T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-α-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.
IntroductionART2.2 is a glycosylphosphatidylinositol (GPI)-anchored ectoenzyme expressed on the surfaces of most terminally differentiated T cells. 1 ART2.2 is related in structure and function to adenosine diphosphate (ADP)-ribosylating bacterial toxins. [2][3][4] After the release of the ADP-ribosyltransferase (ART) substrate nicotinamide adenine dinucleotide (NAD) from cells by lytic or nonlytic mechanisms, ART2.2 catalyzes the transfer of ADP-ribose from NAD onto arginine residues onto leukocyte function-associated antigen (LFA-1), the P2X7 purinoceptor, and other cell-surface proteins. 5,6 Akin to protein phosphorylation, protein ADPribosylation usually profoundly affects the function of the modified target protein. 7,8 ADP-ribosylation activates P2X7, triggering calcium flux, phosphatidylserine flashing, and nonselective pore formation in the cell membrane. 6 ADP-ribosylation of LFA-1 and other cell-surface proteins inhibits the binding of T cells to target cells and interferes with the clustering of the T-cell receptor (TCR). 5,9 Lipid rafts, also known as glycosphingolipid-enriched membranes (GEMs) or detergent-insoluble glycosphingolipid-enriched membranes (DIGs), are plasma membrane microdomains enriched in gangliosides and cholesterol that form liquid-ordered domains. [10][11][12] It is postulated that lipid rafts segregate molecules in the plasma membrane and regulate signaling through the spatial coordination of intermolecular interactions. Lipid rafts are characterized by insolubility in nonionic detergents and low buoyancy in sucrose density gradients. The posttranslational modification of proteins with saturated acyl groups can result in their localization within lipid rafts. Thus, these microdomains are enriched in many signaling molecules, such as the lck protein kinase, the adaptor protein LAT, heterotrimeric G proteins, and GPI-anchored proteins. 13 In addition, TCR and other transmembrane receptors, including interleukin-2 (IL-2) receptor and LFA-1, are inducibly recruited or stabilized in lipid rafts. [14][15][16] Activation of signaling molecules in lipid rafts is thought to facilitate signaling through the TCR and other immunoreceptors. 17 Some cell-surface proteins lack a transmembrane-spanning domain and are anchored in the outer leaflet of the plasma membrane by a GPI moiety. The physiologic significance of the GPI anchor is unknown. Despite the fact that GPI-anchored proteins are restricted to the outer leaflet of the membrane lipid bilayer and lack a cytoplasmic domain, many GPI-anchored proteins can mediate signaling events after the binding of specific antibodies. 18 With Qa-2, CD55, and CD59, it has been shown that exchange of the GPI anchor by a transmembrane anchor abrogates antibody-induced signaling. [19][20][21] The association of GPI-anchored proteins with Src family kinases in lipid rafts may explain the involvement of GPI-anchored proteins in signaling.Considering that ART2.2 and other members of the ART family carry a GPI anchor, 22,23 we hypothesized that the GPI anchor ...
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ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6–8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.
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