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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. [ABSTRACT FROM AUTHOR]

In yeast, the N-linked oligosaccharide modification in the Golgi apparatus is initiated by alpha1,6-mannosyltransferase (encoded by the OCH1 gene) with the addition of mannose to the Man(8)GlcNAc(2) or Man(9)GlcNAc(2) endoplasmic reticulum intermediates. In order to characterize its enzymatic properties, the soluble form of the recombinant Och1p was expressed in the methylotrophic yeast Pichia pastoris as a secreted protein, after truncation of its transmembrane region and fusion with myc and histidine tags at the C-terminus, and purified using a metal chelating column. The enzymatic reaction was performed using various kinds of pyridylaminated (PA) sugar chains as acceptor, and the products were separated by high performance liquid chromatography. The recombinant Och1p efficiently transferred a mannose to Man(8)GlcNAc(2)-PA and Man(9)GlcNAc(2)-PA acceptors, while Man(5)GlcNAc(2)-PA, which completely lacks alpha1,2-linked mannose residues, was not used as an acceptor. At high enzyme concentrations, a novel product was detected by HPLC. Analysis of the product revealed that a second mannose was attached at the 6-O-position of alpha1,3-linked mannose branching from the alpha1,6-linked mannose that is attached to beta1,4-linked mannose of Man(10)GlcNAc(2)-PA produced by the original activity of Och1p. Our results indicate that Och1p has the potential to transfer two mannoses from GDP-mannose, and strictly recognizes the overall structure of high mannose type oligosaccharide.