Abstract
The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1-->3(Galbeta1-->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1-->3GalNAc) and 2 (Galbeta1-->3(GlcNAcbeta1-->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.
Highlights
The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries
The eggs are highly immunogenic and are involved in interactions with the gut to facilitate the passage of eggs through these tissues and in the liver, driving granuloma formation that initiates the pathology of schistosomiasis [4, 5]
When mammalian hosts are exposed to radiation-attenuated cercariae, delivering a very much larger antigenic load than the maximum feasible dose of normal cercariae, virtually the entire antibody response is directed against glycan epitopes common to both cercarial and egg secretions [6]
Summary
Collection of Cercarial Secretions (0 –3-h RPs)—The 0 –3-h RPs were prepared as described previously [9]. ESP spots were identified by tandem mass spectrometry using a 4700 Proteomics Analyzer with TOF-TOF optics (Applied Biosystems) as described previously [9]. The reaction was terminated by lyophilization, and the products were purified using a propanol, 5% (v/v) acetic acid reversephase C18 Sep-Pak (Waters Corp.) system as described previously [24]. Glycopeptides remaining after the PNGase F digestion were further digested with 0.2 milliunits of PNGase A (Roche Applied Science; peptide-N4-(N-acetyl--glucosaminyl)asparagine amidase, EC 3.5.1.52) for 24 h at 37 °C, and products were purified on a C18 Sep-Pak (Waters Corp.) as described previously [24]. MS/MS spectra were acquired and processed using the Analyst QS data system (MDS Sciex, Toronto, Canada) Both instruments were precalibrated using [Glu1]fibrinopeptide B in acetonitrile in 5% aqueous acetic acid (1:3, v/v). The sample was injected into the column at 60 °C, and the temperature was steadily increased at a rate of 8 °C/min to a temperature of 300 °C
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