Human small-intestinal apolipoprotein B-48 oligosaccharide chains

Abstract

Hepatic apolipoprotein (apo) B-100 isolated from human plasma is known to contain N-linked oligosaccharides of high-mannose-type and complex-type structures. Sequencing data have revealed that apo B-48 of small-intestinal origin, which represents about 48% of apo B-100 polypeptide from the N-terminus, possesses six potential sites for N-linked oligosaccharides, of which five are likely to be glycosylated. The characterization of the carbohydrate moiety of apo B-48 is the focus of this study. Apo B-48 was labelled with L-[35S]methionine and D-[3H]glucosamine in organ culture of human small-intestinal explants. N-Glycanase treatment resulted in loss of radioactivity from D-[3H]glucosamine-labelled but not L-[35S]methionine-labelled apo B-48 secreted into the medium, and caused no distinct change in mobility of apo B-48 upon electrophoresis on 5% polyacrylamide gel. Analysis of monosaccharide content revealed the presence of 16.8, 17.8, 13.4, 3.4, 2.4 and 2.3 residues of N-acetylglucosamine, mannose, galactose, fucose, xylose and N-acetylgalactosamine respectively. Small-intestinal apo B-48 from human lymph chylomicrons bound to [14C]concanavalin A, and the binding could be inhibited with methyl alpha-D-mannoside. In addition, wheat-germ, peanut, Limulus, soya-bean and Ulex lectins bound apo B-48 specifically. To characterize the carbohydrate moiety further, N-linked oligosaccharides were released by N-Glycanase treatment and reduced with NaB3H4. Labelled oligosaccharides were separated on a concanavalin A-Sepharose column. The majority (78%) were biantennary complex-type structures, 16% were high-mannose type and 6% (not retained by the column) most probably represented higher-branched oligosaccharides. These results suggest the presence of one high-mannose-type and four biantennary complex-type oligosaccharides, as well as probable O-linked sugars in apo B-48. By the use of h.p.l.c., exoglycosidase treatments and ion-exchange chromatography, a mixture of high-mannose-type species with predominant Man8GlcNAc2 as well as monosialylated, desialylated and fucosylated forms of complex-type oligosaccharides were detected.