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Studies on the Carbohydrate Units of Thyroglobulin

STRUCTURE OF THE MANNOSE-N-ACETYLGLUCOSAMINE UNIT (UNIT A) OF THE HUMAN AND CALF PROTEINS

From the ¹ From the Departments of Biological Chemistry and Medicine, Harvard Medical School, the Elliott P. Joslin Research Laboratory, and the Peter Bent Brigham Hospital, Boston, Massachusetts 02215

An investigation of the structure and peptide attachment of the mannose-N-acetylglucosamine unit (unit A) from human and calf thyroglobulin has been carried out on glycopeptides containing large (8 to 10 mannose plus 2 glucosamine residues) and small (5 to 6 mannose plus 2 glucosamine residues) variants of this unit, as well as on an oligosaccharide fraction (8 mannose plus 2 glucosamine residues).

All of the mannose residues could be released by -mannosidase, after which 1 N-acetylglucosamine could be removed by ß-N-acetyglucosaminidase. This permitted the isolation from the glycopeptides of 2-acetamido-4-O-(2acetamido-2-deoxy-ß-d-glucopyranosyl)-1-N-ß-l-aspartyl-2deoxy-ß-d-glucopyranosylamine and 2-acetamido-1-N-ß-laspartyl-2-deoxy-ß-d-glucopyranosylamine, which could be split to aspartic acid plus di-N-acetylchitobiose or N-acetylglucosamine, respectively, by the action of glycosyl asparaginase. Incubation of the unit A oligosaccharide with -mannosidase resulted in the production of a di-N-acetylglucosamine disaccharide in high yield which was shown to be identical with di-N-acetylchitobiose.

Serial periodate oxidation of the glycopeptides caused destruction of all but approximately 2 of the mannose residues, but spared both N-acetylglucosamine residues in the first Smith degradation. These 2 surviving mannose residues were destroyed in the second degradation. One glucosamine was oxidized in the third treatment, while the other glucosamine still remained intact. Glycerol was the only alcohol released during the three Smith degradations.

Periodate oxidation and sodium borohydride reduction of the reduced unit A oligosaccharide, in which the terminal glucosamine had been converted to glucosaminitol, yielded 1 residue of xylosaminitol.

Methylation of the glycopeptides by the method of Hakamori yielded tetra- and di-O-methyl mannose ethers from glycopeptides with small carbohydrate units, while tetra-, tri-, and di-O-methyl ethers were obtained from the glycopeptides with large carbohydrate units.

All glycopeptides yielded only 1 O-methyl ether of N-methylglucosamine, which was identified by ion exchange chromatography to be 3,6-di-O-methyl-N-methylglucosamine.

The results of these studies suggest the following structure for carbohydrate unit A.

[see PDF for sequence]

The smallest unit found was a tightly branched structure containing 5 mannose residues, and the larger units are believed to contain additional mannose residues, designated as (Man)_x linked to the terminal residues of the three chains by 1-2 bonds.

No significant species variations were observed between the calf and human proteins.

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