Metalloprotease-Mediated Shedding of Enzymatically Active Mouse ecto-ADP-ribosyltransferase ART2.2 Upon T Cell Activation

Abstract

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.

NAD+ and ATP Released from Injured Cells Induce P2X7-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells

“…ART2.2 catalyzes ADP-ribosylation of CD8, the integrin LFA-1, the P2X 7 receptor, and several other target proteins (12)(13)(14)(15). T cell activation induces the metalloprotease-mediated shedding of a soluble, enzymatically active isoform of ART2.2 (16). ART2-deficient mice (17) exhibit reduced sensitivities to Con-A-induced hepatitis (18).…”

Changing patterns of cell surface mono (ADP-ribosyl) transferase antigen ART2.2 on resting versus cytopathically-activated T cells in NOD/Lt mice

“…Using flow cytometry to assess ART2.2 expression on T cells from adult NOD/ Lt mice, we found that 30 to 40 % of splenic CD4 + cells and 70 to 90 % of CD8 + cells expressed this antigen on their surfaces, comparable to percentages observed in non-Type I diabetes-prone C57BL/6 J and C57BLKS/J strains [6]. This antigen apparently marked resting T cells in the periphery, since activation elicited rapid metalloprotease-mediated shedding with the same kinetics exhibited by another surface marker, L-selectin [6,12]. This shedding was inversely correlated with induction of CD25 (IL2R) expression [6].…”

“…To test whether CD38 high B lymphocytes could affect mono-ADP ribosylation mediated by ART2.2 and others ARTs, we incubated spleen cells with etheno-NAD and stained them wit 1G4 mAb which recognizes etheno-ADP-ribosyl moieties [12,15]. As shown on Figure 9, ADP-ribosylation was more effective in B-lymphocyte-depleted spleen cell preparations (panel C).…”

“…Rat anti-mouse ART2.2 monoclonal antibody (Nika 102) was purified from hybridoma culture supernatants and was used for immunohistochemistry (unlabelled) and flow cytometry (FITC-conjugated). Mouse anti-etheno-NAD monoclonal antibody 1G4 [15], kindly provided by Dr. R. Santella, Columbia University, New York, N. Y., USA, was used to assess the degree of T-cell surface mono-ADP ribosylation [12]. Goat anti-mouse Ig antibody conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were from Fisher Scientific, (Pittsburgh, Pa., USA).…”

“…This shedding was inversely correlated with induction of CD25 (IL2R) expression [6]. Islet autoreactive NOD T cells shed ART2.2 within 2 h after T cell receptor cross-linking or activation of protein kinase C by phorbol ester [12]. Activated T cells that had shed ART2.2 were markedly less sensitive to ADP-ribosylation.…”