Background

Glycosylation of asparagine residues, which is also referred to as N-glycosylation, is one of the most ubiquitous protein modifications (Cherepanova et al., 2016). N-glycosylation occurs not only in Eukarya, but also in most Archaea (Larkin and Imperiali, 2011). N-glycosylation is also found in some species in Eubacteria. In the entire complex N-glycosylation process, the transfer of an oligosaccharide chain from a sugar donor to asparagine residues in proteins is the defining event (Figure 1). This reaction is catalyzed by a membrane-bound enzyme, oligosaccharyltransferase (OST or OTase). The acceptor asparagine residues, in principle, reside in the consensus sequence Asn-X-Thr or Asn-X-Ser (or Asn-X-Cys in rare cases), where X can be any amino acid residue except Pro. Eubacterial OST uses an extended version of the 5-residue sequon, Asp-X-Asn-X-Ser/Thr or Glu-X-Asn-X-Ser/Thr (X ≠ Pro). Asp is preferable to Glu at the -2 position, even though the acidic residue at the -2 position is not absolutely required in some eubacterial species (Schwarz et al., 2011). The sugar donor is a type of glycolipid, referred to as a lipid-linked oligosaccharide (LLO). The chemical structure of LLO is an oligosaccharide chain attached to a lipid-phospho carrier. The lipid part is dolichol in Eukarya and Archaea, and undecaprenol in Eubacteria. The phosphate group is basically diphosphate, but some archaeal species belonging to Euryarchaeota (an ancient phylum of Archaea) use monophosphate (Taguchi et al., 2016).


Figure 1. Overview of the biosynthesis of N-glycans attached to proteins. The oligosaccharyltransferase catalyzes the transfer of the oligosaccharide chain from LLO to asparagine residues in nascent proteins on the luminal side of the eukaryotic ER. OST: oligosaccharyltransferase. LLO: lipid-linked oligosaccharide.

The in vivo oligosaccharyl transfer activity is measurable by analyzing the extent of oligosaccharide modification of a specified protein in cells. The amino-acid variants of model glycoproteins can be used to analyze the efficiency of the oligosaccharyl transfer reaction around the N-glycosylation site. However, it is difficult to analyze the effects of amino-acid variation on native oligosaccharyl transfer activity in cells, because the N-glycosylation is essential in eukaryotic cells and rather vital in prokaryotic cells. In general, in-cell studies are difficult to set up. In an exceptional case, the whole N-glycosylation system (the LLO biosynthetic pathway, the OST enzyme, and the substrate protein) of the eubacterium Campylobacter was transferred to Escherichia coli cells (Wacker et al., 2002).

The reaction solution for in vitro oligosaccharyl transfer assay is a mixture of a detergent-solubilized OST enzyme, a sugar donor LLO, and a sugar acceptor peptide. Chromatographically purified LLO is preferred, but crude fractions after two-phase partitioning with a chloroform/methanol/water system are usable. In principle, OST strictly recognizes the chemical structure of LLO. As for eukaryotic OSTs, the chemical structure of LLO (Glc₃Man₉GlcNAc₂-PP-dolichol) is common, and thus the human OST enzyme can utilize LLO isolated from yeast cells. In contrast, the archaeal LLOs are quite structurally diverse and must be isolated from the same or closely related species for the oligosaccharyl transfer assay.

One previous assay method is based on the lectin binding of radioactive glycopeptide products after the oligosaccharyl transfer reaction with a ¹²⁵I-labeled peptide substrate (Geetha-Habib et al., 1990). Another assay method is based on the recovery of radioactive glycopeptide products in the upper water phase, in two-phase partitioning after the oligosaccharyl transfer reaction with ¹⁴C-labeled LLO (Bause and Hettkamp, 1979; Sharma et al., 1981). The two methods are problematic, in terms of the use of radioactive compounds and the cumbersome separation procedures.

Therefore, we have developed a new oligosaccharyl transfer assay method, which is radioisotope-free and highly efficient, by using PAGE as the separation mechanism (Figure 2) (Kohda et al., 2007). The bulky oligosaccharide chain in glycopeptides decreases the rate of migration during electrophoresis, due to the sieving action of the polyacrylamide gel. The glycopeptide products are detected and quantified by fluorescent imaging of the PAGE (polyacrylamide gel electrophoresis) gels. A fluorescent dye for detection is attached to a peptide during peptide synthesis. The minimal detectable quantity of the glycopeptide is 1 fmol in routine assay conditions. The excellent linearity over three orders of magnitude for fluorescently labeled peptide amounts up to 1,000 fmol enabled us to quantify the glycopeptide product accurately, for the determination of the K_m and V_max values for peptide substrates (Igura and Kohda, 2011a). At 12 years after its development, the radioisotope-free oligosaccharyltransferase assay method is now widely used in many laboratories.


Figure 2. Radioisotope-free assay for the oligosaccharyltransferase activity. Oligosaccharyl transfer assay method using a fluorescently labeled peptide for detection and a separation mechanism based on PAGE (polyacrylamide gel electrophoresis). OST: oligosaccharyltransferase. LLO: lipid-linked oligosaccharide. Dol-PP: dolichol-pyrophosphate