Analysis of the site on a TNF-alpha molecule which affects type II TNF receptor binding in human cells.
The epitope region on the TNF-alpha molecule recognized by monoclonal antibody (mAb) 3-D-6, which neutralizes the cytotoxic activity on murine LM cells, has been determined as Gly24-Gln-Leu-Gln-Trp-Leu-Asn-Arg31. To examine whether this region participates in TNF receptor binding in human cell lines, four kinds of TNF-alpha mutants (Gln25 --> Glu, Gln27 --> Glu, Leu29 --> Val, and Arg31 --> Ser) were prepared using site-directed mutagenesis. One mutant, mRS31, which has a nonconserative mutation at position 31 (Arg --> Ser), showed markedly reduced binding in U-937 cells and in HL-60 cells compared with the wild-type recombinant TNF-alpha (rTNF-alpha). These two cell lines have been reported to have both type I and type II TNF receptors. mRS31 also showed reduced cytotoxicity on U-937 cells. Another mutant, mLV29, which has a conservative mutation at position 29 (Leu --> Val), showed, to a lesser extent, reduced binding in U-937 cells and HL-60 cells and reduced cytotoxic activity in U-937 cells. However, all four TNF-alpha mutants showed a similar binding in HEp-2 cells and in HeLa cells, which have been reported to have only the type I TNF receptor. These results suggest that Leu29 may be involved in direct contact with the type II receptor and that the nonconservative mutation at position 31 may induce a local conformational change in the site involved in type II TNF receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Ikegami
H
H
Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan.
Arai
N
N
Moriyasu
J
J
Taniguchi
M
M
Tsusaki
K
K
Honji
N
N
Kohno
K
K
Ando
S
S
Kurimoto
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D004247:DNA