WO, PO, EO, LO, RO, non-retained, and Cn, Wn, Pn, En, Ln, Rn (n = 1-4), retarded or bound glycopeptide fractions on columns of immobilized ConA, WGA, PEA, E-PHA, L-PHA and RCA,, respectively. The fraction names are also used sequentially, e.g. C1 P 1, which indicates the fraction of glycopeptides eluted from ConA-Sepharose on position C1 and subsequently eluted from PEA-agarose on position P 1.