Studies on the Carbohydrate Units of Thyroglobulin: EVALUATION OF THEIR MICROHETEROGENEITY IN THE HUMAN AND CALF PROTEINS

Abstract

Information has been obtained in regard to the carbohydrate units of human and calf thyroglobulin through a study of the glycopeptides obtained after extensive Pronase digestion of these proteins. Human thyroglobulin was shown to contain, as has been previously reported for the calf protein, carbohydrate units consisting only of mannose and N-acetylglucosamine (unit A) as well as more complex units consisting of mannose, Nacetylglucosamine, galactose, N-acetylneuraminic acid, and fucose (unit B). The units A of the human and calf thyroglobulin were similar, consisting on an average of 8 to 9 residues of mannose and 2 of N-acetylglucosamine. There were 7 to 8 units of this type in the human protein and 5 to 6 in the calf. Chromatography of human and calf unit A glycopeptides on Dowex 50-X2 revealed extensive microheterogeneity, with the mannose content varying from 5 to 11 residues per unit, but with the glucosamine content remaining constant at 2 residues in each. The average unit B of the human thyroglobulin was found to consist of 3 mannose, 4 N-acetylglucosamine, 2 to 3 galactose, 1 fucose, and 1 sialic acid residues. It differed from that previously reported for the calf in containing fewer residues of sialic acid, galactose, and glucosamine. There were found to be 22 units of the B type in the human thyroglobulin compared with 13 to 14 of these units in the calf protein. The human unit B was also found to exhibit pronounced microheterogeneity, as was revealed by fractionation of its glycopeptides on DEAE-cellulose. The variation was primarily evident in the sialic acid, galactose, and glucosamine residues, with the mannose of the core portion remaining constant at 3 residues. The galactosamine residues of the human thyroglobulin were shown, by density gradient centrifugation, to be an integral part of this protein and to occur in units distinct from carbohydrate units A and B, which have been designated as unit C. Alkaline sulfite treatment of glycopeptides containing galactosamine suggested that the C type of unit is linked to serine and threonine residues by O-glycosidic bonds, in contrast to the glycosylamine type of linkage to asparagine which attaches units A and B to the peptide chain. During Pronase digestion, some of the glycopeptide bonds linking unit A were split, yielding neutral oligosaccharides containing 7 to 8 mannose and 2 N-acetylglucosamine residues, with 1 of the latter in a reducing terminal position.