Chemo-enzymatic synthesis of lipid-linked GlcNAc2Man5 oligosaccharides using recombinant Alg1, Alg2 and Alg11 proteins

Abstract

The biosynthesis of eukaryotic lipid-linked oligosaccharides (LLOs) that act as donor substrates in eukaryotic protein N-glycosylation starts on the cytoplasmic side of the endoplasmic reticulum and includes the sequential addition of five mannose units to dolichol-pyrophosphate-GlcNAc2. These reactions are catalyzed by the Alg1, Alg2 and Alg11 gene products and yield Dol-PP-GlcNAc2Man5, an LLO intermediate that is subsequently flipped to the lumen of the endoplasmic reticulum. While the purification of active Alg1 has previously been described, Alg11 and Alg2 have been mostly studied in vivo. We here describe the expression and purification of functional, full length Alg2 protein. Along with the purified soluble domains Alg1 and Alg11, we used Alg2 to chemo-enzymatically generate Dol-PP-GlcNAc2Man5 analogs starting from synthetic LLOs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25). We found that while the addition of the first mannose unit by Alg1 was successful with all of the LLO molecules, the Alg2-catalyzed reaction was only efficient if the acceptor LLOs contained a sufficiently long lipid tail of four or five isoprenyl units (C20 and C25). Following conversion with Alg11, the resulting C20 or C25 -containing GlcNAc2Man5 LLO analogs were successfully used as donor substrates of purified single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei. Our results provide a chemo-enzymatic method for the generation of eukaryotic LLO analogs and are the basis of subsequent mechanistic studies of the enigmatic Alg2 reaction mechanism.

Alternative routes for synthesis of N‐linked glycans by Alg2 mannosyltransferase

“…In contrast to yeast Alg2, lipids are not required for the in vitro activity of purified human Alg2 (37). Human Alg2 contains only a single transmembrane domain at its N terminus (37), so one explanation for the different lipid dependencies of yeast and human Alg2 may stem from their different membrane topologies. A controversial issue we addressed in this study concerned the role of the conserved C-terminal EX 7 E motif in Alg2 function.…”

“…Net‐neutral bilayer lipids PC and DGDG are likely to be necessary to create a bilayer thickness and ionic balance that is optimal for yeast Alg2 topology and function in vitro. In contrast to yeast Alg2, lipids are not required for the in vitro activity of purified human Alg2 (37). Human Alg2 contains only a single transmembrane domain at its N terminus (37), so one explanation for the different lipid dependencies of yeast and human Alg2 may stem from their different membrane topologies.…”

Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide

“…Whereas the OST of higher eukaryotes is a multiprotein complex with STT3 being the catalytic subunit, archaea, kinetoplastids and bacteria have single-subunit OST (ssOST) enzymes that are homologous to STT3 4 , 12 – 14 . Although OSTs have been found to show some relaxed specificity with respect to the oligosaccharide donor, bacterial and archaeal enzymes can transfer a much vaster array of glycans compared to their eukaryotic counterparts 3 , 12 , 15 – 18 .…”

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

“…In order to in vitro N ‐glycosylate proteins by enzymatic reactions, lipid‐linked oligosaccharides (LLO) are needed as substrates (Ramírez, Boilevin, Biswas, et al, ; Ramírez, Boilevin, Lin, et al, ). The ER associated biosynthesis of the LLO is a highly conserved process in eukaryotic cells.…”