Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway

Abstract

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc₂-pyrophosphate-dolichol (M₁Gn₂-PDol) in either order to generate the branched M₃Gn₂-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M₁Gn₂ before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M₁Gn₂ substrate, both of which trigger production of the M₂Gn₂(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M₂Gn₂ (α-1,6) intermediate.

Despite the conservation of N-glycosylation, human and yeast Alg2 structures have diverged with distinct ER-binding topologies. The human enzyme is more stable than the yeast orthologue, and its activity is modulated by the concentration of donor or acceptor substrate.