Title : O-glycosylated species of natural human
tumor-necrosis factor-alpha
Abstract :
- Tumor-necrosis factor-alpha , produced by human B-cell lymphoblastoid cell line BALL-1, was expressed as four protein bands on SDS /PAGE analysis
- It may have been glycosylated, based on the fact that the heavier two of the four bands disappeared after neuraminidase treatment
- Sugar com position analyses revealed that the tumor necrosis factor-alpha contained galactose, N-acetylgalactosamine and N-acetylneuraminic acid as sugar components.
- To prepare sugar chains , tumor necrosis factor-alpha was treated with alkaline borodeuteride and the oligosaccharide-alditols liberated were fractionated by gel-filtration chromatography on a Bio-Gel P-4 column, followed by normal-phase HPLC
- Three oligosaccharide-alditols were obtained, and the structures of two of them were identified by methylation analysis and exoglycosidase digestion
- The structures of these oligosaccharide-alditols were Gal beta 1-3(NeuAc alpha 2-6)GalNAcol and Gal beta 1-3GalNAcol (GalNAcol, N-acetylgalactosaminitol)
- The structure of the remaining oligosaccharide-alditol was determined to be NeuAc alpha 2-3Gal1-3GalNAcol by com position and methylation analyses
- About 20% of tumor necrosis factor-alpha was found to be 0-glycosylated, based on the results of the sugar composition and structure analyses
- An amino acid sequence analysis of the glycosylated peptides was performed after Staphylococcus aureus V8 protease digestion of tumor necrosis factor-alpha had been completed, and it was proved that the 0-glycosylation site of tumor necrosis factor-alpha was Ser 4