Excretory-secretory products of the fish-borne parasiteAnisakis simplexL3 larvae possess allergens and unusual glycan modifications

Anisakis simplexis 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 L3Anisakislarvae. SeveralAnisakisexcretory/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 toA. simplexsomatic antigens, the N-glycosylation pattern ofAnisakisand 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 fromAnisakisE/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 ofAnisakisand highlights the needs for investigating whether and which N-glycans are indubitably involved in the modulation of allergic responses.