ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

P Lang,L Guizani,I Vitté-Mony,R Stancou,O Dorseuil,G Gacon,J Bertoglio

doi:10.1016/s0021-9258(19)49747-0

P Lang, L Guizani + Show 5 more

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https://doi.org/10.1016/s0021-9258(19)49747-0

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Abstract

The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.

Highlights

94805 Villejuif, and SINSERM Unite 257, Institut Cochin 8)
These results indicate that the low molecular weight GTP-binding protein RhoA
purified from normal blood donorperipheral lymphocytes (PBL) were depleted of the majority of T cells by rosetting with sheep red blood cells at 29 "C for 1 h [18].Enriched natural killer (NK) cell preparations were recovered from the interface of a Ficoll-Hypaque gradient and further purified by incubating with a mixtureof monoclonal antibodies against T and B cell antigens.After two washes, cells were incubated with anti

Summary

Introduction

94805 Villejuif, and SINSERM Unite 257, Institut Cochin 8). Little information is available about the cellular de Ginetique Moleculaire, 75014 Paris, France functions of ras-related G-proteins inlymphocytes. Plasma membranes were recovered at the top conditions where over 75% of the cells are able tfounctionally of the gradient, and cytotoxic granules, containing perforin as identified by Western blotting, sedimented at the 40-50% sucrose interface.

Results

Conclusion