Abstract
Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. Metazoan organisms express two oligosaccharyltransferase complexes that are composed of a catalytic subunit (STT3A or STT3B) assembled with a shared set of accessory subunits and one to two complex specific subunits. siRNA mediated knockdowns of STT3A and STT3B in HeLa cells have shown that the two OST complexes have partially non-overlapping roles in N-linked glycosylation. However, incomplete siRNA mediated depletion of STT3A or STT3B reduces the impact of OST complex loss, thereby complicating the interpretation of experimental results. Here, we have used the CRISPR/Cas9 gene editing technology to create viable HEK293 derived cells lines that are deficient for a single catalytic subunit (STT3A or STT3B) or two STT3B-specific accessory subunits (MagT1 and TUSC3). Analysis of protein glycosylation in the STT3A, STT3B and MagT1/TUSC3 null cell lines revealed that these cell lines are superior tools for investigating the in vivo role and substrate preferences of the STT3A and STT3B complexes.
Highlights
Is associated with the protein translocation channel[6,9], and mediates cotranslational glycosylation of NXT/S sites on nascent polypeptides[10,11]
The analysis of glycosylation in STT3B depleted cells indicates that the major cellular role for the STT3B complex is to maximize sequon occupancy in glycoproteins by modification of sites that are skipped by the STT3A complex[11]
The cultured cell lines we have tested to date (HeLa, CHO, HepG2, U97, NSC-34 and human fibroblasts) express MagT1 protein, but do not express sufficient TUSC3 to readily detect by protein immunoblotting using anti-TUSC3 sera[5], (Cherepanova, Shrimal and Gilmore, unpublished observations)
Summary
Is associated with the protein translocation channel[6,9], and mediates cotranslational glycosylation of NXT/S sites on nascent polypeptides[10,11]. HEK293 cells with our previously validated siRNAs for human STT3A, STT3B or MagT1 caused roughly a 2-fold reduction in expression levels of the targeted proteins as detected on protein immunoblots (Fig. 1a). Expression of the catalytic subunits (STT3A or STT3B), shared OST subunits (e.g., ribophorin I), or the protein translocation channel (Sec61) was not reduced by treating cells with the MagT1 siRNA (Fig. 1a,b).
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