Biology subjects

Paschinger, K.

Publications and source records attributed to Paschinger, K..

5 recordsLinked to original sources

bioRxiv2025

Excretory-secretory products of the fish-borne parasite Anisakis simplex L3 larvae possess allergens and unusual glycan modifications

Anisakis simplex is a parasitic aquatic nematode, which may cause mild-to-severe gastrointestinal allergic reactions (Anisakiasis) with clinical symptoms, such as rhinitis and urticaria in humans who accidentally consume raw or undercooked marine products contaminated with infective L3 Anisakis larvae. Several Anisakis excretory/secretory (E/S) products and somatic proteins are known to be involved in IgE-mediated allergic reactions. In comparison to vertebrates, nematodes have a distinct machinery to glycosylate their proteins and unusual glycan structures have been reported previously, many of which play immunogenic and immunomodulatory roles in host-parasite interactions. While an early study indicated that O-glycans participate the cross-reactivity of antibodies in allergy patients to A. simplex somatic antigens, the N-glycosylation pattern of Anisakis and the potential role of N-glycans in allergic reactions remained unknown. The aim of this study was to characterise N-glycans and the associated glycoproteins from Anisakis E/S products using mass spectrometry. We collected E/S products from larvae culture and released N-glycans from trypsinised proteins using PNGase Ar. Native glycans were pyridylaminated prior to HPLC separation and MALDI-TOF-MS/MS analysis. In addition, hydrofluoric acid and glycosidase digestions were performed to aid structural characterisation. MS data of 5h and 24h E/S products indicated the presence of pauci-mannose and core fucosylated N-glycans as major species; tri-fucosylated and methylated glycans as well as complex-type and phosphorylcholine-substituted glycans were also detected. In addition, E/S products were subject to proteomics analysis which revealed a set of proteins with conserved domains associated with allergens. Our study provides the first insight into the N-glycosylation machinery of Anisakis and highlights the needs for investigating whether and which N-glycans are indubitably involved in the modulation of allergic responses.

biochemistry↗

bioRxiv2024

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

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 1,3- and 1,6-linked fucose by FUT-1 and FUT-8 respectively, the distal GlcNAc residue is 1,3-fucosylated solely by FUT-6. Interestingly, FUT-6 can only fucosylate N-glycan structures lacking the 1,6-mannose upper arm, indicating that a specific -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 1,3-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 -mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent 1,6-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. BackgroundTri-fucosylation of N-glycan core is a conserved feature seen in the N-glycomes of several nematode species. However, beyond the three core fucosyltransferases, we know very little about the biosynthesis and biological function of these core modifications. ResultsComparative glycomics data revealed that aman-3 mutants possess underfucosylated N-glycomes. Biochemical characterisation of AMAN-3 clarified its optimal reaction conditions and substrate specificity and, we demonstrated a Golgi localisation. Thereafter in vitro reconstruction of biosynthesis of a core tri-fucosylated N-glycan was achieved using 8 recombinant C. elegans glycoenzymes. Notably, aman-3 deficient worms exhibited significant developmental and behavioural changes. ConclusionAMAN-3 is a Golgi -mannosidase required for core fucosylation of the distal N-acetylglucosamine of N-glycoproteins. SignificanceThis study elucidates the key role of a novel Golgi -mannosidase in the biosynthesis of the unusual N-glycans of C. elegans and related nematodes, thereby setting the stage for new approaches to study the roles of glycan in the biology and immunology of nematode glycoproteins.

biochemistry↗

bioRxiv2023

Bioinformatic, enzymatic and structural characterization of Trichuris suis hexosaminidase HEX-2

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 {beta}-hexosaminidase subclade only present in the Dorylaimia. We have expressed one of these, HEX-2 from Trichuris suis, a porcine parasite, and shown that it prefers an aryl {beta}-N-acetylgalactosaminide in vitro. HEX-2 has an almost neutral pH optimum and is best inhibited by GalNAc-isofagomine. Towards N-glycan substrates, it displays a preference for the removal of GalNAc residues from LacdiNAc motifs as well as the GlcNAc attached to the 1,3-linked core mannose. Thereby, it has a broader specificity than insect fused lobes (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.

biochemistry↗

bioRxiv2023

Recognition of highly branched N-glycans of the porcine whipworm by the immune system

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array we explored the interactions of glycans with C-type lectins, C-reactive protein and sera from T. suis infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties, but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems, and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

biochemistry↗

bioRxiv2020

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the hosts immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.

biochemistry↗