Heterotrimers formed by tumor necrosis factors of different species or muteins

Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentra tions results in partial dissociation of the trimers, coinciding with a dec rease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincu bation with an excess of unlabeled mTNF but also with unlabeled human TNF ( hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF com plexes were revealed by native polyacrylamide gel electrophoresis, viz. hom otrimeric mTNF and hTNF, as well as two complexes with an intermediate migr ation pattern. Analytical gel filtration in combination with native polyacr ylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two differ ent species of TNF, which results in homotrimeric and heterotrimeric TNF. T o assess receptor interaction in vitro, TNF heterotrimeric molecules were u sed as obtained after incubation of mTNF with labeled hTNF (which only bind s to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF rece ptor II). These heterotrimers were retained by both mTNF receptors, which m eans that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease i n mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cy totoxicity assay, indicating that heterotrimerization can influence the ove rall bioactivity.