Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155

• Published in: Glycobiology (Oxford) 1999-08, Vol.9 (8), p.787-795
• Main Authors: Wugeditsch, Thomas, Zachara, Natasha E., Puchberger, Michael, Kosma, Paul, Gooley, Andrew A., Messner, Paul
• Format: Article
• Language: English
• Subjects: Bacillus - chemistry; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Carbohydrate Conformation; Carbohydrate Sequence; Chromatography, Gel; Chromatography, High Pressure Liquid; core oligosaccharide; eubacteria; Glycopeptides - chemistry; Glycopeptides - isolation & purification; glycoprotein; heterogeneity; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - isolation & purification; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Oligosaccharides - chemistry; Oligosaccharides - isolation & purification; Pronase; Spectrometry, Mass, Secondary Ion; surface-layer (S-layer)
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/9.8.787

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 by Pronase 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)-α-L-Rhap-(1→3)-β-D-glycero-D-manno-Hepp-(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 α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GalpNAc-(1→O)-Thr/Ser and the truncated forms α-L-Rhap-(1→3)-β-D-GalpNAc-(1→O)-Thr/Ser and β-D-GalpNAc-(1→O)-Thr/Ser. All glycopeptides possessed the novel linkage types β-D-GalpNAc-(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.