Studies on the unique presence of an N-acetylgalactosamine residue in the carbohydrate moieties of human follicle-stimulating hormone

Abstract

Glycopeptides located at asparagine residues at position 52 in the α-subunit and at position 7 in the β-subunit of human follicle-stimulating hormone (FSH) were obtained by pronase digestion of the intact human FSH molecule. The glycopeptide fractions were isolated by gel filtration and by high-voltage electrophoresis on paper at pH 6 and/or pH 3.5. Alkaline degradation studies performed on the α- and β-subunits of human follicle-stimulating hormone confirmed that carbohydrate moieties linked O-glycosidically to threonine or to serine are not present in either subunit. Carbohydrate composition of both α-52 and β-7 glycopeptides was similar. Sialic acid, mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine and fucose were present in a ratio of 3:4:3:4:1:1 in both glycopeptides. The results of sequential enzymatic digestion revealed that the peripheral monosaccharide sequence of both the α-52 and β-7 glycopeptides consisted of three chains each containing the following sequence: sialic acid ▪ galactose ▪ β- N-acetylglucosamine ▪ mannose. The composition of the undigested ‘inner core’ of both carbohydrate moieties was found to be similar and consisted of one residue/mol glycopeptide each of mannose, N-acetylglucosamine, N-acetylgalactosamine, and fucose. The unique presence of GalNAc attached to an N-glycosidically linked GlcNAc was confirmed by hexosamine analysis on an amino acid analyzer, by thin-layer chromatography, by digestion with enzymes such as exo- α- N-acetylgalactosaminidase and endoglycosidases D and H, and by ion-exchange chromatography on DEAE-Sephacel. The monosaccharide sequence of the α-52 and the β-7 glycopeptides of human follicle-stimulating hormone is shown below and is identical to the one at the α-78 position. ▪