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

粉蝶素-1是一种存在于菜粉蝶中的98-kDa的细胞毒性蛋白，与霍乱毒素等ADP-核糖基化毒素具有序列相似性。为了鉴定粉蝶菌素-1的ADP-核糖基转移酶活性的底物蛋白，将[腺苷酸-β <32>P]-NAD、HeLa细胞提取物和粉蝶菌素-1的混合物孵育并通过SDS/PAGE和放射自显影进行分析。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ADP核糖基化的底物。当具有不同序列的多种寡核苷酸用于受体时，效率与每种寡核苷酸中鸟嘌呤碱基的比例强烈相关。为了通过粉蝶素-1测定反应产物的结构，使用2 ′-脱氧鸟苷作为受体分子。UV光谱和ESI-MS表明存在ADP-核糖基化dG。几种NMR分析显示其结构为N^2-（ADP-核糖-1-基）-2 '-脱氧鸟苷的α和β形式。最后，我们独立地进行了化学合成，并确认了与酶反应产物的结构一致性。当使用高分子量双链DNA作为受体分子时，鸟嘌呤碱基的相同位置被ADP核糖基化，这一点通过包括^1H-MNR的结构分析证实。用于<32>检测ADP-核糖基化dG的13 P-后裂解法表明，在用pierisin-1处理的HeLa细胞中也发生鸟嘌呤碱基N-2的ADP-核糖基化。从以上结果可以得出结论，粉蝶素-1将ADP-核糖基部分从NAD转移到细胞DNA的鸟嘌呤碱基的N-2，这导致细胞的凋亡诱导。

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.