IPSE/alpha‐1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core‐difucosylated N‐glycans

Abstract

Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin‐4‐inducing factor from schistosome eggs (IPSE/alpha‐1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin‐4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC‐MS(/MS) and MALDI‐TOF(/TOF)‐MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha‐1 contains two N‐glycosylation sites, which are each occupied for a large proportion with core‐difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein‐specific glycosylation analysis of schistosome eggs.

Interleukin-4-Inducing Principle from Schistosoma mansoni Eggs Contains a Functional C-Terminal Nuclear Localization Signal Necessary for Nuclear Translocation in Mammalian Cells but Not for Its Uptake

“…However, the results also indicate that deletion of the C terminus, including the NLS, abrogates this function. As an explanation for this, our recent studies have shown that dimerization of native IPSE/alpha-1 requires a cysteine residue (C132) at the C terminus of IPSE, which forms an interchain disulfide bond (54). This cysteine is removed in IPSE/alpha-1 ⌬NLS but is present in the IPSE/ alpha-1 3R mutant.…”

“…Native IPSE/alpha-1 released by schistosome eggs is a dimer as a consequence of an interchain disulfide bond involving the C-terminal cysteine residue C132 (54). Despite the relatively low molecular mass (33 to 35 kDa) of IPSE/alpha-1 dimers, the mutants defective for NLS activity were excluded from the nucleus (Fig.…”

“…C-type lectins are carbohydrate-binding receptors expressed on both immune and structural cells and are known to depend on calcium for binding to and internalization of glycosylated molecules. Given that native IPSE/alpha-1 is glycosylated (54), it is likely that in dendritic cells and possibly other cell types, such as macrophages, IPSE/alpha-1 can gain access to the cytosolic and nuclear compartments through C-type lectin-dependent internalization. Previous work demonstrated uptake of SEA (containing IPSE/ alpha-1) by human MDDCs via the MR, macrophage galactosetype lectin (MGL), and DC-SIGN (52).…”

Local Antiglycan Antibody Responses to Skin Stage and Migratory Schistosomula of Schistosoma japonicum

“…LC fractions were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using Ultraflex II and Ultraflextreme mass spectrometers (Bruker Daltonics, Bremen, Germany) in negative-ion reflectron mode with 2,5-dihydroxybenzoic acid (DHB) (Bruker Daltonics) as the matrix. The detected masses were translated into putative glycan structures using Glycopeakfinder (http://glyco -peakfinder.org), the literature (9,(19)(20)(21)(22)(23)(24)(25)(26)(27)(36)(37)(38)(39)(40)(41), and our own unpublished tandem-MS (MS-MS) data. The three most abundant signals in each mass spectrum were translated into glycan structures, unless the individual signals made up less than 10% of the total intensity of all glycan masses in the spectrum of a glycan sample.…”

Site Specific Identification of N-Linked Glycosylation in Proteins by Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

“…Several approaches have become commonly used to address this issue: (i) analysis of the N-linked carbohydrates released from the protein by enzymatic procedures followed by derivatization of the glycan pool [14], or (ii) analysis of the glycopeptides formed by proteolytic degradation of the protein [15,16]. While the former provides a global picture of the glycans decorating the protein, the second strategy discloses information about the carbohydrate attachment site, which can be obtained using various tandem mass spectrometric methods (reviewed in [17]).…”