High-mannose structure of apolipoprotein-B from low-density lipoproteins of human plasma

Abstract

Human plasma low-density lipoproteins were purified by flotation followed by gel filtration. The protein moiety of the lipoproteins, apolipoprotein-B, which was detected by polyacrylamide gel electrophoresis as the only protein component, contained 4.4% (by weight) carbohydrate. Glycopeptides liberated from apolipoprotein-B by pronase were fractionated by affinity chromatography on concanavalin-A--Sepharose. The results indicated that high-mannose glycopeptides interacting strongly with the lectin comprise about 37% of the total monosaccharides of apolipoprotein-B. Thus, as compared to the total serum glycoproteins having about 5% of their monosaccharides in high-mannose glycopeptides, low-density lipoproteins are relatively enriched in these structures amounting up to about 10% of the total high-mannose oligosaccharides in serum. The rest of the carbohydrates in low-density lipoproteins are suggested to be mainly biantennary acidic oligosaccharides interacting weakly with concanavalin A. The oligomannosidic chains from native low-density lipoproteins and isolated glycopeptides were released by digestion with endo-beta-N-acetylglucosaminidase H. Thin-layer chromatography of the released oligosaccharides indicated that apolipoprotein-B contains five different oligomannosidic structures varying in the number of the mannose residues from Man5GlcNAc to Man9GlcNAc. Separation of the per-O-benzoylated high-mannose oligosaccharides by high-pressure liquid chromatography revealed the same polymeric structures in a molar ratio (from Man5 to Man9) of 10:2:3:2:3. Apolipoprotein-B in low-density lipoproteins was calculated to contain five high-mannose chains in total. The different high-mannose oligosaccharides liberated by endo-beta-N-acetylglucosaminidase H were isolated with high-pressure liquid chromatography after reduction with NaBH4, and subjected to methylation analysis with gas-liquid chromatography--mass spectrometry. The data of these studies and the results of exoglycosidase treatment suggest the following structure for the main high-mannose oligosaccharide: (formula: see text) The higher polymeric structures are composed of chains in which the Man5GlcNAc structure is continued by one to four Man(alpha 1-2) residues.