1. Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.
- Author
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Wugeditsch T, Zachara NE, Puchberger M, Kosma P, Gooley AA, and Messner P
- Subjects
- Bacterial Proteins isolation & purification, Carbohydrate Conformation, Carbohydrate Sequence, Chromatography, Gel, Chromatography, High Pressure Liquid, Glycopeptides chemistry, Glycopeptides isolation & purification, Membrane Glycoproteins isolation & purification, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Oligosaccharides isolation & purification, Pronase, Spectrometry, Mass, Secondary Ion, Bacillus chemistry, Bacterial Proteins chemistry, Membrane Glycoproteins chemistry, Oligosaccharides chemistry
- Abstract
The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.
- Published
- 1999
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