IDENTIFICATION OF 2 FORMS (31-33-KD AND 48-KD) OF THE URINARY SOLUBLEP55 TUMOR-NECROSIS-FACTOR RECEPTOR THAT ARE DIFFERENTIALLY N-GLYCOSYLATED 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 cha racterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escheri chia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD an d in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sT NF-R1 was resolved in two bands of 29 and 31 kD. All bands were recogn ized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in wes tern and ligand blotting assays. No cross-reaction was observed with a nti-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 u rine also contains O-linked sugars. The urinary sTNF-R1 antigen mixtur e 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. co li 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-glyc osylated form of 48 kD that appears to be a minor constituent of the u rinary sTNF-R1 antigen.