Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.
The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.
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Department of Cellular and Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Miller
W J
WJ
Kubek
D J
DJ
Strang
A M
AM
van Halbeek
H
H
Piesecki
S J
SJ
Alhadeff
J A
JA
D000581:Amidohydrolases; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006514:Hepatitis B Surface Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000032:analysis*; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology; D014761:Viral Hepatitis Vaccines / Q000737:chemistry
D006514:Hepatitis B Surface Antigens; D011134:Polysaccharides; D011498:Protein Precursors; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014761:Viral Hepatitis Vaccines; C050807:presurface protein 2, hepatitis B surface antigen; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase