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)