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 an 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, nano-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 nondenaturing 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.
Isobe
Minoru
M
Laboratory of Organic Chemistry Graduate School of Bioagricultural Sciences, Nagoya University Chikusa, Nagoya 464-8601, Japan. isobem@agr.nagoya-u.ac.jp
Kai
Hidenori
H
Kurahashi
Takuya
T
Suwan
Sathorn
S
Pitchayawasin-Thapphasaraphong
Suthasinee
S
Franz
Thomas
T
Tani
Naoki
N
Higashi
Kenichiro
K
Nishida
Hideo
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001683:Biological Clocks / Q000502:physiology; D012831:Bombyx / Q000378:metabolism / Q000502:physiology*; D002240:Carbohydrate Sequence; D002417:Cattle; D003300:Copper / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008018:Life Cycle Stages; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010063:Ovum / Q000737:chemistry*; D011487:Protein Conformation; D016415:Sequence Alignment; D013482:Superoxide Dismutase
D006023:Glycoproteins; D019476:Insect Proteins; D008667:Metalloproteins; D003300:Copper; D013482:Superoxide Dismutase