O‐Glycosylated Species of Natural Human Tumor‐Necrosis Factor‐α

Abstract

Tumor-necrosis factor-a, produced by human B-cell lymphoblastoid cell line BALL-I, 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 composition analyses revealed that the tumor necrosis factor-a contained galactose, N-acetylgalactosamine and N-acetylneuraminic acid as sugar components. To prepare sugar chains, tumor necrosis factor-a 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~l-3(NeuAca2-6)GalNAcol and GalPl-3GalNAcol (GalNAcol, N-acetylgalactosaminitol). The structure of the remaining oligosaccharidealditol was determined to be NeuAca2-3Gall-3GalNAcol by composition and methylation analyses. About 20 % of tumor necrosis factor-a 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-a had been completed, and it was proved that the 0-glycosylation site of tumor necrosis factor-a was Ser 4.

Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation

“…N- and O-linked glycosylation are common post-translational modifications that have been described for a variety of ADAM substrates besides the IL-6R, e.g., for TNFα [46] or transforming growth factor alpha (TGFα) [47]. Substrates with and without glycans have also been used to generate novel ADAM17-specific inhibitors [48].…”

TNF Trafficking to Human Mast Cell Granules: Mature Chain-Dependent Endocytosis

“…1, C and D). It has to be noted, however, that posttranslational modifications may be cell lineage specific because O-glycosylation of TNF in the human B cell lymphoblastoid cell line BALL-1 has been reported (29). This might reflect involvement of differential, cell line-specific trafficking mechanisms in delivery of the protein cargo to the target compartment such as mast cell granules or in release to extracellular space.…”

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

“…O-glycosylation has been proposed to specifically affect ADAM-mediated ectodomain shedding (15,16). Tumor necrosis factor alpha (TNF-α), a type 2 transmembrane precursor protein that undergoes ectodomain shedding to produce a soluble truncated form, was shown to be O-glycosylated near a known ADAM cleavage site in lymphoblastic leukemia B cells (16), and Minond and colleagues showed that glycosylation of synthetic peptides covering the TNF-α cleavage site affected in vitro ADAM processing (15).…”

“…Tumor necrosis factor alpha (TNF-α), a type 2 transmembrane precursor protein that undergoes ectodomain shedding to produce a soluble truncated form, was shown to be O-glycosylated near a known ADAM cleavage site in lymphoblastic leukemia B cells (16), and Minond and colleagues showed that glycosylation of synthetic peptides covering the TNF-α cleavage site affected in vitro ADAM processing (15). Furthermore, it was recently found that also ADAM mediated cleavage of the extracellular N-terminal tail of the GPCR β1-adrenergic receptor may be affected by O-glycosylation (17).…”