Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Abstract

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.

Figure 1. N-linked glycosylation across the three domains of life

The diphosphate-dependent pathway used by eukaryotes and bacteria is initiated by a phosphoglycosyl transferase, which generates a polyprenyl-PP-linked monosaccharide (Pren-PP-monosaccharide) (top). In this reaction, UMP is released as a byproduct. In contrast, the monophosphate-dependent pathway found in selected archaea begins with the action of a retaining glycosyltransferase to generate a polyprenyl-P-linked monosaccharide (Pren-P-monosaccharide) (bottom). Here, UDP is released as a byproduct. In both pathways, glycosyltransferases then complete assembly of the glycan, which is then transferred by an OTase to an acceptor protein. The sugar molecule is variable depending on the pathway and is depicted with blue shading to symbolize various carbohydrates.

Figure 2. N-linked glycosylation in M. voltae

(a) The M. voltae N-linked glycan is ManNAc(6Thr)A-β1,4-Glc-2,3-diNAcA-β1,3-GlcNAc, where Glc-2,3-diNAcA is 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid and the stereochemistry of the threonine residue is currently unknown. (b) N-linked glycan biosynthesis in M. voltae is initiated in the cytoplasm by the AglK-catalyzed transfer of GlcNAc to the membrane bound acceptor Dol-P. AglC transfers Glc-2,3-diNAcA to Dol-P-GlcNAc to form a β-1,3 linkage, presumably followed by the action of AglA. The resulting Dol-P linked trisaccharide is flipped from the cytoplasm to the exterior of the cell by a currently unidentified flippase and then transferred en bloc to a recipient protein by the OTase, AglB.

Figure 3. AglK is a Dol-P-GlcNAc synthase

(a) AglK catalyzes the transfer of GlcNAc from the donor substrate UDP-GlcNAc to Dol-P. (b) Glycosyl donor specificity of AglK and AglH using (C55-60) Dol-P and radiolabeled nucleotide sugars. Assay monitors the formation of radiolabeled Dol-P-monosaccharide over time. In this experiment, AglH activity was tested using only UDP-[³H]GlcNAc and (C55-60) Dol-P. Traces represent AglK function unless otherwise indicated. (c) Metal dependency of AglK with UDP-[³H]GlcNAc as the glycosyl donor and (C55-60) Dol-P as the polyprenyl-phosphate acceptor. (d) Polyprenyl-phosphate specificity of AglK with UDP-[³H]GlcNAc. Assay data shown in (b–d) are representative traces from at least three independent experiments. Variability seen between runs was less than 5%.

Figure 4. AglC is a UDP-Glc-2,3-diNAcA glycosyltransferase

(a) AglC assembles Dol-PGlcNAc-Glc-2,3-diNAcA from UDP-Glc-2,3-diNAcA and the AglK product Dol-P-GlcNAc. (b) Glycosyl donor specificity of AglC using Dol-P-GlcNAc and radiolabeled nucleotide sugars. Assay monitors the formation of radiolabeled Dol-P-disaccharide over time. (c) Polyprenyl-phosphate acceptor specificity of AglC using UDP-[³H]Glc-2,3-diNAcA as the donor substrate. Assay data shown in (b,c) are representative traces from at least three independent experiments. Variability seen between runs was less than 5%.

Figure 5. The OTase AglB utilizes Dol-P-disaccharide to generate an N-linked glycopeptide

(a) AglB catalyzes the transfer of the disaccharide GlcNAc-Glc-2,3-diNAcA from the AglC product Dol-P-GlcNAc-Glc-2,3-diNAcA to an acceptor peptide. (b) AglB activity assay using the peptide YKYNESSYK, based on the M. voltae flagellum protein FlaB2, as the acceptor substrate and Dol-P-GlcNAc (MS) or Dol-P-disaccharide (DS) as the donor substrates. A peptide lacking the key asparagine residue, YKYQESSYK, was also screened. (c) AglB assay performed with either the addition of MnCl₂, no exogenous MnCl₂, or EDTA. Assay data shown in (b,c) are representative traces from at least three independent experiments. Variability seen between runs was less than 5%. (d) RP-HPLC purification of the peptide and the glycopeptide (280 nm).