Characterization of a new apoptosis-inducing protein from the cabbage butterfly

Pierisin-1是在卷心菜蝴蝶中发现的一种98 kda的细胞毒蛋白，其序列与adp核糖基化毒素（如霍乱毒素）相似。为了鉴定pierisin-1 adp -核糖基转移酶活性的底物蛋白，将[腺苷酸-^<32>P]- nad、HeLa细胞提取物和pierisin-1的混合物孵育，并通过SDS/PAGE和放射自显像进行分析。标记物对蛋白酶有抗性，但对dna酶敏感。这一结果表明DNA可能是粉孢素介导adp核糖基化的底物。当使用不同序列的多种寡核苷酸作为受体时，其效率与各寡核苷酸中鸟嘌呤碱基的比例密切相关。对反应产物的结构进行了分析，并以吡咯烷酮- 1,2′-脱氧鸟苷为受体分子。紫外光谱和ESI-MS表明存在adp核基化dG。核磁共振分析显示其结构为N^2-(ADP-ribos-1-yl)-2'-脱氧鸟苷的α和β形式。最后，我们独立进行了化学合成，并与酶反应产物进行了结构上的同一性验证。当使用高分子量的dsDNA作为受体分子时，鸟嘌呤碱基的相同位置被adp核糖化，包括^1H-MNR的结构分析证实了这一点。用^<32> p -后分层法检测adp -核糖化dG，表明鸟嘌呤碱基N-2的adp -核糖化也发生在pierisin-1处理的HeLa细胞中。综上所述，pierisin-1将一个adp -核糖基片段从NAD转移到细胞DNA鸟嘌呤碱基的N-2上，从而诱导细胞凋亡。

Pierisin-1 is a 98-kDa cytotoxic protein found in the cabbage butterfly and shows sequence similarity with ADP-ribosylating toxins such as cholera toxin. To identify a substrate protein for ADP-ribosyltransferase activity of pierisin-1, a mixture of[adenylate-^<32>P]-NAD, HeLa cell extract and pierisin-1 was incubated and analyzed by SDS/PAGE and autoradiography. The labeled material was protease resistant but DNase sensitive. This result suggested that DNA might be the substrate for ADP-ribosylation by pierisin. When a variety of oligonucleotides having different sequences were used for acceptors from the efficiency strongly correlated with the proportion of guanine base in each oligonucleotide. For structural determination of the reaction product by pierisin-1, 2'-deoxyguanosine was used as an acceptor molecule. The UV spectrum and ESI-MS suggested the presence of ADP-ribosylated dG. Several NMR analyses revealed the structure as α and β forms of N^2-(ADP-ribos-1-yl)-2'-deoxyguanosine. Finally, we independently synthesized chemically and confirmed the structural identity with the enzyme reaction product. When high-molecular-weight dsDNA was used as acceptor molecule, the same position of guanine base was ADP-ribosylated, as confirmed by structural analyses including ^1H-MNR. The ^<32>P-postlaveling method for detection of ADP-ribosylated dG indicated that ADP-ribosylation of N-2 of guanine base also occurred in HeLa cells treated with pierisin-1. From the above results, it is concluded that pierisin-1 transfers an ADP-ribosyl moiety from NAD to N-2 of guanine base of cellular DNA, and this causes apoptosis induction of the cells.