TY - JOUR
T1 - The biological role of core 1β1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori
AU - Morio, Akihiro
AU - Lee, Jae Man
AU - Fujii, Tsuguru
AU - Mon, Hiroaki
AU - Masuda, Akitsu
AU - Kakino, Kohei
AU - Xu, Jian
AU - Banno, Yutaka
AU - Kusakabe, Takahiro
N1 - Funding Information:
mAG (monomeric Azami Green)-fused T-synthase and dsRed-fused BmGolgin84 (Golgi marker protein) were co-overexpressed in BmN4 cells and the cells were observed using confocal laser microscopy. The pIE2-T-synthase-mAG-DEST vector was prepared as described above and the pIE2-BmGolgin84-dsRed-DEST vector was established in a previous study (Xu et al., 2018). Four days after transfection of the expression vector using Avalanche-Everyday transfection reagent (EZ Biosystems, USA), the cells were fixed on glass slides as previously described (Hino et al., 2022) and observed under a confocal laser microscope (TCS SP8, Leica, Germany), which was provided by the Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University.
Publisher Copyright:
© 2023 Elsevier Ltd
PY - 2023/5
Y1 - 2023/5
N2 - O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC–MS. We found GalNAc or GlcNAc monosaccharide and core 1 disaccharide (Galβ1-3-GalNAcα1-Ser/Thr) were major components of the O-glycan attached to secreted proteins produced in silkworms. Furthermore, we characterized the 1 b1,3-galactosyltransferase (T-synthase) required for synthesis of the core 1 structure, common to many animals. Five transcriptional variants and four protein isoforms were identified in silkworms, and the biological functions of these isoforms were investigated. We found that BmT-synthase isoforms 1 and 2 were localized in the Golgi apparatus in cultured BmN4 cells and functioned both in cultured cells and silkworms. Additionally, a specific functional domain of T-synthase, called the stem domain, was found to be essential for activity and is presumed to be needed for dimer formation and galactosyltransferase activity. Altogether, our results elucidated the O-glycan profile and function of T-synthase in the silkworm. Our findings allow the practical comprehension of O-glycosylation required for employing silkworms as a productive expression system.
AB - O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC–MS. We found GalNAc or GlcNAc monosaccharide and core 1 disaccharide (Galβ1-3-GalNAcα1-Ser/Thr) were major components of the O-glycan attached to secreted proteins produced in silkworms. Furthermore, we characterized the 1 b1,3-galactosyltransferase (T-synthase) required for synthesis of the core 1 structure, common to many animals. Five transcriptional variants and four protein isoforms were identified in silkworms, and the biological functions of these isoforms were investigated. We found that BmT-synthase isoforms 1 and 2 were localized in the Golgi apparatus in cultured BmN4 cells and functioned both in cultured cells and silkworms. Additionally, a specific functional domain of T-synthase, called the stem domain, was found to be essential for activity and is presumed to be needed for dimer formation and galactosyltransferase activity. Altogether, our results elucidated the O-glycan profile and function of T-synthase in the silkworm. Our findings allow the practical comprehension of O-glycosylation required for employing silkworms as a productive expression system.
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U2 - 10.1016/j.ibmb.2023.103936
DO - 10.1016/j.ibmb.2023.103936
M3 - Article
C2 - 36990248
AN - SCOPUS:85151530725
SN - 0965-1748
VL - 156
JO - Insect Biochemistry and Molecular Biology
JF - Insect Biochemistry and Molecular Biology
M1 - 103936
ER -