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

Abstract

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.

Seasonal phenotype‐specific transcriptional reprogramming during metamorphosis in the European map butterfly Araschnia levana

“…2006). The identification of a differentially expressed ATPase, which is known to be responsible for measuring the duration of diapause in B. mori eggs, provides a plausible mechanism to explain how the enhanced expression of the A. levana homolog under SD conditions (Fig.…”

Molecular Heterogeneity of TIME-EA4, a Timer Protein in Silkworm Diapause Eggs

“…In previous studies, the whole sequence of TIME-EA4 has been established as a metallo-glycoprotein by combined methods of peptide sequence analysis, nano-HPLC-ESI-Q-TOF-MS (electrospray ionization quadrupole time-of-flight mass spectrometry) and -MS/MS, and cDNA dictation. 1) The TIME-EA4 protein showed a 46-55% identity from an amino acid sequence homology search (the BLAST algorithm) with Cu,Zn-SODs (superoxide dismutase); in particular the SOD active site (core domain) included metal-holding amino acid ligands and a disulfide bond, and these structures were completely identical in Bombyx SOD, bovine SOD and TIME-EA4 proteins. 1) We have also proposed a computer-generated 3D structure on the basis of the fact that only one folding structure was found by overlaying all the main chains from the 25 reported X-ray crystal structures as Cu,Zn-SOD.…”

“…1) The TIME-EA4 protein showed a 46-55% identity from an amino acid sequence homology search (the BLAST algorithm) with Cu,Zn-SODs (superoxide dismutase); in particular the SOD active site (core domain) included metal-holding amino acid ligands and a disulfide bond, and these structures were completely identical in Bombyx SOD, bovine SOD and TIME-EA4 proteins. 1) We have also proposed a computer-generated 3D structure on the basis of the fact that only one folding structure was found by overlaying all the main chains from the 25 reported X-ray crystal structures as Cu,Zn-SOD. This seems identical to the computer-generated structure 1) and crystal structure forming the gene-overexpressed one reported recently by Hiraki et al, except for the sugar chain (not shown in the X-ray data).…”

“…1) We have also proposed a computer-generated 3D structure on the basis of the fact that only one folding structure was found by overlaying all the main chains from the 25 reported X-ray crystal structures as Cu,Zn-SOD. This seems identical to the computer-generated structure 1) and crystal structure forming the gene-overexpressed one reported recently by Hiraki et al, except for the sugar chain (not shown in the X-ray data).…”

“…6) Furthermore, we have already confirmed that the TIME-EA4 sugar chain had an affinity to ConA lectin and had been cleaved with PNGase F. 6) In the case of the silkworm C108 pure strain, digesting TIME-EA4 with trypsin produced 14 peptide fragments (respectively named T1 to T14 according to the amino-acid sequence) which was determined by a nano-HPLC-ESI-Q-TOF-MS analysis. 1) Since the T5 and T14 peptides were observed as one T5-S-S-T14 fragment connected by a disulfide bond (Cys 61 -Cys 150 ), this was reduced to each T5 and T14 by treating with dithiothreitol (DTT). Since T12 contained only one lysine residue, T12 was observed as T12-T13.…”