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.
Highlights
N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER)
To determine if hAlg[2] behaved as an integral membrane protein versus one that is peripherally associated with the ER, ER membrane fractions were treated with Na2CO3 and 0.2% sodium dodecyl sulfate (SDS) detergent
To explore the biological relevance and molecular basis of these alternative M3Gn2 biosynthetic routes during N-linked glycosylation, we developed a method that enabled a kinetic analysis of these reactions
Summary
N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). N-linked glycan structures is immense, all these different glycans are derived from a common lipid-linked oligosaccharide (LLO) precursor This Glc3Man9GlcNAc2 precursor (G3M9Gn2) is made by the ordered sequential addition of sugars to dolichol pyrophosphate (PDol) at the ER membrane[1,2] by twelve Alg (Asparagine-linked glycosylation) glycosyltransferases (GTases)[3,4]. A key intermediate in LLO synthesis is M5Gn2-PDol. The five mannoses on this oligosaccharide are added by the Alg[1], Alg[2] and Alg[11] mannosyltransferases (MTases). Among the different Alg GTases, Alg[2] is unusual in that it displays two distinct activities; the addition of a mannose in an α1,3 and in an α1,6 linkage Evidence for these dual activities comes from both in vivo and in vitro experiments. Quantitative MS analyses of these reconstituted reactions demonstrate that the α1,3- and α1,6-Man can be added to
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