Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxins alpha and beta.
The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. 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 precisely known but is believed to be involved in the development of the peripheral 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-expressed with LT-alpha using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-alpha3, LT-alpha2/beta1, and LT-alpha1/beta2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-alpha3-like components in the LT-alpha1/beta2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha3 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-alpha3 based cytotoxic activity was used to show that the LT-alpha1/beta2 form cannot readily scramble into a mixture of forms following various 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 analysis of the specificity of the LT-bet a and TNF receptors for the various LT forms.
Browning
J L
JL
Department of Protein Engineering, Biogen, Cambridge, Massachusetts 02142, USA.
Miatkowski
K
K
Griffiths
D A
DA
Bourdon
P R
PR
Hession
C
C
Ambrose
C M
CM
Meier
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D003603:Cytotoxins / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008233:Lymphotoxin-alpha / Q000737:chemistry*; D053308:Lymphotoxin-beta; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses; D011994:Recombinant Proteins; D012995:Solubility; D018411:Spodoptera
D003603:Cytotoxins; D017931:DNA Primers; C505886:LTB protein, human; C505887:Ltb protein, mouse; D008233:Lymphotoxin-alpha; D053308:Lymphotoxin-beta; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins