Thyroglobulin glycosylation: location and nature of the N-linked oligosaccharide units in bovine thyroglobulin.
The cDNA sequence of bovine thyroglobulin (bTg) predicts 14 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of bovine thyroglobulin in order to establish actual glycosylation sites. The distribution of oligosaccharide types within the known sites of N-linked glycosylation has also been examined. Each glycopeptide was subjected to neutral and amino sugar analysis, as well as sialic acid analysis. Thirteen of the 14 putative N-linked glycosylation sites in bTg were confirmed as glycopeptides in the mature protein. Nine of these confirmed glycosylation sites appear to bear complex or hybrid type oligosaccharide units and four contain oligosaccharide structures in which only mannose and glucosamine were seen. Complex or hybrid type glycosylation was confirmed at asparagine residues 91, 464, 476, 834, 928, 1121, 1757, 1851, and 2232, while oligosaccharides containing only mannose and glucosamine were found at asparagine residues 1346, 1995, 2104, and 2277. Analysis of the amino acid sequence in the region of each putative glycosylation site predicts a high probability of a beta turn at 10 of the 13 sites. While glycosylation was distributed through most of the length of the thyroglobulin sequence, no oligosaccharides containing only mannose and glucosamine were found in the N-terminal half of the molecule. Several of the glycosylated peptides showed microheterogeneity and occurred in two or more discrete peaks on HPLC. A single putative site at asparagine residue 179 was not recovered in the purified glycopeptide population.
Rawitch
A B
AB
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160.
Pollock
H G
HG
Yang
S X
SX
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D010449:Peptide Mapping; D011487:Protein Conformation; D013954:Thyroglobulin / Q000737:chemistry* / Q000302:isolation & purification; D013961:Thyroid Gland / Q000737:chemistry
D006020:Glycopeptides; D007202:Indicators and Reagents; D009844:Oligosaccharides; D013954:Thyroglobulin