Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans

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.

Fig. 1

Mannosylation in the eukaryotic LLO biosynthesis pathway. Alg mannosyltransferases (MTases) catalyze formation of M9GN2-PP-Dol by sequentially adding mannoses to GN2-PP-Dol. Cytosolic reactions use the nucleotide sugar GDP-Man donor, while luminal enzymes use Man-P-Dol, synthesized by Dpm1. Linkages of each sugar are indicated on the right, as are the A, B and C arms of the triantennary oligosaccharide

Fig. 2

In vitro M5GN2-PP-Phy synthesis. a Schematic diagram of sequential mannosylation reactions catalyzed by Alg1ΔTM, Trx-Alg2, and Alg11ΔTM. b UPLC chromatograms of hydrolyzed glycans from reactions with various combinations of purified MTases. Each segment of sequential reactions was incubated for 12 h, as described in Methods. Reactions that included GN2-PP-Phy, GDP-Man, and Alg1ΔTM produced M1GN2 (Alg1ΔTM); sequential addition of Alg1ΔTM and Trx-Alg2 produced M3GN2 [(Alg1ΔTM)+Trx-Alg2]; sequential addition of Alg1ΔTM, Trx-Alg2, and Alg11ΔTM generated M5GN2 [(Alg1ΔTM+Trx-Alg2)+Alg11ΔTM]. c UPLC chromatogram of hydrolyzed glycans from a one-pot reaction containing GN2-PP-Phy, GDP-Man, and a membrane fraction purified from E. coli that co-expressed Alg1ΔTM, Trx-Alg2, and Alg11ΔTM

Fig. 3

In vitro assembly of the M9GN2-PP-Phy. a Schematic diagram of sequential mannosylation reactions catalyzed by Mistic-Alg3, Mistic-Alg9, and Alg12. b UPLC chromatograms of hydrolyzed glycans from reactions with various combinations of membrane fraction purified from E. coli expressing either Alg3, Alg9, or Alg12. In the presence of M5GN2-PP-Phy and Man-P-Phy, addition of Mistic-Alg3 produced M6GN2 (Mistic-Alg3); sequential addition of Mistic-Alg3 and Mistic-Alg9 produced M7GN2 (Mistic-Alg3+Mistic-Alg9); the reaction with Mistic-Alg3 and Mistic-Alg9 was stopped by heating, then adding Alg12 produced M8GN2 [(Mistic-Alg3+Mistic-Alg9)/heat+Alg12]; sequential addition of Mistic-Alg3 for 12 h, Mistic-Alg9 for 12 h, Alg12 for 12 h, and Mistic-Alg9 for 12 h generated M9GN2 (Mistic-Alg3+Mistic-Alg9+Alg12). c Mass spectra of glycans released from Phy-PP-linked oligosaccharide products. Mass analyses showed the peaks eluted (b) at ~15.5, ~16.0, ~16.6, and ~17.2 min correspond to M6GN2 ([M6GN2+Na]⁺), M7GN2 ([M7GN2+Na]⁺), M8GN2 ([M8GN2+Na]⁺), and M9GN2 ([M9GN2+Na]⁺), respectively. d UPLC chromatogram of hydrolyzed glycans from reactions in which Mistic-Alg3, Mistic-Alg9, and Alg12 were added simultaneously for 20 h in one pot to generate M9GN2 from M5GN2-PP-Phy and Man-P-Phy

Fig. 4

UPLC–MS analyses of mannosidase digestion of M9GN2 and precursors. Each of the glycans generated in experiments shown in Fig. 3b were digested with linkage-specific mannosidases, including: α1,2-mannosidase, which removes terminal α1,2 mannoses; α1,2-3-mannosidase, which removes terminal α1,2 mannoses and terminal α1,3 mannoses; α1,6-mannosidase, which removes terminal non-branched α1,6 mannoses; β-mannosidase, which removes terminal β-mannoses. Digestion products and their deduced structure are depicted schematically. a Digestion of M6GN2 with α1,2-3-mannosidase produced M2AGN2; while its digestion with α1,2-mannosidase produced M4A2BC2GN2. b Digestion of M7GN2 with α1,2-mannosidase produced M4A2BC2GN2. c Digestion of M8GN2 with α1,2-3-mannosidase produced M3A3B2CGN2; while its digestion with α1,2-3-mannosidase and α1,6-mannosidase produced M1GN2. d Digestion of M9GN2 with α1,2-mannosidase produced M5BCGN2; digestion with α1,2–3-mannosidase produced M3A3B2CGN2; digestion with both α1,2-3-mannosidase and α1,6-mannosidase produced M1GN2; further treatment with β-mannosidase produced GN2

Fig. 5

Substrate specificity of Alg MTases. a Substrate specificity of Alg1, Alg2, and Alg11. Reactions contained GN2-PP-Phy and GDP-Man and the indicated combination of Alg1∆TM, Trx-Alg2, and/or Alg11∆TM. Glycan products were analyzed by UPLC–MS. b Substrate specificity of Alg3, Alg9, and Alg12. Each reaction contained M5GN2-PP-Phy and Man-P-Phy and the indicated combination of Mistic-Alg3, Mistic-Alg9, and Alg12. Glycan products were analyzed by UPLC–MS. Reaction products are indicated by the arrows, and their deduced structure is depicted schematically

Fig. 6

Specificity of the ER luminal MTases for non-physiological substrates. a Reactions were performed in the presence of GN2-PP-Phy, M1GN2-PP-Phy, or a mixture of M1GN2-PP-Phy/M2GN2-PP-Phy, and Man-P-Phy. Products were analyzed by UPLC–MS. Addition of Mistic-Alg3, Mistic-Alg9, and Alg12 (Mistic-Alg3+Mistic-Alg9+Alg12) failed to elongate any of those substrates. b Reactions were performed in the presence of M3GN2-PP-Phy and Man-P-Phy. Stepwise addition of the membrane fractions of Mistic-Alg3, Mistic-Alg9, and Alg12 produced a variety of unusual LLOs, whose deduced structure is shown schematically. Addition of Mistic-Alg3 alone generated M4A2BC2GN2 (Mistic-Alg3); sequential addition of Mistic-Alg3 and Mistic-Alg9 generated M5A2C2GN2 [(Mistic-Alg3)+Mistic-Alg9]; sequential addition of Mistic-Alg3, Mistic-Alg9, and Alg12 generated M6A2CGN2 and M7AGN2 [(Mistic-Alg3+Mistic-Alg9)+Alg12]. Products are indicated by the arrows