Activity and specificity of toxin-related mouse T cell ecto-ADP-ribosyltransferase ART2.2 depends on its association with lipid rafts.
Adenosine diphosphate (ADP)-ribosyl-transferases (ARTs) transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto target proteins. T cells express ART2.2, a toxin-related, glycosylphosphatidylinositol (GPI)-anchored ecto-enzyme. After the release of NAD from cells, ART2.2 ADP-ribosylates the P2X7 purinoceptor, lymphocyte function-associated antigen (LFA-1), and other membrane. Using lymphoma transfectants expressing either ART2.2 with its native GPI anchor (ART2.2-GPI) or ART2.2 with a grafted transmembrane anchor (ART2.2-Tm), we demonstrated that ART2.2-GPI but not ART2.2-Tm associated with glycosphingolipid-enriched microdomains (lipid rafts). At limiting substrate concentrations, ART2.2-GPI exhibited more than 10-fold higher activity than ART2.2-Tm. On intact cells, ART2.2-GPI ADP-ribosylated a small number of distinct target proteins. Strikingly, the disruption of lipid rafts by cyclodextrin or membrane solubilization by Triton X-100 increased the spectrum of modified target proteins. However, ART2.2 itself was a prominent target for ADP-ribosylation only when GPI anchored. Furthermore, cholesterol depletion or detergent solubilization abolished the auto-ADP-ribosylation of ART2.2. These findings imply that ART2.2-GPI, but not ART2.2-Tm, molecules are closely associated on the plasma membrane and lend support to the hypothesis that lipid rafts exist on living cells as platforms to which certain proteins are admitted and others are excluded. Our results further suggest that raft association focuses ART2.2 on specific targets that constitutively or inducibly associate with lipid rafts.
Bannas
Peter
P
Institute of Immunology, University Hospital, Martinistr 52, D-20246 Hamburg, Germany.
Adriouch
Sahil
S
Kahl
Sarah
S
Braasch
Fenja
F
Haag
Friedrich
F
Koch-Nolte
Friedrich
F
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002784:Cholesterol; D017261:Glycosylphosphatidylinositols / Q000378:metabolism* / Q000502:physiology; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014118:Toxins, Biological; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D011506:Proteins; D014118:Toxins, Biological; D000244:Adenosine Diphosphate; D002784:Cholesterol; D036002:ADP Ribose Transferases; C449339:Art2b protein, mouse