Krebs Christian, Koestner Wolfgang, Nissen Marion, Welge Vivienne, Parusel Ines, Malavasi Fabio, Leiter Edward H, Santella Regina M, Haag Friedrich, Koch-Nolte Friedrich
Institute of Immunology, University Hospital, Martinistrasse 52, D-20246, Hamburg, Germany.
Anal Biochem. 2003 Mar 1;314(1):108-15. doi: 10.1016/s0003-2697(02)00640-1.
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.
NAD 依赖性 ADP 核糖基化是蛋白质翻译后修饰之一。在哺乳动物细胞上，糖基磷脂酰肌醇锚定的细胞表面 ADP 核糖基转移酶（ARTs）会对其他细胞表面蛋白进行 ADP 核糖基化，从而影响重要的细胞功能。在此，我们描述了便捷的流式细胞术和免疫印迹分析方法，通过利用 ART 以乙烯基-NAD 作为底物的能力来监测活细胞表面蛋白的 ADP 核糖基化。细胞表面蛋白的乙烯基 ADP 核糖基化可以通过使用 1G4（一种对乙烯基腺苷特异的单克隆抗体）进行流式细胞术检测。用 1G4 标记细胞取决于细胞表面 ART 的表达，并且仅在表达 ART 的细胞与乙烯基-NAD 而非乙烯基-ADP 核糖孵育后才会发生。剂量反应分析表明，在微摩尔浓度的乙烯基-NAD 下，表达 ART 的细胞能被 1G4 有效染色。在 1 - 2 μM 乙烯基-NAD 时达到半数最大染色，在 5 - 20 μM 乙烯基-NAD 时达到饱和。免疫印迹分析证实，表达 ART 的细胞将乙烯基腺苷共价（即抗 SDS）掺入几种细胞表面蛋白中。流式细胞术 1G4 染色分析可用于鉴定表达细胞表面 ART 活性的细胞亚群，并选择 ART(高表达)细胞变体。免疫印迹 1G4 染色分析也可用于鉴定乙烯基 ADP 核糖基化的靶蛋白。这些新方法在生物化学和细胞生物学的许多有趣应用中具有前景。
Zotero 插件