Identification of Two Forms (31-33 and 48 kD) of the Urinary Soluble p55 Tumor Necrosis Factor Receptor That Are Differentially N- and O-Glycosylated

Abstract

The structure and the activity of urinary soluble TNF receptor type 1 (sTNF-R1), isolated from the urine of normal individuals, has been characterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escherichia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD and in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sTNF-R1 was resolved in two bands of 29 and 31 kD. All bands were recognized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in western and ligand blotting assays. No cross-reaction was observed with anti-TNF-R2 antibodies. N- and O-glycosylation studies indicated that (1) the 29-31 kD recombinant form as well as the 31-33 kD urinary form are N-glycosylated; (2) the differences between the 29-31 and 31-33 kD recombinant and natural products are mainly related to differences in the N-linked sugar content; and (3) the 48 kD sTNF-R1 isolated from urine also contains O-linked sugars. The urinary sTNF-R1 antigen mixture was able to inhibit TNF-alpha cytotoxicity with a potency comparable to that of nonglycosylated E. coli sTNF-R1. At variance, urinary sTNF-R1 was able to inhibit TNF-beta sevenfold more efficiently than E. coli sTNF-R1. In conclusion, two subtypes of sTNF-R1 have been isolated from urine: a main N-glycosylated form of 31-33 kD and a N- and O-glycosylated form of 48 kD that appears to be a minor constituent of the urinary sTNF-R1 antigen.

ST6Gal-I Regulates Macrophage Apoptosis via α2-6 Sialylation of the TNFR1 Death Receptor

“…To date there is very little information concerning the glycosylation of TNFR1. Analyses of the secreted form of TNFR1 present in human urine suggest that TNFR1 is elabo- rated with both N-and O-linked glycans (24), and a soluble chimeric TNFR1-IgG protein was also found to be N-glycosylated (25). However, to our knowledge, the glycan profile on the native transmembrane form of TNFR1 has not been previously characterized.…”

Synergistic Damage of Tumor Vessels with Ultra Low-Dose Endothelial-Monocyte Activating Polypeptide-II and Neovasculature-Targeted Tumor Necrosis Factor-α

“…When we measured serum soluble TNF receptors after administration of increasing doses of EMAP-II, we observed a significant increase of sTNF-R1. Given that small changes in soluble TNF receptor levels are sufficient to significantly inhibit TNF (25)(26)(27), this mechanism could have contributed to NGR-TNF inhibition when we used highdose EMAP-II.…”

Poliovirus Protein 3A Inhibits Tumor Necrosis Factor (TNF)-Induced Apoptosis by Eliminating the TNF Receptor from the Cell Surface

“…7c). This could be explained by the fact that more than 95% of TNFR1 molecules have only N-linked oligosaccharides, which are added in the ER, and less than 5% of TNFR1 species have Golgi-derived long O-linked oligosaccharide chains (15).…”