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.

Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation.

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.

CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins.

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.

A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens.

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.