Identification of differentially glycosylated forms of the soluble p75 tumor-necrosis-factor (TNF) receptor in human urine

Human urine is known to contain a 30 kDa soluble form of the p75-TNF receptor (sTNF-R2), In this work we have purified sTNF-R2 from the urine of normal subjects and further characterized its structure and activity. sTNF-R2 was resolved by reducing SDS-PAGE in a major band of 30 kDa, similar in size to the previously described urinary sTNFR2, and in a minor band of 45 kDa, ''Western'' blotting analysis with anti-TNF-R1 and anti-TNF-R2 antibodies showed that both bands were immunologically related to the membrane TNF-R2. Glycosylation studies indicated that the 30 kDa is N-glycosylated while the 45 kDa form is N- and O-glycosylated, and suggested that both forms contain terminally linked sialic acid that is differentially recognized by lectins. These results indicate that human urine contains, besides the 30 kDa form, a new form of 45 kDa characterized by different glycosylation type and degree.