Structure of pentasaccharide of glycopeptide from TIME-EA4, N-glycoprotein in silkworm diapause eggs

Pitchayawasin, Suthasinee; Isobe, Mitsuaki; Tani, Naoki; Huang, Kai

doi:10.1016/j.bmcl.2004.02.093

Cited by 6 publications

(10 citation statements)

References 10 publications

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“…7) In this study, Bombyx diapause eggs of the Showa-Kinshu strain, which is an F1 hybrid between the Showa and Kinshu strains, showed both tetra-and penta-saccharides of TIME-EA4. We next examined whether or not the tetra-saccharide of the Showa-Kinshu strain was similar to that of the C108 pure strain with special reference to the 1-6 linkage between the terminal and internal Man residues.…”

Section: Analysis Of a Tetra-saccharide Of Time-ea4 By Using Smith Dementioning

confidence: 59%

“…We have already reported the established procedure of Smith degradation combined with LC-MS and tandem MS (MS/MS). 7,8) In addition, the structure of the penta-saccharide in the glycopeptide from TIME-EA4 has been determined by utilizing our methodology. 7) Therefore, in this study, we examined under similar conditions according to the method of Pitchayawasin and Isobe.…”

Section: Methodsmentioning

confidence: 99%

“…1,6) With our combined methodology of Smith degradation (SD) and LC-MS and -MS/MS, we have established that the terminal Man residue was always observed as the 134 Da mass loss after SD, instead of the 162 Da mass loss before SD. 7,8) The TIME-EA4 protein from the C108 strain was digested with trypsin and the mass spectra compared from samples with SD and without SD by means of nano-HPLC-ESI-Q-TOF-MS. We found the molecular ions of the T3-tetra-saccharide fragment at m=z 1019.47 (½M þ 2H 2þ ) without SD and at m=z 991.49 (½M þ 2H 2þ ) with SD as shown in Fig. 6.…”

Section: Analysis Of a Tetra-saccharide Of Time-ea4 By Using Smith Dementioning

confidence: 99%

“…We have subsequently established a new methodology for determining the sugar linkage on the basis of Smith degradation (NaIO 4 and then NaBH 4 ) combined with nano-HPLC-ESI-Q-TOF-MS and -MS/ MS. 7,8) We then examined the penta-saccharide-T3 peptide of TIME-EA4, which was also obtained from another silkworm of the Showa strain, by using the same methodology. 7) This methodology led us to conclude that the penta-saccharide of TIME-EA4 was branched as Man (1)(2)(3)(4)(5)(6)[Man(1-3)]Man(1-4)GlcNAc(1-4)GlcNAc. 7) In that study, we determined the tetra-saccharide structure of TIME-EA4 by using our methodology of LC-MS and -MS/MS in a comparison of samples without and with the Smith degradation.…”

mentioning

confidence: 99%

“…7) This methodology led us to conclude that the penta-saccharide of TIME-EA4 was branched as Man (1)(2)(3)(4)(5)(6)[Man(1-3)]Man(1-4)GlcNAc(1-4)GlcNAc. 7) In that study, we determined the tetra-saccharide structure of TIME-EA4 by using our methodology of LC-MS and -MS/MS in a comparison of samples without and with the Smith degradation. In our current studies, we report that the sugar chain of TIME-EA4 had many varieties among different silkworm strains.…”

mentioning

confidence: 99%

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

Pitchayawasin-Thapphasaraphong

Tani

Isobe

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Section: Analysis Of a Tetra-saccharide Of Time-ea4 By Using Smith Dementioning

confidence: 59%

Section: Methodsmentioning

confidence: 99%

Section: Analysis Of a Tetra-saccharide Of Time-ea4 By Using Smith Dementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Pitchayawasin-Thapphasaraphong

Tani

Isobe

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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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

et al. 2006

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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.

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