DETERMINATION OF TUMOR-NECROSIS-FACTOR BINDING-PROTEIN DISULFIDE STRUCTURE - DEVIATION OF THE 4TH DOMAIN-STRUCTURE FROM THE TNFR NGFR FAMILY CYSTEINE-RICH REGION SIGNATURE/

Tumor necrosis factor binding protein is a soluble molecule derived fr om the extracellular domain of the 55 kDa human tumor necrosis factor receptor, which can block the biological function of tumor necrosis fa ctor by binding to the growth factor. This cysteine-rich molecule is s ubdivided into four domains, each containing six conserved cysteines t hat form three intrachain disulfide linkages known as the tumor necros is factor receptor/nerve growth factor receptor family cysteine-rich r egion signature structure. In an effort to elucidate the molecular int egrity of the molecule, we performed detailed analysis and searched fo r strategies to elucidate the complete disulfide structure of the E. c oli-derived tumor necrosis factor binding protein and to determine the disulfide arrangement In the fourth domain of Chinese hamster ovary c ell-derived molecule, The methods employed included various proteolyti c digestions, peptide mapping, partial reduction, and assignment of di sulfides by N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry with past-source decay, The first three domains of the molecule were confirmed to have disulfide structures id entical to the cysteine-rich region signature structure found in the a bove-mentioned receptor superfamily. The fourth domain has a different structure from the first three domains where the last four cysteines form two disulfide bonds in opposite positions.