Characterization and affinity cross-linking of receptors for human recombinant lymphotoxin (tumor necrosis factor-beta) on a human histiocytic lymphoma cell line, U-937.
Recombinant human lymphotoxin (rhLT) expressed in a mammalian cell line was purified and used to examine its receptors on the human histiocytic lymphoma cell line U-937. rhLT was radioiodinated by the IODO-GEN method to a specific activity of 60 microCi/micrograms; the labeled protein was biologically active in the cytolytic assay, and displaceable binding to U-937 cells was observed. The specific binding reached a plateau within 10, 60, and 180 min at 37, 23, and 4 degrees C, respectively. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 0.6 nM and a capacity of 33,000 +/- 7,000 binding sites/cell. The binding of 125I-rhLT to U-937 cells could be inhibited by excess unlabeled rhLT or recombinant human tumor necrosis factor (rhTNF), suggesting a common receptor for both molecules. As competitive inhibitor of the binding, rhTNF was equal in its potency to rhLT. Bacterial derived rhLT lacking carbohydrate was also found equipotent to cell line-derived rhLT for cell binding, indicating that carbohydrate plays no significant role in receptor interaction. Additionally, 125I-rhLT was covalently attached to the cell surface via a bifunctional cross-linking reagent, ethylene glycol bis(succinimidyl succinate), solubilized, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cross-linking of the receptor to rhLT revealed two distinct bands at approximate molecular masses of 100,000 and 120,000 daltons. Both bands were absent when unlabeled rhLT or rhTNF was used for competition, indicating the specificity. Affinity cross-linking of U-937 cells with 125I-rhTNF, however, provided only a single band with a molecular mass of about 100,000 daltons. These results suggest that the manner in which rhLT interacts with its receptor is perhaps somewhat different from that of rhTNF.
Stauber
G B
GB
Department of Protein Biochemistry, Genentech, Inc., South San Francisco, California 94080.
Aggarwal
B B
BB
D000345:Affinity Labels; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D016403:Lymphoma, Large B-Cell, Diffuse / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000378:metabolism*; D013388:Succinimides; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D013388:Succinimides; D014409:Tumor Necrosis Factor-alpha; C028722:ethylene glycolylbis(succinimidyl succinate)