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.
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.
Corti
A
A
Molecular Immunology and Biochemistry Unit, Tecnogen ScpA, Piana di Monte Verna, Italy.
Merli
S
S
Bagnasco
L
L
D'Ambrosio
F
F
Marino
M
M
Cassani
G
G
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000276:immunology; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008233:Lymphotoxin-alpha / Q000276:immunology; D051379:Mice; D009584:Nitrogen / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000276:immunology; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D014556:Urine / Q000737:chemistry
D015703:Antigens, CD; D008233:Lymphotoxin-alpha; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009584:Nitrogen; D010100:Oxygen