Abstract
The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Understanding how its structure contributes to these functions has been stymied by our inability to produce this glycan as a homogenous structure of sufficient quantities for study. Here, we report the high yield chemoenzymatic synthesis of Man9GlcNAc2 and its biosynthetic intermediates by reconstituting the eukaryotic lipid-linked oligosaccharide (LLO) pathway. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2. These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. Indeed, non-physiological glycans are produced only when the luminal MTases are challenged with cytosolic substrates. Reconstitution of the LLO pathway to synthesize Man9GlcNAc2 in vitro provides an important tool for functional studies of the N-linked glycoprotein biosynthesis pathway.
Highlights
The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion
Five mannoses were added to GN2 by recombinant Alg[1], Alg[2], and Alg[11] to produce M5GN2
This M5GN2 served as the substrate for extension by recombinant Alg[3], Alg[9], and Alg[12] to produce M9GN2
Summary
The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2 These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. The N-linked glycan is processed very differently in species-specific, tissue-specific, and cell-specific ways, leading to an immensely complex glycome Despite their heterogeneity, most of N-glycans share a common Glc3Man9GlcNAc2 precursor oligosaccharide that is pre-assembled on the endoplasmic reticulum (ER) membrane before it is transferred to protein. In contrast to the cytosolic mannosylations of M5GN2 that use GDP-Man as sugar donor, the luminal mannosylations use dolichyl phosphate mannose, whose synthesis is catalyzed by dolichol phosphate mannose synthase (Dpm[1], Fig. 1)[31]. Biosynthesis of M9GN2-PP-Dol requires expression of nine different Alg GTases and three different donor sugar substrates
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