Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans

Abstract

The modification of N-glycans by α-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for α1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-α1,6-mannosidase activity consistent with specificity for N-linked glycans having their α1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (α/α)(6)-fold, recognize and hydrolyze the α1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-α1,6-mannosidases.

FIGURE 1.

Analysis of GH125 specificity by HPAEC-PAD (A) and capillary electrophoresis (B). Panel A, HPAEC-PADS traces i–v show the elution profiles of mannose, α1,2-mannobiose, α1,3-mannobiose, α1,6-mannobiose, and α(1,3)(1,6)-mannotriose standards, respectively. Traces vi–ix show the elution profiles of α1,2-mannobiose, α1,3-mannobiose, α1,6-mannobiose, and α(1,3)(1,6)-mannotriose, respectively, treated with SpGH125. Panel B, CE traces of Man9 treated with SpGH125 (i), CpGH125 (ii), Man5 treated with SpGH125 (iii), CpGH125 (iv), and Man3a treated with SpGH125 (v), CpGH125 (vi), α(1,2)(1,3)-mannosidase (vii), SpGH125 + α(1,2)(1,3)-mannosidase (viii), and CpGH125 + α(1,2)(1,3)-mannosidase (ix). The structures of the N-glycans are shown above the traces. The Man2a product indicated with an asterisk is inferred. The identities of the peaks were determined from the mobilities of standards (see supplemental Fig. S2).

FIGURE 2.

The structure of GH125. Divergent stereo schematic representation of SpGH125 showing its overall fold with N and C termini labeled and putative catalytic residues labeled and shown in yellow stick representation.

FIGURE 3.

Carbohydrate recognition by GH125. A, active site representations of inhibitor binding by SpGH125. The green mesh shows the maximum likelihood (15)/σ_a-weighted (40) electron density maps contoured at 1σ (0.47 e/ų). Key active site residues are shown in stick representation and colored yellow and DMJ colored in gray. B, electrostatic surface potential of the SpGH125 catalytic site with DMJ bound, shown in gray. C, active site representations of methyl-S-(α-d-mannopyranosyl)-(1–6)-α-d-mannopyranose binding by CpGH125. The electron density map is contoured at 1σ (0.32 e/ų) (40). Key active site residues are colored blue and methyl-S-(α-d-mannopyranosyl)-(1–6)-α-d-mannopyranose colored purple. D, electrostatic surface potential of CpGH125 catalytic site with methyl-S-(α-d-mannopyranosyl)-(1–6)-α-d-mannopyranose bound, shown in purple. E, active site representations of α(1,6)-mannobiose binding by SpGH125 at pH 9. The electron density map is contoured at 1σ (0.40 e/ų). Key active site residues are colored yellow and α(1,6)-mannobiose are colored green. F, electrostatic surface potential of the SpGH125 catalytic site with α(1,6)-mannobiose shown in green; the methyl-S-(α-d-mannopyranosyl)-(1–6)-α-d-mannopyranose from CpGH125 in the −1 and +1 subsites is superimposed and shown in purple for reference. In all panels active site subsites are labeled according to the convention established by Davies et al. (41). Electrostatic surface potentials are shown with red as negative charge and blue as positive charge. Hydrogen bonds between the protein and compound are shown as dashed blue lines identified using the criteria of proper geometry and a distance cutoff of 3.2 Å. Panels A, C, and E are shown in divergent stereo.

FIGURE 4.

Similarities between GH family 125 and family 15. A, a structural overlay of CpGH125 (yellow), SpGH125 (blue), and the GH15 A. globiformis glucodextranase G1d (PDB code 1ulv) (purple). Bound ligands are shown in stick representation. Overlay of the active sites of CpGH125 and G1d showing the −1 subsite (B, shown in divergent stereo) and the +1 subsite (C). CpGH125 is colored yellow (residue labels in black) and G1d colored purple (residue labels in purple). Hydrogen bonds are shown as dashed lines. Putative catalytic waters are shown as spheres. Subsites are labeled in green. D, divergent stereo view of the relative orientations of the catalytic residues in CpGH125 (blue) and SpGH125 (yellow). Hydrogen bonds and distances are indicated. Labels and lines are color coded black and gray for CpGH125 and SpGH125, respectively. The putative catalytic waters, shown as spheres, do not hydrogen bond with C1 of the substrate, the dashed line is intended to indicate the putative trajectory of nucleophilic attack.

FIGURE 5.

SpGH125 catalyzed cleavage of methyl 6-O-(α-d-mannopyranosyl)-β-d-mannopyranoside (Manα1–6Manβ1-OMe) proceeds by an inverting catalytic mechanism. A, methyl 6-O-(α-d-mannopyranosyl)-β-d-mannopyranoside (Manα1–6Manβ1-OMe), the substrate monitored by ¹H NMR spectroscopy. B, mannose, the product monitored by ¹H NMR spectroscopy. C, the SpGH125 catalyzed cleavage of Manα1–6Manβ1-OMe was monitored as a function of time by ¹H NMR spectroscopy. A stacked plot shows hydrolysis of the substrate, with S_H1α representing the resonance of the anomeric proton of the non-reducing, terminal mannose unit of Manα1–6Manβ1-OMe, to first form the mannose hemiacetal having the β-configuration at the anomeric center, indicated by P_H1β. P_H1β represents the anomeric proton of the α-anomer of the mannose product and arises from spontaneous mutarotation of the first formed β-anomer. D, graphical representation of the anomeric ratio of S_H1α (▴), P_H1β (■), and P_H1α (♦) with respect to time illustrate that the β-anomer is formed first.

FIGURE 6.

Phylogenetic analysis of GH family 125. A, the phylogenetic tree for family GH125 was inferred using the Minimum Evolution method. Phylogenetic analyses were conducted in MEGA4 (–45). B and C, conservation of surface residues in family GH125. The SpGH125 structure is shown in surface representation from back (B) to front (C) with the substrate in the active site, which was modeled based on the SpGH125-dMNJ complex and the CpGH125-substrate complex. The surface is colored according to conservation scores for the surface residues (variable in blue to highly conserved in purple; see legend below panel C). Conservation of residues was scored based on the alignment used for phylogenetic tree construction and calculated using Consurf (26, 27).