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

Asparagine (N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosac-charide (LLO) precursor Glc₃Man₉GlcNAc₂-pyrophosphate-dolichol (Glc₃Man₉Gn₂-PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg₂, an unusual mannosyltransferase (MTase) with two different MTase activities; Alg₂ adds both an α1,3- and α1,6-mannose onto ManGlcNAc₂-PDol to form the trimannosyl core Man₃GlcNAc₂-PDol. The biochemical properties of Alg2 are controversial and remain undefined. In this study, a liquid chromatography/mass spectrometry-based quantitative assay was established and usedto analyze the MTase activities of purified yeast Alg2. Alg2-dependent Man₃GlcNAc₂-PDol production relied on net-neutral lipids with a propensity to form bilayers. We further showed addition of the α1,3- and α1,6-mannose can occur independently in either order but at differing rates. The conserved C-terminal EX7E motif, N-terminal cytosolic tail, and 3 G-rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. These findings provide insight into the unique bifunctionality of Alg2 during LLO synthesis and lead to a new model in which alternative, independent routes exist for Alg2 catalysis of the trimannosyl core oligosaccharide.