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J. Biol. Chem., Vol. 265, Issue 33, 20131-20138, Nov, 1990

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

H Loetscher, EJ Schlaeger, HW Lahm, YC Pan, W Lesslauer and M Brockhaus
Central Research Units, F. Hoffmann-La Roche Ltd., Basel, Switzerland.

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65- kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65- kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.
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