The molecular mechanism of the termination of insect diapause, part 1:: A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein
The molecular mechanism of the termination of insect diapause, part 1:: A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein
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DOI：
10.1002/cbic.200600138
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发表时间：
2006-10-01
期刊：
影响因子：
3.2
通讯作者：
Nishida, Hideo
中科院分区：
文献类型：
--
作者：
Isobe, Minoru;Kai, Hidenori;Nishida, Hideo
TIME-EA4 位于测量时间间隔的 ATP 酶上，充当家蚕滞育卵中的滞育持续时间时钟。 TIME-EA4 的初级和高级结构的表征有助于阐明蛋白质测量时间间隔的机制。在我们目前的研究中，通过肽序列分析、nono-HPLC-ESI-Q-TOF-MS 和 MS/MS 以及 cDNA 听写等组合手段，已将 TIME-EA4 的整个序列确定为金属糖蛋白的序列。 TIME-EA4的氨基酸序列与已报道的Cu,Zn-SOD（超氧化物歧化酶）家族蛋白有46-55%的同源性；特别是，SOD活性位点（核心结构域）包括金属结合氨基酸配体和二硫键，这些结构在Bombyx SOD、牛SOD和TIME-EA4蛋白中完全相同。然而，我们发现 TIME-EA4 比其他 SOD 多含有一个铜离子，这在中性非变性条件下得到了证明。 ESI质谱分析显示定时器功能不在SOD核心域中。此外，TIME-EA4 具有附加糖链，这对于其作为计时器蛋白的功能是不可或缺的。
TIME-EA4 is on ATPase that measures time intervals, functioning as a diapause duration clock in diapause eggs of the silkworm, Bombyx mori. Characterization of the primary and higher structures of the TIME-EA4 would be desirable to clarify the mechanism by, which the protein measures the time intervals. In our current studies, the whole sequence of TIME-EA4 has been established as that of a metallo-glycoprotein by combinational means involving peptide sequence analysis, nono-HPLC-ESI-Q-TOF-MS and MS/MS, and cDNA dictation. The amino acid sequence of TIME-EA4 showed 46-55% homology with the reported proteins of the Cu,Zn-SOD (superoxide dismutase) family; in particular, the SOD active site (core domain) includes metal-binding amino acid ligands and a disulfide bond, and these structures are completely identical in Bombyx SOD, bovine SOD, and TIME-EA4 proteins. We found, however that TIME-EA4 contains one more copper ion than other SODs, as was proven under neutral non-denaturing conditions. ESI mass spectrometry revealed that the timer function was not in the SOD core domain. In addition, TIME-EA4 has an attached sugar chain, which is indispensable to its functioning as a timer protein.