PREPARATION AND CHARACTERIZATION OF SOLUBLE RECOMBINANT HETEROTRIMERIC COMPLEXES OF HUMAN LYMPHOTOXIN-ALPHA AND LYMPHOTOXIN-BETA

The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha , also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) cha ins anchored to the membrane surface by the transmembrane domain of th e LT-beta portion, Both proteins belong to the TNF family of ligands a nd receptors that regulate aspects of the immune and inflammatory syst ems, The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells, The signaling function of this receptor-ligand pair is not prec isely known but is believed to be involved in the development of the p eripheral lymphoid organs, To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired , The LT-beta molecule was engineered into a secreted form and co-expr essed with LT-alpha using baculovirus/insect cell technology, By explo iting receptor affinity columns, the LT-alpha 3, LT-alpha 2/beta 1, an d LT-alpha 1/beta 2 forms were purified, All three molecules were trim ers, and their biochemical properties are described, The level of LT-a lpha 3-like components in the LT-alpha 1/beta 2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha 3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors, Heteromeric LT complexes were prepared with this mutant LT-alpha form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors, A LT-alph a 3 based cytotoxic activity was used to show that the LT-alpha 1/beta 2 form cannot readily scramble into a mixture of forms following vari ous treatments and storage periods, This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analys is of the specificity of the LT-beta and TNF receptors for the various LT forms.