Determination of tumor necrosis factor binding protein disulfide structure: deviation of the fourth domain structure from the TNFR/NGFR family cysteine-rich region signature.
Tumor necrosis factor binding protein is a soluble molecule derived from the extracellular domain of the 55 kDa human tumor necrosis factor receptor, which can block the biological function of tumor necrosis factor by binding to the growth factor. This cysteine-rich molecule is subdivided into four domains, each containing six conserved cysteines that form three intrachain disulfide linkages known as the tumor necrosis factor receptor/nerve growth factor receptor family cysteine-rich region signature structure. In an effort to elucidate the molecular integrity of the molecule, we performed detailed analysis and searched for strategies to elucidate the complete disulfide structure of the E. coli-derived tumor necrosis factor binding protein and to determine the disulfide arrangement in the fourth domain of Chinese hamster ovary cell-derived molecule. The methods employed included various proteolytic digestions, peptide mapping, partial reduction, and assignment of disulfides by N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry with post-source decay. The first three domains of the molecule were confirmed to have disulfide structures identical to the cysteine-rich region signature structure found in the above-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.
Jones
M D
MD
Department of Protein Structure, Amgen Inc., Amgen Center, Thousand Oaks, California 91320, USA.
Hunt
J
J
Liu
J L
JL
Patterson
S D
SD
Kohno
T
T
Lu
H S
HS
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis
D004220:Disulfides; D010446:Peptide Fragments; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D003545:Cysteine