Title : Glycosylation in human
thyroglobulin : location of
the N-linked oligosaccharide units and comparison with bovine
thyroglobulin
Abstract :
- The amino acid sequence established for human thyroglobulin ( hTG ) from its cDNA sequence contains 20 putative N-linked glycosylation sites
- We have characterized the glycopeptides contained in a tryptic digest of hTG in order to determine which sites are actually linked to carbohydrate
- In addition, the distribution of oligosaccharide type(s) at these confirmed sites of N-linked glycosylation has been examined
- Glycopeptides were purified using gel permeation chromatography followed by several steps of HPLC
- The purified tryptic glycopeptides were characterized by gas phase sequencing and carbohydrate analysis and located within the amino acid sequence of thyroglobulin
- Each of the recovered glycopeptides contained a consensus sequence for N-linked glycosylation
- Of the 20 putative N-linked glycosylation sites in the human thyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the mature protein
- Eight of these confirmed glycosylation sites (at positions 57, 465, 510, 729, 797, 1696, 1754, and 2230 ) appear to be linked to complex-type oligosaccharide units containing fucose and galactose in addition to mannose and glucosamine.
- Five sites (at positions 1200, 1329, 1993, 2275, and 2562 ) contain high mannose type units and two sites (at positions 179 and 1345 ) are linked to oligosaccharide units containing galactose in addition to mannose and glucosamine but no fucose and may be either hybrid or complex structures
- In addition, position 928 was found to be degenerate in oligosaccharide structure and very different oligosaccharide composition types were found associated with peptides containing the same amino acid sequence
- A high probability of a beta turn which would include the glycosylated asparagine residue was predicted for the amino acid sequence found at 13 of the 16 sites
- The glycosylation pattern in hTG was also compared with the data recently reported for bovine thyroglobulin ( bTG ) (27) and as has been recently reported for bTG , no oligosaccharides of the high mannose type were found in the N-terminal portion of hTG
- Only four of the 20 putative sites the sequence of hTG , at asparagine residues 91, 477, 1849, and 2102 were not represented in the purified glycopeptide population and are presumed to escape significant glycosylation