Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins

Abstract

Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.

Fig 1. Schematic structure of an N-glycan.

The N-glycans contain different carbohydrates depending on the type, but share a common core structure (gray box): branch a, complex N-glycan consisting of two to four branches with mixed sugar residues; branch b, high-mannose N-glycan contains only mannose residues beyond the core structure; mixed a+b, a combination of complex and high-mannose type N-glycan. Color scheme: blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose; purple diamond, sialic acid; red triangle, fucose.

Fig 2. Proposed C. acnes 266 N-glycan-processing locus 1.

Gene organization of the proposed N-glycan processing locus in the genome of C. acnes 266. GH genes predicted as mannosidases are colored green, and GH genes with predicted N-acetylhexosaminidase activity are blue. Other associated genes are colored light gray and include: predicted sugar ABC-transporter substrate-binding protein (SBP), sugar ABC-transporter permease (PERM), transcriptional-regulator gene (REG), and ABC-transporter ATP-binding protein (ATPB). Accession numbers (GenBank, or RefSeq when GenBank was not available) are shown below each gene.

Fig 3. Proposed degradation pathway for host N-glycans by C. acnes.

(A) Proposed cytoplasmic pathway involving the enzymes GH38, GH5_18 and GH20, and (B) a hypothetical extracellular pathway. Symbols: blue square: N-acetylglucosamine; green circle, mannose; yellow circle, galactose; purple diamond, sialic acid; red triangle, fucose.

Fig 4. Ribbon representation of the CaMan5_18 subunit structure.

The subunit structure of CaMan5_18 features a (β/α)₈ TIM-barrel fold typical for members of the GH5 family. The catalytic acid/base Glu140 and the nucleophile Glu259 are represented as stick models. Secondary-structure elements are represented as: α-helices, blue cylinders; β-sheets, red arrows; and loops, gray coils.

Fig 5. Close up of the active site in CaMan5_18.

(A) Interactions made by the catalytic residues Glu140 and Glu259 with Arg41, Asn139, His215, Trp217 and Trp295. (B) Hypothetical binding of Man-GlcNAc₂ and possible interactions with Ser88, Asn139 and His151. Purple dashed lines highlight interactions.

Fig 6. Dimerization of CaMan5_18.

The dimer interface between subunit A (cyan) and subunit B (brown) with the side chains highlighted that stabilize the dimeric state through intersubunit salt links (Asp19-Arg57 and Arg53-Asp306). Loops L2 (red; residues 89–92) and L3 (blue; residues 92–102) participate in dimer formation, as well as in forming the blockage of the active site required for exo-mode activity. Inset: Interactions formed by loops L2 and L3 in the active site. The steric blockage is mainly provided by L2, supported by L3 and L5 (residues 299–314). The catalytic residues Glu140 and Glu259 are colored yellow.

Fig 7. CaMan5_18 activity on pNP-βMan.

Dependency of CaMan5_18 activity with pNP-βMan as substrate on (A) temperature, and on (B) pH. (C) Kinetic stability of CaMan5_18 hydrolysis of pNP-βMan. Symbols: filled circles, 40°C; empty circles, 50°C; filled triangles, 60°C; empty triangles, 70°C. (D) ThermoFluor analysis of thermal stability to unfolding as a function of pH.

Fig 8. TLC analysis of product formation.

Separation of the hydrolysis products from time-dependent hydrolysis of mannooligosaccharides and Man-GlcNAc by wild-type CaMan5_18. (A) M3 and M4; (B) M6 and M5 with the mannooligosaccharide standards M1-M6 at the left; (C) Man-GlcNAc hydrolysis by CaMan5_18 wild type and the E140Q/E259Q double mutant.

Fig 9. MALDI—TOF-MS analysis of Man-GlcNAc hydrolysis.

(A) Enzymatic reaction products using Man-GlcNAc as substrate after 1 h of incubation. Controls: (B) Man-GlcNAc; (C) GlcNAc; and (D) Man.