title: Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine.
abstract: NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.
authors.affiliation: Institute of Immunology, University Hospital, Martinistrasse 52, D-20246, Hamburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Krebs
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Koestner
authors.forename: Marion
authors.initials: M
authors.lastname: Nissen
authors.forename: Vivienne
authors.initials: V
authors.lastname: Welge
authors.forename: Ines
authors.initials: I
authors.lastname: Parusel
authors.forename: Fabio
authors.initials: F
authors.lastname: Malavasi
authors.forename: Edward H
authors.initials: EH
authors.lastname: Leiter
authors.forename: Regina M
authors.initials: RM
authors.lastname: Santella
authors.forename: Friedrich
authors.initials: F
authors.lastname: Haag
authors.forename: Friedrich
authors.initials: F
authors.lastname: Koch-Nolte
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000241:Adenosine / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D005030:Ethylenes / Q000032:analysis* / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D005030:Ethylenes; D008565:Membrane Proteins; C036216:ethylene; D036002:ADP Ribose Transferases; D000241:Adenosine