Structure of carbohydrate units of ovine luteinizing hormone

The detailed structures of the three asparagine-linked carbohydrate units of ovine lutropin subunits alpha and beta (oLH-alpha and oLH-beta) have been determined by carrying out the structural analysis on the three glycopeptides alpha GP-1, alpha GP-2, and beta GP-3, and the oligosaccharide obtained by alkaline sodium borohydride treatment of oLH and the individual subunits. Based on the results of methylation, periodate oxidation, deamination, acetolysis, and enzymatic studies with exoglycosidases, the following structure is proposed for the carbohydrate units of oLH. Methylation studies indicated that the 3 N-acetylglucosaminyl residues were substituted at 1-, 1,4-, and 1,4,6-positions. Two of the three mannosyl residues were 1,2-linked and 3rd mannosyl residue was 1,3,6-linked. Fucose and galactose were terminally located and N-acetylgalactosamine was substituted at C-4. Periodate oxidation studies were consistent with the methylation data. The isolation of (formula, see text) 2,5-anhydromannose by the deamination of the oLH oligosaccharide established the branched structure for the mannosyl residues as well as the linkage of the trimannose unit to N-acetylglucosamine. The nonreducing terminal position of N-acetylgalactosamine was indicated by the formation of N-acetylgalactosaminyl glyceraldehyde on Smith degradation of oLH. The presence of a substituent "X" on N-acetylgalactosamine was established by the isolation of X-2,5-anhydrotalitol from the deamination products of oLH oligosaccharide. X was found to be an acid-labile substituent and was identified as a sulfate ester. Last but not least, since oLH oligosaccharide, obtained by hydrolysis of the hormone with NaOH + NaBH4, contained N-acetylglucosaminitol at the reducing end, the carbohydrate must be linked to the protein through N-acetylglucosamine.