Rationally Designed Short Polyisoprenol-Linked PglB Substrates for Engineered Polypeptide and Protein N-Glycosylation

Abstract

The lipid carrier specificity of the protein N-glycosylation enzyme C. jejuni PglB was tested using a logical, synthetic array of natural and unnatural C10, C20, C30, and C40 polyisoprenol sugar pyrophosphates, including those bearing repeating cis-prenyl units. Unusual, short, synthetically accessible C20 prenols (nerylnerol 1d and geranylnerol 1e) were shown to be effective lipid carriers for PglB sugar substrates. Kinetic analyses for PglB revealed clear K(M)-only modulation with lipid chain length, thereby implicating successful in vitro application at appropriate concentrations. This was confirmed by optimized, efficient in vitro synthesis allowing >90% of Asn-linked β-N-GlcNAc-ylated peptide and proteins. This reveals a simple, flexible biocatalytic method for glycoconjugate synthesis using PglB N-glycosylation machinery and varied chemically synthesized glycosylation donor precursors.

Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs

“…Following previously reported assay designs used for in vitro studies of bacterial PglB and archaeal AglB (Kohda et al 2007; Gerber et al 2013; Liu et al 2014), we synthesized a range of peptides containing a glycosylation sequon and a 5-carboxyfluorescein label that was attached to the N-terminus for quantitation purposes. Starting from the peptide sequence DANYTK, which was previously used to study PglB and the cytoplasmic glycosyltransferase NGT (Schwarz et al 2011; Liu et al 2014), we explored how distinct side chains in and around the sequon influenced the activity of TbSTT3A (Figure 2A and B).…”

“…Starting from the peptide sequence DANYTK, which was previously used to study PglB and the cytoplasmic glycosyltransferase NGT (Schwarz et al 2011; Liu et al 2014), we explored how distinct side chains in and around the sequon influenced the activity of TbSTT3A (Figure 2A and B). In vitro glycosylation experiments were performed with purified TbSTT3A and farnesyl-PP-chitobiose 1b (see below) as a donor substrate (Figure 2A and B).…”

“…In contrast to the bacterial PglB enzyme, for which milligram amounts of pure LLO can be isolated by introducing the C. jejuni pgl operon into Escherichia coli SCM6 cells (Kowarik et al 2006), such an approach is not available for the production of eukaryotic LLO. We therefore synthesized LLO analogs as donor substrates, starting from procedures reported earlier (Fang et al 1995; Kočovský et al 1999; Liu et al 2014) but improving reaction conditions (see below). We synthesized four distinct analogs of the eukaryotic LLO and systematically varied the length of the lipid moiety (C 10 , C 15 , C 20 and C 25 ) as well as the double bond stereochemistry (Figure 3A).…”

“…Mechanistic studies using purified eukaryotic OST enzymes are scarce, which is in part due to the challenges associated with their expression and purification. Therefore, much of the structural and mechanistic insight has been derived from the bacterial (PglB) and archaeal (AglB) ssOST enzymes (Lizak et al 2011, 2014; Gerber et al 2013; Matsumoto et al 2013; Liu et al 2014). Protist ssOST enzymes offer an opportunity to study eukaryotic OST with the reduced complexity of a single membrane protein.…”

Structure and mechanism of an active lipid-linked oligosaccharide flippase

“…Truncated LLOs synthesis. Farnesyl-and geranyl-geranyl-PP-GlcNAc were synthesized in seven steps from commercially available lipids and sugar according to the previously published strategy with slight modifications 61 . As the procedure involves the formation of highly acid sensitive phosphorylated intermediates, flash column chromatography were performed on silica gel basified with an ammonium hydroxide solution to avoid product degradation during purification.…”