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

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 101 55 has a total carbohydrate content of 15% (by mass), consisting of O-linke d oligosaccharide chains. After proteolytic digestion of the S-layer glycop rotein by Pronase E and subsequent purification of the digestion products b y gel permeation chromatography, chromatofocusing and high-performance liqu id chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-L-Rhap-(1-->3)-beta-D-glycero-D-m anno-Hepp-(1--> (Kosma et al,, 1995b, Glycobiology, 5, 791-796) were obtain ed. Combined evidence from modified]Edman-degradation in combination with l iquid chromatography electrospray mass-spectrometry and nuclear magnetic re sonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-L-Rhap-(1 --> 3)-alpha-L-Rhap-(1 --> 3)-beta-D-GalpNAc-(1 --> O)-Thr/Ser and the truncated forms alpha-L-Rhap-(1 --> 3)-beta-D-GalpNAc(1 --> O)-Thr/Ser and P-D-GalpNAc-(1 --> O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-D-GalpNAc(1 --> O) -Thr/Ser. The different cores were substituted with varying numbers of disa ccharide repeating units, By 300 MHz proton nuclear magnetic resonance spec troscopy the complete carbohydrate core structure of the fluorescently labe led glyco-peptide B was determined after Smith-degradation of its glycan ch ain. The NMR data confirmed and complemented the results of the mass spectr oscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.