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

Suthasinee PITCHAYAWASIN-THAPPHASARAPHONG; Naoki TANI; Minoru ISOBE; Hidenori KAI; Takuya KURAHASHI; Junko KATO; Saikwan TRISUNAN

doi:10.1271/bbb.90066

Organic Chemistry Regular Papers

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

Suthasinee PITCHAYAWASIN-THAPPHASARAPHONG, Naoki TANI, Minoru ISOBE, Hidenori KAI, Takuya KURAHASHI, Junko KATO, Saikwan TRISUNAN

Author information

Suthasinee PITCHAYAWASIN-THAPPHASARAPHONG
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University
Naoki TANI
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University
Laboratory of Insect Biochemistry and Biotechnology, Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University
Minoru ISOBE
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University
Laboratory of Organic Chemistry, Institute of Advanced Research, Nagoya University
Hidenori KAI
Laboratory of Insect Biochemistry and Biotechnology, Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University
Takuya KURAHASHI
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University
Junko KATO
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University
Saikwan TRISUNAN
Chul Thai Silk Co., Ltd.

Keywords: molecular heterogeneity, metallo-glycoprotein, glycoside bond heterogeneity, Smith degradation, timer protein

JOURNAL FREE ACCESS

2009 Volume 73 Issue 7 Pages 1578-1585

DOI https://doi.org/10.1271/bbb.90066

View "Advance Publication" version (July 7, 2009).

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Abstract

TIME-EA4 is an ATPase that measures time intervals as a diapause-duration clock found in diapause eggs of the silkworm, Bombyx mori. In the current studies, we report the molecular heterogeneity of TIME-EA4 protein regarding not only amino acid L62V, but also the numbers and linkage patterns of the sugar chain attached to the Asn²² residue. These sugar chain structures were determined in a pico-molar amount of the protein by combining the methods of chemical modification (Smith degradation) and nano-HPLC-electrospray ionization-quadrupole-time of fight-mass spectrometry (ESI-Q-TOF-MS) and -MS/MS. The Japanese and bi-voltine Thai silkworm strains were compared to show the heterogeneity represented by four kinds of molecular species. Judicious choice of the combination methods led us to find the first example of a linkage-position difference in the glycosidic bonds even in sugar moieties of the same molecular weight; thus, the Man(1-6)Man(1-4)GlcNAc(1-4)GlcNAc structure in the C108 pure strain and Man(1-3)Man(1-4)GlcNAc(1-4)GlcNAc structure in the Kinshu-Showa hybrid. A total of five kinds of molecular heterogeneity was determined, including the amino acids in TIME-EA4 protein. This paper describes the details for determining the sugar chain linkage in TIME-EA4 from the diapause eggs of various silkworm strains.

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