Excretory–secretory products of the fish-borne parasite <i>Anisakis simplex</i> 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 with 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 in 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 the present 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 5 h and 24 h 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 need for investigating whether and which N-glycans are indubitably involved in the modulation of allergic responses.