Solubilization and structural determination of a glycoconjugate which is assembled into the sheath of Leptothrix cholodnii

The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already determined, its modifications with amino acids and other molecules are not fully resolved. In this study, we aimed to determine the chemical structure of the glycoconjugate as a whole. Enantiomeric determination of cysteine in the sheath was performed and as a result, l-cysteine was detected. NMR spectroscopy was endeavored to determine overall structure of the glycoconjugate. Prior to NMR analysis, solubilization of the glycoconjugate was attempted by adding denaturing reagents or by derivatization. As far as tested, sulfonation by performic acid oxidation was suitable for solubilization, but further improvement was achieved by N-acetylation. The approximate molecular weight of the derivative was estimated to be 4.5 × 104 by size-exclusion chromatography. The NMR studies for the sulfonated glycoconjugate and its N-acetylated derivative revealed that the sheath glycoconjugate is a glycosaminoglycan consisting of a pentasaccharide repeating unit which is substoichiometrically esterified with 3-hydroxypropionic acid and stoichiometrically amidated with acetic acid and glycyl-l-cysteine.