Plant N-glycan breakdown by human gut Bacteroides

Abstract

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.

Keywords: glycobiology; glycoside hydrolase; microbiota; plant complex N-glycans.

Fig. 1.

PNGases in species of Bacteroides. (A) Different types of N-glycans. High-mannose N-glycans (HMNGs) have mannose sugars decorating both arms usually to give a total of between five and nine mannose sugars, dubbed Man5 and Man9, respectively, for example. HMNGs do not vary between different organisms, whereas complex N-glycans do have differences according to the source. In mammals, complex N-glycans have LacNAc disaccharides (Galβ1,4GlcNAc) attached to the mannose arms through a β1,2-linkage. The galactose sugars are typically decorated with sialic acids, but these can also decorate the antenna GlcNAc. Mammalian complex N-glycans can have additional antenna through a β1,4-linkage on the α1,3-mannose arm and a β1,6-linkage on the α1,6-mannose arm to produce tri- and tetra-antennary structures, respectively. An α1,6-fucose is a common decoration on the first core GlcNAc in mammals, but α1,3/4-linked fucose is also found to decorate the antenna GlcNAc. In contrast, plant N-glycans typically have Lewis A epitopes (Galβ1,3[Fucα1,4]GlcNAc) as their antenna, a core α1,3-fucose, and a bisecting$$β1,2-xylose. Insect N-glycan structures typically have both α1,3- and α1,6-fucose decorating the core GlcNAc. (B) Active sites of the two PNGases from E. meningoseptica. The key active site residues are shown as sticks and chitobiose is present in the PNGaseF/EMTypeI structure. (C) Phylogenetic tree of the PNGase enzymes from Bacteroides species, which broadly split into two groups. The members of group 1 have quite variable identity between them, as low as 52% in one instance, but generally between 67 and 99%. The members of group 2 have 75 to 97% identity between them. The codes used for the different species is explained Fig. S2. (D) A sequence alignment to show residues that are key to the specificity of accommodating the core α1,3-fucose often present on plant N-glycans. The residue blocking the α1,3-fucose binding in the group I PNGases is highlighted in blue (E118 in PNGaseF/EMTypeI; Asp in the Bacteroides enzymes) and the glycine replacing this residue in the group 2 PNGases is highlighted in pink (G380 in EMTypeII). The glutamic acid replacing the function of E118 is highlighted in green (E419 in EMTypeII).

Fig. 2.

Activity of PNGase B035DRAFT_03341 from B. massiliensis and PNGase BF0811 from B. fragilis against different substrates. (A) α₁acid glycoprotein. (B) Fetuin. (C) Horseradish peroxidase. (D) RNaseB. (E) Soya protein. (F) Papaya protein. B035DRAFT_03341 (green), PNGaseF (black), and BF0811 (red). The time window shown for the different chromatograms varies between the panels to provide clarity of the main peaks. The glycan products for A–E were labeled with procainamide and analyzed by LC-FLD-ESI-MS. The glycan products for F were labeled with 2-aminobenzamide (2-AB) and analyzed by UPLC.

Fig. 3.

Structure of the group 2 PNGase from B. massiliensis. (A) Structure of the group 2 PNGase from B. massiliensis with two different domains. The protein is shown in a rainbow gradient with the N and C termini going from blue to red, respectively. The position of the active site is indicated (gray arrow). (B) Key catalytic residues of the active site. (C) Surface representation of the active sites of three PNGases to show the space to accommodate α-1,3-fucose in EMTypeII and B035DRAFT_03441^PNGase. The arrows indicate the C3 of the GlcNAc where the α1,3-fucose would be attached, and the direction indicates the space it would approximately occupy. For EMTypeI there is no equivalent space where the α1,3-fucose would sit. EMTypeI had chitobiose crystallized in the active site and this is overlaid. (D) Two NBL domains from EMTypeII and B035DRAFT_03441^PNGase to show their structural similarity.

Fig. 4.

Activity of CAZymes identified from the functional association analysis against plant N-glycan structures. (A) Genes identified encoding the putative CAZymes highlighted by the functional association analysis carried out in search of enzymes involved in the degradation of plant N-glycans. Predicted signal peptide types are shown below the CAZymes. (B) Horseradish peroxidase was incubated with different combinations of enzymes, and the products were labeled with procainamide at the reducing end and analyzed by LC-FLD-ESI-MS (Left). The mass spectra of the different peaks are provided (Right). (C) Activity of B. massiliensis GH29 putative α-fucosidases against soya bean N-glycans in the presence (orange) and absence (red) of B035DRAFT_00996^GH2 galactosidase (gray indicates galactosidase only).

Fig. 5.

Crystal structure of the α1,3-mannosidase B035DRAFT_03340^GH92. (A) Structures of B035DRAFT_03340^GH92 shown in cartoon and surface (Top and Bottom, respectively). The N-terminal β-sandwich domain, the two connecting helices, and the C-terminal (α/α)₆-barrel are shown in marine, magenta, and lime, respectively. The metal ion is shown in orange, and catalytic residues are shown in red. (B) Details of the active site with likely important residues shown as sticks. Those labeled in bold are conserved throughout the GH92 family, and those not in bold are unique to this enzyme. (C) Diagram showing the sugar subsites for this enzyme. (D) Surface representation of the active site of the GH92 with partial transparency so the residues in sticks can also be viewed. Thiomannobioside from BT3990 (PDB 2WW1) has been overlaid (blue sticks) to show the approximate −1 and +1 subsites. The likely +1′ xylose and +1″ mannose subsites are also labeled. (E) This is the same as D, but showing a cross-section through the front of the enzyme to show the extent of the binding pockets for the +1′ xylose and +1″ mannose subsites.

Fig. 6.

Likely degradation pathway for plant N-glycans by B. massiliensis. Schematic of the order in which B. massiliensis likely degrades plant N-glycans based on the biochemical data presented in this study.