Synthesis and Antibody Binding Studies of Schistosome-Derived Oligo-α-(1-2)-l-Fucosides

Abstract

Schistosomiasis is caused by blood-dwelling parasitic trematodes of the genus Schistosoma and is classified by the WHO as the second most socioeconomically devastating parasitic disease, second only to malaria. Schistosoma expresses a complex array of glycans as part of glycoproteins and glycolipids that can be targeted by both the adaptive and the innate part of the immune system. Some of these glycans can be used for diagnostic purposes. A subgroup of schistosome glycans is decorated with unique α-(1-2)-fucosides and it has been shown that these often multi-fucosylated fragments are prime targets for antibodies generated during infection. Since these α-(1-2)-fucosides cannot be obtained in sufficient purity from biological sources, we set out to develop an effective route of synthesis towards α-(1-2)-oligofucosides of varying length. Here we describe the exploration of two different approaches, starting from either end of the fucose chains. The oligosaccharides have been attached to gold nanoparticles and used in an enzyme-linked immunosorbent assay ELISA and a microarray format to probe antibody binding. We show that binding to the oligofucosides of antibodies in sera of infected people depends on the length of the oligofucose chains, with the largest glycans showing most binding.

Schistosoma mansoni egg-derived thioredoxin and Sm14 drive the development of IL-10 producing regulatory B cells

“…As the affinity chromatography procedure may have affected the activity of the glycosylated substances and may have failed to select an active subset of fucosylated molecules, we decided to explore an alternative approach to study the role of glycosylated molecules present in SEA in Breg cell development, by using glycans coupled to gold nanoparticles (AuNP). The presence and bio-recognition by mAbs of these glycans on the AuNP was confirmed as previously reported [49]. We applied simple representations of fucosylated glycan motifs present in SEA glycoproteins [48,56] to generate fucosylated AuNP, including the Fucα1-2Fucα1-R difucosyl motif that is recognized by mAb 114-4D12.…”

“…Membranes were incubated with primary antibody diluted in TBSTM for 1h at room temperature (RT), washed in TBSTM and incubated with the secondary antibody (rabbit polyclonal anti-mouse IgG-HRP, Dako), diluted 1:10,000 in TBSTM for 1h at RT. Monoclonal antibodies used for the detection of glycans include mAb 291-4D10-A (recognizes Lewis X structures [46]), mAb 291-5D5-A (recognizes F-LDN(-F) structures [47]), mAb 114-4D12-AA (recognizes Fucα1-2Fucα1-R structures [48,49]). Detection was performed using Pierce ECL Plus Western blotting substrate (Thermo Fisher Scientific) and Super RX-N X-Ray films (Fujifilm), developed with the X-ray film processor (Huqju Imaging Technology).…”

“…Mono-, di-, tri-and tetra-fucosylated 5nm gold nanoparticles were generated as described previously [49].…”

“…In particular, fractions F2 to F4 were strongly recognized by the FF-reactive monoclonal antibody (mAb) 114-4D12 compared to antibodies against F-LDN or Lewis X , indicating the abundant presence of difucosylated motifs in the high MW molecules. Previous studies have shown that such difucosyl motifs bound by mAb 114-4D12 often are part of larger multi-fucosylated structures [48,49,56].To investigate if these glycan structures are involved in SEA-induced Breg cell development, high and medium MW fractions were either mock treated or treated with sodium metaperiodate in order to oxidize glycans and prevent their recognition by lectin receptors [57][58][59][60]. As glycosylated molecules in high MW fractions rich in multi-fucosylated glycans seem to play a role in IL-10 induction in splenic B cells, we next aimed to separate these multi-fucosylated molecules from the other SEA components using mAb 114-4D12 affinity chromatography.…”