Human al-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 150/,) using concanavalin A (ConA) -Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively.The occurrence of N-linked di-, tri-and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties.The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri-and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA).With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri-and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP.The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHAagarose and Ricinus communis agglutinin-I -agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri-and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri-and 65% of the tetraantennary chains.It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed. Abbreviations. AGP, ccl-acid glycoprotein; ConA, concanavalin A; L-PHA, Phaseolus vulgaris leukoagglutinating lectin; RCAI, Ricinus communis agglutinin I; CO, LO, RO, non-retarded or nonbound, and Cn, Ln, Rn (n = 1-4) retarded or bound glycopeptide fractions on ConA-Sepharose, L-PHA-agarose and RCA,-agarose, respectively. The fraction names are sometimes used sequentially, e.g. COLlR3, which indicates the fraction of glycopeptides that was not bound on ConA-Sepharose but retarded sequentially on position L1 of L-PHA-agarose and bound on position R3 of RCAI-agarose. Use of the prefix 'a' denotes that the fraction is desialylated by mild acid hydrolysis. fini...
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.
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