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Construction of green fluorescence protein mutant to monitor STT3B-dependent N-glycosylation.
Kitajima, Toshihiko; Xue, Wei; Liu, Yi-Shi; Wang, Chun-Di; Liu, Si-Si; Fujita, Morihisa; Gao, Xiao-Dong.
Afiliación
  • Kitajima T; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
  • Xue W; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
  • Liu YS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
  • Wang CD; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
  • Liu SS; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
  • Fujita M; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
  • Gao XD; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
FEBS J ; 285(5): 915-928, 2018 03.
Article en En | MEDLINE | ID: mdl-29282902
Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Mutación Puntual / Mutación Missense / Hexosiltransferasas / Proteínas de la Membrana Límite: Humans Idioma: En Revista: FEBS J Asunto de la revista: BIOQUIMICA Año: 2018 Tipo del documento: Article País de afiliación: China Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Mutación Puntual / Mutación Missense / Hexosiltransferasas / Proteínas de la Membrana Límite: Humans Idioma: En Revista: FEBS J Asunto de la revista: BIOQUIMICA Año: 2018 Tipo del documento: Article País de afiliación: China Pais de publicación: Reino Unido