Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates.

L Grazioli,D Casero,A Restivo,E Cozzi,F Marcucci

doi:10.1016/s0021-9258(17)31790-8

Abstract

We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been partially purified by gel filtration chromatography yielded two prominent bands of M(r) 60,000 and 75,000 corresponding to the two known TNFR (types I and II, respectively). In addition to these, types I and II TNFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting that TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that had been partially purified by gel filtration chromatography and incubated with TNF before electrophoretic separation. Indeed, type II TNFR aggregates, but not type I TNFR aggregates, were generated at optimal TNF concentrations. Formation of type II TNFR aggregates in this last experimental setting and of both type I and type II TNFR aggregates during affinity purification could be prevented if an alkylating agent (N-ethylmaleimide) was added during the TNFR-TNF incubation step. Similar results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggregates were generated at TNF concentrations ranging from 10(-9) to 10(-10) M and their formation was prevented in the presence of N-ethylmaleimide.

Highlights

Preparations that hadbeen partially purifiedby gel fil- intracellular signaling cascadeleadingto the biological retration chromatography yielded two prominent bands sponse
Tumor necrosis factor (TNF)'R aggregates in this last experimentalsetting and of with intact transmembrane/cytoplasmic domains form, upon both typeI and typeI1 TNFR aggregates during affinity interaction with TNF, disulfide-linked aggregates. This implies purification could be prevented if an alkylating agent that initial binding of natural TNFR to their trimeric ligand (N-ethylmaleimide) was added duringTNthFeR-TNFin- generates receptor aggregates that are stabilized through the cubation step
Soluble TNFR-Affinity-purified with N-glycanase, neuraminidase, andLiganBd lotting on TNFR after Gel FiltratioCnhro

Summary

Introduction

Preparations that hadbeen partially purifiedby gel fil- intracellular signaling cascadeleadingto the biological retration chromatography yielded two prominent bands sponse. Digestion with N-glycanase (5 units) was per- matography-To detect TNFR by ligand blottingwe employed, formed in 200 mM sodium acetate buffer, pH 7.8, containing 10 mM initially, solubilized membranes from K562 cells that hadbeen o-phenanthroline, 1 mM PMSF at 37 "C for 18 h.

Results

Conclusion