Characterization of ligand binding by the human p55 tumour-necrosis-factor receptor. Involvement of individual cysteine-rich repeats.
Two soluble tumour-necrosis-factor-alpha(TNF)-binding proteins are derived from the extracellular domains of the p55 and p75 TNF receptors. They are considered to play a pivotal regulatory role in TNF-mediated inflammatory processes, including diseases such as rheumatoid arthritis, by competing with the cell surface receptors for TNF and lymphotoxin (LT, tumour-necrosis factor beta). The extracellular domains of the two receptors each contain four similar cysteine-rich repeats of about 40 amino acids, in common with several other cell surface proteins including the p75 nerve-growth-factor receptor and the CD40 and Fas antigens. The aim of this study was to characterize the involvement of the four cysteine-rich repeats of the human p55 TNF receptor in TNF and LT binding by both membrane-bound and soluble forms of the receptor. Individual repeats were systematically deleted by PCR mutagenesis and the variants transiently expressed in COS cells. Immunoprecipitated receptor variants exhibited the expected sizes on SDS/PAGE gels, and bound a panel of conformation-dependent anti-(TNF receptor) antibodies. Binding of TNF by the four soluble derivatives was compared with binding by the wild-type soluble receptor using a TNF-affinity column and a BIAcore Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and 125I-TNF binding to U937 cells. delta 4, which lacks the fourth cysteine-rich repeat, bound TNF comparably with the full-length soluble receptor. TNF-binding affinity was unaltered by deletion of the fourth membrane-proximal cysteine-rich repeat, as determined by Scatchard analysis of the transmembrane derivatives. We conclude that the fourth cysteine-rich repeat is not required for TNF binding. In contrast, both the soluble and the transmembrane derivatives lacking any one of the first, second or third repeats failed to bind TNF. Although we cannot entirely exclude the possibility that this may be due to indirect conformational change, rather than the removal of essential epitopes, our results suggest that the first three repeats are each required for TNF binding by both the soluble and the cell-surface receptor.
Corcoran
A E
AE
Mathilda and Terence Kennedy Institute of Rheumatology, Sunley Division, London, England.
Barrett
K
K
Turner
M
M
Brown
A
A
Kissonerghis
A M
AM
Gadnell
M
M
Gray
P W
PW
Chernajovsky
Y
Y
Feldmann
M
M
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001483:Base Sequence; D001681:Biological Assay; D015374:Biosensing Techniques; D003545:Cysteine / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D004252:DNA Mutational Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008024:Ligands; D008233:Lymphotoxin-alpha / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D008024:Ligands; D008233:Lymphotoxin-alpha; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D003545:Cysteine