Recognition of Highly Branched N-Glycans of the Porcine Whipworm by the Immune System

Eckmair, Barbara; Gao, Chao; Mehta, Akul Y.; Dutkiewicz, Zuzanna; Vanbeselaere, Jorick; Cummings, Richard D.; Paschinger, Katharina; Wilson, Iain B.H.

doi:10.1016/j.mcpro.2024.100711

Cited by 4 publications

(7 citation statements)

References 82 publications

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“…Thus, the fine biosynthetic control mechanism involving LacdiNAc-containing glycans may be lacking in T. suis and so may be a partial explanation for its species-specific glycome . On the other hand, the catabolism of GalNAc- and GlcNAc-containing N-glycans at acidic pH may be performed in T.…”

Section: Discussionmentioning

confidence: 99%

“…21 Thus, the fine biosynthetic control mechanism involving LacdiNAc-containing glycans may be lacking in T. suis and so may be a partial explanation for its species-specific glycome. 47 On the other hand, the catabolism of GalNAc-and GlcNAc-containing N-glycans at acidic pH may be performed in T. suis by HEX-1, which belongs to GH20 subfamily 2; at least, the potentially related T. spiralis enzyme can degrade such structures. 22 The GH20C enzyme from Streptococcus pneumoniae, 39 which is the closest related protein (29% identity) with a crystal structure, has some bias toward GalNAc, but far less than T. suis HEX-2.…”

Section: Discussionmentioning

confidence: 99%

“…Inhibitors were prepared as previously reported. 30,34,60−63 For tests with remodelled glycopeptides 12 or 2D-HPLC fractions, 35,47 a 1 μL aliquot was mixed with 0.2 μL enzyme and 0.8 μL of 50 mM ammonium acetate solution, pH 6.5. After overnight incubation at 37 °C, 0.5 μL of the mixture was analyzed by MALDI-TOF-MS (Autoflex Speed, Bruker, Bremen) with 6-aza-2-thiothymine (ATT) as the matrix; data were analyzed with the Flexanalysis (Bruker) program.…”

Section: Discussionmentioning

confidence: 99%

“…Typically, a mixture of 2.5 μL of pNP-β-GalNAc (100 mM in dimethyl sulfoxide), 46.5 μL of McIlvaine buffer pH 6.5, and 1 μL of enzyme was incubated for 1 h at 37 °C; 200 μL of stop solution (0.4 M glycine/NaOH, pH 10.4) was added and the absorbance measured with an Infinite 200 PRO instrument (Tecan). Inhibitors were prepared as previously reported. ,,− For tests with remodelled glycopeptides or 2D-HPLC fractions, , a 1 μL aliquot was mixed with 0.2 μL enzyme and 0.8 μL of 50 mM ammonium acetate solution, pH 6.5. After overnight incubation at 37 °C, 0.5 μL of the mixture was analyzed by MALDI-TOF-MS (Autoflex Speed, Bruker, Bremen) with 6-aza-2-thiothymine (ATT) as the matrix; data were analyzed with the Flexanalysis (Bruker) program.…”

Section: Methodsmentioning

confidence: 99%

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Bioinformatic, Enzymatic, and Structural Characterization of Trichuris suis Hexosaminidase HEX-2

Dutkiewicz,

Varrot,

Breese

et al. 2024

Biochemistry

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Hexosaminidases are key enzymes in glycoconjugate metabolism and occur in all kingdoms of life. Here, we have investigated the phylogeny of the GH20 glycosyl hydrolase family in nematodes and identified a β-hexosaminidase subclade present only in the Dorylaimia. We have expressed one of these, HEX-2 from Trichuris suis, a porcine parasite, and shown that it prefers an aryl β-N-acetylgalactosaminide in vitro. HEX-2 has an almost neutral pH optimum and is best inhibited by GalNAc-isofagomine. Toward N-glycan substrates, it displays a preference for the removal of GalNAc residues from LacdiNAc motifs as well as the GlcNAc attached to the α1,3-linked core mannose. Therefore, it has a broader specificity than insect fused lobe (FDL) hexosaminidases but one narrower than distant homologues from plants. Its X-ray crystal structure, the first of any subfamily 1 GH20 hexosaminidase to be determined, is closest to Streptococcus pneumoniae GH20C and the active site is predicted to be compatible with accommodating both GalNAc and GlcNAc. The new structure extends our knowledge about this large enzyme family, particularly as T. suis HEX-2 also possesses the key glutamate residue found in human hexosaminidases of either GH20 subfamily, including HEXD whose biological function remains elusive.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Bioinformatic, Enzymatic, and Structural Characterization of Trichuris suis Hexosaminidase HEX-2

Dutkiewicz,

Varrot,

Breese

et al. 2024

Biochemistry

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“…In contrast to mammals, it has been shown that C. elegans can express complicated core modified N-glycan structures and a portion of these structural elements (glyco-epitopes) can also be found in parasitic nematodes [7]. Some nematode glyco-epitopes are obviously immunogenic in mammals, for instance the anti-HRP epitope (core α1,3-fucose) is recognised by IgE and IgG antibodies of the host [8,9].…”

Section: Introductionmentioning

confidence: 99%

Core Tri-fucosylation of Nematode N-glycans Requires Golgi α-mannosidase III Activity that Impacts Animal Growth and Behaviours

Kendler,

Wöls,

Thapliyal

et al. 2024

Preprint

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Many nematodes possess N-glycans with complex core chitobiose modifications, which is a feature observed in various free-living and parasitic nematodes but is absent in mammals. Using Caenorhabditis elegans as a model to study N-glycan biosynthesis, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues can be modified by three fucosyltransferases in the Golgi, namely FUT-1, FUT-6 and FUT-8. While the asparagine-linked GlcNAc is modified with a α1,3- and α1,6-linked fucose by FUT-1 and FUT-8 respectively, the distal GlcNAc residue is α1,3-fucosylated solely by FUT-6. Interestingly, FUT-6 can only fucosylate N-glycan structures lacking the α1,6-mannose upper arm, indicating that a specific α-mannosidase is required to generate substrates for subsequent FUT-6 activity. By analysing the N-glycomes of aman-3 mutants (tm5400 and a CRISPR/Cas9 knockout, hex-2;hex-3;aman-3) using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of the aman-3 gene abolishes α1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. To further investigate it, we recombinantly expressed AMAN-3 in insect cells and characterised its enzymatic activity in vitro. In contrast to the classical Golgi α-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent α1,6-mannosidase activity towards N-glycans. Using AMAN-3 and other recombinant C. elegans glycoenzymes, we remodelled a fluorescein conjugated-Man5GlcNAc2 structure; we were able to mimic N-glycan biosynthesis in the Golgi and generate a tri-fucosylated glycan in vitro. We performed confocal microscopy studies using a knock-in strain (aman-3::eGFP) and could show the Golgi localisation of AMAN-3. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that AMAN-3 deficient worms display significant developmental delays, morphological and behavioural alterations in comparison to the wild type. Therefore, our data suggested that AMAN-3 participates in nematode N-glycan biosynthesis in the Golgi and generates substrates for FUT-6; thereby, this enzyme is essential for the formation of the unusual tri-fucosylated chitobiose cores of nematode N-glycans, which may play important roles in nematode development and behaviour.

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N-glycan core tri-fucosylation requires Golgi α-mannosidase III activity that impacts nematode growth and behavior

Kendler,

Wӧls,

Thapliyal

et al. 2024

Journal of Biological Chemistry

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Recognition of Highly Branched N-Glycans of the Porcine Whipworm by the Immune System

Cited by 4 publications

References 82 publications

Bioinformatic, Enzymatic, and Structural Characterization of Trichuris suis Hexosaminidase HEX-2

Bioinformatic, Enzymatic, and Structural Characterization of Trichuris suis Hexosaminidase HEX-2

Core Tri-fucosylation of Nematode N-glycans Requires Golgi α-mannosidase III Activity that Impacts Animal Growth and Behaviours

N-glycan core tri-fucosylation requires Golgi α-mannosidase III activity that impacts nematode growth and behavior

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