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

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.