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-alpha-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.
Subject(s)
ADP Ribose Transferases , Histocompatibility Antigens/metabolism , Lymphocyte Activation , Membrane Glycoproteins , Metalloendopeptidases/physiology , Poly(ADP-ribose) Polymerases/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Adenosine Diphosphate Ribose/metabolism , Animals , Antigens, Differentiation, T-Lymphocyte , Cell Membrane/enzymology , Cell-Free System/immunology , Dose-Response Relationship, Immunologic , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Glycosylphosphatidylinositols/metabolism , Hydrolysis , Kinetics , L-Selectin/metabolism , Lymphocyte Activation/drug effects , Membrane Proteins/metabolism , Metalloendopeptidases/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Molecular Weight , Phosphatidylinositol Diacylglycerol-Lyase , Precipitin Tests , Protein Kinase C/metabolism , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, Cultured , Type C Phospholipases/metabolismABSTRACT
A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1-ART7) with activities similar to bacterial toxins have also been cloned. We show here that PSIBLAST, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by PSIBLAST from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the PSIBLAST program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.
