Biochemical characterization of the extracellular domain of the 75-kilodalton tumor necrosis factor receptor.
An expression plasmid encoding the extracellular domain of the 75-kDa human tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable cell line secreting soluble TNF-R2 (sTNF-R2). Purified sTNF-R2 was resolved by SDS-PAGE into one band of approximate M(r) 43,000, consistent with a molecular weight of 36,000 +/- 4800 obtained by sedimentation equilibrium analysis. The apparent molecular weight observed by gel filtration chromatography was approximately 136,000. Glycosylation analysis revealed that Asn-149 is fully glycosylated, while Asn-171 is incompletely glycosylated (approximately 50%), and that a proline-, serine-, and threonine-rich region (residues 175-234) contains O-linked carbohydrate structures. Scatchard analysis of [125I]TNF-alpha and [125I]TNF-beta binding to sTNF-R2 gave dissociation constants (Kd) of 0.3 and 0.75 nM, respectively, comparable to those observed for intact cell-surface TNF-R2. The sTNF-R2 was found to block the cytotoxicity of both TNF-alpha and TNF-beta in a murine L-M cell assay. The sizes of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes determined by gel filtration chromatography were approximately 322 and 204 kDa, respectively. The stoichiometry of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes were examined by size-exclusion chromatography, sedimentation equilibrium, and cross-linking. The data from these studies suggest that at least two molecules of sTNF-R2 can bind to a single TNF-alpha or TNF-beta trimer.
Pennica
D
D
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Lam
V T
VT
Weber
R F
RF
Kohr
W J
WJ
Basa
L J
LJ
Spellman
M W
MW
Ashkenazi
A
A
Shire
S J
SJ
Goeddel
D V
DV
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D008233:Lymphotoxin-alpha / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D008233:Lymphotoxin-alpha; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha