Construction Of A Screening System For Genes Involved In STT3B-dependent N-glycosylation

Asparagine linked glycosylation(N-glycosylation)of proteins is an essential protein modification in most eukaryotic organisms.The process of N-glycosylation starts with the formation of dolichol-linked precursor glycan in the endoplasmic reticulum(ER),then oligosaccharyltransferase(OST)transfers the precursor glycan to polypeptide acceptor sites NXS/T(sequons,where X ? Pro).The oligosaccharide will form a covalent bond with the nitrogen atom on the asparagine side chain.The OST is a heteroligomeric membrane proteins composed of four to eight subunits.The highly conserved STT3 subunit contains the OST active site.Yeast has a unique STT3 subunit.Mammalian cells express two OST complexs that have different catalytic subunits(STT3A or STT3B).The STT3 A isoform is associated with protein translocation channel,and mediates cotranslational glycosylation of NXT/S sites on nascent polypeptides,with similar fuction of STT3 in yeast.Sequons close to the terminal of peptide or NCS/T sites are easily skipped by STT3 A.One special fuction of STT3 B complex is to maximize sequon occupancy in glycoproteins by modification of sites that are skipped by STT3 A complex.Researches indicate that STT3 B has a unique glycosylation system Mutations in STT3 B or genes involved in STT3B-dependent glycosylation cause serious diseases.The mechanism in STT3B-dependent N-glycosylation is still not so clear.To construct a screening system for genes involved in STT3B-dependent N-glycosylation,we introduced one N-glycosylation consensus sequence at different position of monomer enhanced green fluorescent protein(mEGFP).We fused the signal peptide of calreticulin to the N terminal of mEGFP and tag the m EGFP with KDEL at the C terminal to loacate it to the ER.Then we expressed these N-glycosylatable mEGFP(Ng-mEGFP)in STT3 A knock-out and STT3 B knock-out Human Embryonic Kidney 293 cell(HEK293)lines to screen for mutants that can only be glycosylated by STT3 B.We successfully identified three mutants mEGFPQ185 N,mEGFPK215T and mEGFPQ185N/N186 C,which can only glycosylated by STT3 B and give stronger fluorescence.Furtherly,we integrated mEGFPQ185N/N186 C,which has the biggest peak shift in STT3 B knocked out cell line,into the genome of HEK293 and got a single green fluorescent cell line whose fluorescence is sensitive to STT3B-dependent N-glycosylation.Our results suggests that this cell line can be used for high throughput screening of genes involved in STT3B-dependent N-glycosylation.