Authors

Discussion

Previously studies have characterized the Fasciola hepatica newly excysted juveniles (NEJs) excretory-secretory (ES) products as a complex mixture of ~100 different proteins, which come into direct contact with their host’s tissues and cells and aid the parasite to infect and survive within the host. Despite the fact that glycosylation is one of the most common posttranslational modification associated to secreted proteins, there is significant dearth of knowledge regarding the glycosylation of those proteins found in the NEJ ES. Since glycans influence the antigenicity, molecular functions and interactions of proteins, in the present study we sought to characterize the N-glycosylation profile of individual ES proteins. Combining proteomic, glycopeptide and glycomic mass spectrometry (MS) analysis we identified 31 proteins N-glycosylated within the ES of the NEJs, including cathepsin proteinases (e.g., FhCL4, FhCB2, FhCB3, and FhCB11), legumain, peptidase inhibitor 16, enzymes involved in carbohydrate metabolism (e.g., glucosyltransferase, lysosomal alpha-mannosidase and alpha glucosidase) and 12 uncharacterized proteins. Bioinformatics analysis indicated that the glycoproteins in the ES range from 17 to 206 kDa and that least half of them possess a signal peptide. Although, depending on the protein, they were predicted by bioinformatic tools to contain between one and 22 N-glycosylation sites, our glycopeptide data showed that these sites are not always decorated. Our analysis demonstrated that the glycosite occupancy of NEJs secreted glycoproteins varies from 4.5% to 100%. By allying glycopeptide and glycan analysis we established that the N-glycosylation of F. hepatica glycoproteins is highly heterogeneous. While proteinases such as FhCL4 occupies its only N-glycosylation site with small pauci-mannosylated glycans of similar composition (e.g., GlcNAcβ1-4GlcNAc or GlcNAcβ1-4(Fucα1-6)GlcNAc), other proteins such as FhCB2, FhCB3, legumain and lysosomal alpha-mannosidase significantly vary the glycan moiety associated with the same site. To take an example, the same glycosylation site (N302) of FhCB2 can be decorated with eight different structures (e.g., Man(2-9)GlcNAc2). In terms of complexity, most of the N-glycans associated to the glycoproteins identified are pauci- or oligo-mannosylated structures. Nonetheless, at least 13 of the glycoproteins carry hybrid and complex structures with truncated or extended branches. Tetraspanin, for example, has two of its glycosylated sites decorated with N-glycans such as Hex5HexNAc3deHex1, Hex5HexNAc4deHex1. Surprisingly, this glycoprotein also carries a complex N-glycan containing bisecting GlcNAc (Hex3HexNAc5deHex1). Altogether, using high throughput MS analyses we mapped and identified the precise N-glycosylation profile of proteins secreted by the invasive F. hepatica NEJ. Our data show that the liver fluke is capable of synthesising and secreting proteins with variable glycosylation patterns, suggesting that the composition of the ES released into the host tissues is highly heterogeneous and much more complex than initially anticipated. As glycans participate in key cellular and molecular interactions that contribute to evasion of host immune responses, our data provide the foundation for the discovery of biomarkers for diagnostics and vaccine candidates to control fasciolosis, as well as glycan-based immuno-biotherapeutics.