Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

Abstract

f Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fuc␣3GalNAc␤4(Fuc␣3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core ␤-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. Schistosomiasis is a major health problem in tropical and subtropical areas where it is endemic, with more than 200 million people actively infected and 800 million at risk of contracting the disease (1-3). Current treatment for disease is limited to the drug praziquantel (4), but cases of drug resistance have been reported (5). Decades of research on schistosomiasis vaccines have yielded only two candidates for clinical trials, and no encouraging results have been published yet (6-9). Thus, there is an urgent need to develop more sensitive diagnostic methods and to identify new vaccine candidates.Recent studies have shown that a major part of the host immune response to infection is directed against carbohydrate (glycan) antigens in glycoproteins and glycolipids (10-17). A wide variety of unusual antigenic determinants include glycans containing the LDN, fucosylated LDN sequences (LDNF, LDN-dF, FLDN, and FLDNF), Lewis X (Le x ), poly-Le x , core ␣3 fucose, and core ␤2 xylose structures (Fig. 1) (11, 14, 17), many of which are expressed by all developmental stages of schistosomes (18). Interestingly, monoclonal antibodies (MAbs) specific to these glycans recognize these antigens on the surface of 3-h-old schistosomula, and some anti-glycan antibodies can mediate killing in vitro in a complement-dependent fashion (18-21). Schistosoma mansoniinfected rhesus monkeys, which are known to self-cure after infection, have IgG to many glycan antigens, including Le x , LDN, LDNF, core fucose, and core xylose determinants, and their sera are effective in complement-mediated cytolysis of cells expressing Le x as well as schistosomulum larvae in ...

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins

“…This strategy is important for being able to profile the innate and adaptive immune response toward the glycan-containing components of the organism. Our studies with Schistosoma mansoni and the human response to this parasitic infection led us to generate a natural shotgun N-glycan microarray from the schistosome egg glycoproteins (Mickum et al, 2016). The schistosome eggs, which were isolated from infected mice, were treated with PNGase F and A to isolate as many N-glycans as possible, separated and printed to create the arrays.…”

“…As noted above, anti-glycan antibodies can be characterized on the array platforms, and many have been studied over the years on the CFG glycan array (Agrawal-Gamse et al, 2011;Zipser et al, 2012;Noble et al, 2013;Falkowska et al, 2014;Chua et al, 2015;Mickum et al, 2016;Tati et al, 2017;Nkurunungi et al, 2019), such as characterization of a new Lewis x antibody (Mandalasi et al, 2013) and multiple iterations of an anti-Tn antigen antibody (Chaturvedi et al, 2008;Tati et al, 2017). While antibody specificities are not the main focus of this review, it is clear from past work that the glycan microarrays have been and will continue to be very useful tools for their characterization, especially as the glycans and the types of linkers diversify.…”

A library of cell-surface and secreted proteins fromSchistosoma mansoniidentifies early serological markers of infection

“…Although the lower immunoreactivity for 37 proteins may suggest incorrect folding, they could also be weakly immunogenic in humans, or not directly exposed to the host immune system. Although a large fraction of the natural host immune response has been reported to be directed against parasite glycans present at different stages of development [44][45][46], the conformation-sensitive immunoreactivity that we observed in our assay is almost certainly directed against the protein backbones since the immunoreactivity against glycans would not be affected by heat-treatment and the glycan groups added on the recombinant proteins in HEK293 cells may differ substantially from the ones present on endogenous proteins. Of the sixteen most immunoreactive proteins observed, eleven had already been identified in the surface and secreted proteome of S. mansoni; by contrast, only six out of the 37 proteins with low serum reactivity (16%) have already been described in proteomics studies.…”

In silico analyses of protein glycosylating genes in the helminth Fasciola hepatica (liver fluke) predict protein-linked glycan simplicity and reveal temporally-dynamic expression profiles

“…hepatica 13 , 14 ; although sialic acid was detected in the latter study its presence was attributed to contamination by host glycans. Sialylation also appears absent from schistosome glycoproteins 41 – 44 , although binding of sialic acid lectins has been described in the cestodes Echinococcus granulosus and Taenia solium 45 , 46 . The general view across invertebrates is that sialic acids are only seen in glycans of the deuterostome lineage 47 .…”