A novel type of carbohydrate-protein linkage region in the tyrosine-bound S-layer glycan of Thermoanaerobacterium thermosaccharolyticum D120-70

The surface-layer (S-layer) protein of Thermoanaerobacterium thermosaccharo lyticum D120-70 contains glycosidically linked glycan chains with the repea ting unit structure --> 4)[alpha-d-Galp-(1 --> 2)]-alpha-l-Rhap-(1 --> 3)[b eta-d-Glcp-(1 --> 6)]-beta-d-Manp-(1 --> 4)-alpha-L-Rhap-(1 --> 3)-alpha-D- Glcp-(1 -->. After proteolytic degradation of the S-layer glycoprotein, thr ee glycopeptide pools were isolated, which were analyzed for their carbohyd rate and amino-acid compositions. In all three pools, tyrosine was identifi ed as the amino-acid constituent, and the carbohydrate compositions corresp onded to the above structure. Native polysaccharide PAGE showed the specifi c heterogeneity of each pool. For examination of the carbohydrate-protein l inkage region, the S-layer glycan chain was partially hydrolyzed with trifl uoroacetic acid. 1D and 2D NMR spectroscopy, including a novel diffusion-ed ited difference experiment, showed the O-glycosidic linkage region beta-D-g lucopyranose --> O-tyrosine. No evidence was found of additional sugars ori ginating from a putative core region between the glycan repeating units and the S-layer polypeptide. For the determination of chain-length variability in the S-layer glycan, the different glycopeptide pools were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrom etry, revealing that the degree of polymerization of the S-layer glycan rep eats varied between three and 10. All masses were assigned to multiples of the repeating units plus the peptide portion. This result implies that no c ore structure is present and thus supports the data from the NMR spectrosco py analyses. This is the first observation of a bacterial S-layer glycan wi thout a core region connecting the carbohydrate moiety with the polypeptide portion.