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Construction of green fluorescence protein mutant to monitor STT 3B‐dependent N ‐glycosylation
Author(s) -
Kitajima Toshihiko,
Xue Wei,
Liu YiShi,
Wang ChunDi,
Liu SiSi,
Fujita Morihisa,
Gao XiaoDong
Publication year - 2018
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14375
Subject(s) - glycosylation , n linked glycosylation , mutant , gene isoform , asparagine , endoplasmic reticulum , biochemistry , unfolded protein response , chemistry , green fluorescent protein , glycoprotein , glycan , biology , microbiology and biotechnology , gene , enzyme
Oligosaccharyltransferases ( OST s) 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 STT 3 is highly conserved as a catalytic core. Metazoan organisms encode two STT 3 genes ( STT 3A and STT 3B ) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT 3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT 3B isoform is involved in cotranslational and post‐translocational glycosylation of sequons that are skipped by the STT 3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein ( mEGFP ), which are susceptible to STT 3B‐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 STT 3A‐ or STT 3B ‐knockout cell lines. Among the mutant constructs that we tested, the ER ‐ mEGFP mutant containing the N 185 ‐C 186 ‐T 187 sequon was the best substrate for the STT 3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER ‐ mEGFP is useful for monitoring STT 3B‐dependent post‐translocational N ‐glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.