Mapping the binding between the tetraspanin molecule (Sjc23) of Schistosoma japonicum and human non-immune IgG

Abstract

Background: Schistosomal parasites can establish parasitization in a human host for decades; evasion of host immunorecognition including surface masking by acquisition of host serum components is one of the strategies explored by the parasites. Parasite molecules anchored on the membrane are the main elements in the interaction. Sjc23, a member of the tetraspanin (TSP) family of Schistosoma japonicum, was previously found to be highly immunogenic and regarded as a vaccine candidate against schistosomiasis. However, studies indicated that immunization with Sjc23 generated rapid antibody responses which were less protective than that with other antigens. The biological function of this membrane-anchored molecule has not been defined after decades of vaccination studies.

Methodology and principal findings: In this study, we explored affinity pull-down and peptide competition assays to investigate the potential binding between Sjc23 molecule and human non-immune IgG. We determined that Sjc23 could bind human non-immune IgG and the binding was through the interaction of the large extra-cellular domain (LED) of Sjc23 (named Sjc23-LED) with the Fc domain of human IgG. Sjc23 had no affinity to other immunoglobulin types. Affinity precipitation (pull-down assay) in the presence of overlapping peptides further pinpointed to a 9-amino acid motif within Sjc23-LED that mediated the binding to human IgG.

Conclusion and significance: S. japonicum parasites cloak themselves through interaction with human non-immune IgG, and a member of the tetraspanin family, Sjc23, mediated the acquisition of human IgG via the interaction of a motif of 9 amino acids with the Fc domain of the IgG molecule. The consequence of this interaction will likely benefit parasitism of S. japonicum by evasion of host immune recognition or immunoresponses. This is the first report that an epitope of schistosomal ligand and its immunoglobulin receptor are defined, which provides further evidence of immune evasion strategy adopted by S. japonicum.

Figure 1. Detection of Sjc23 on the surface of S. japonicun with IFA.

A and C are phase contrast images of cercarie and schistosomulum. B and D are the same parasites stained with specific anti-Sjc23 antibodies. The tail of the cercarie de-attached during washing steps.

Figure 2. Cloning and expression of the large extracellular domain of Sjc23 (Sjc23-LED).

A PCR product of the gene fragment coding for Sjc23-LED. The length of the fragment is 228 bp. B Purified His-tagged recombinant Sjc23-LED. The molecular weight of the His-Sjc23-LED is 12.4 kDa. C Western-blot confirmation of the recombinant protein with an anti-His-tag mAb.

Figure 3. Binding of Sjc23-LED with human non-immune immunoglobulins in ELISA assay.

Purified human plasma IgG and IgM (5 µg/ml) from individuals without background of schistosomiasis were coated on ELISA plates and the affinity of Sjc23, TSP2 and GST to human IgG (A) and IgM (B) was examined. Only Sjc23-LED binds to human IgG with concentration dependency. No adhesion to human IgM and other types of immunoglobulins (data not shown) was observed with any of the antigen.

Figure 4. Binding of Sjc23-LED with immunoglobulins in pull-down assay.

A Human IgG (lane 1), IgA (lane 4), IgE (lane 6), and IgM (lane 8) were incubated with Sjc23-LED bound Sepharose resin, after extensive washing, the proteins were resolved in SDS-PAGE and blotted to nylon film. The immunoglobulins that bound to Sjc23-LED was detected by using mAbs specific for human antibodies (γ-chain, α-chain,ε-chain or µ-chain specific). Only human IgG could specifically bind to Sjc23-LED, whereas the other antibody types did not. GST did not bind human IgG (lane 2). Lanes 3, 5, 7 and 9 were corresponding antibodies as controls for detection. B Porcine and bovine IgG was respectively incubated with Sjc23-LED bound Sepharose resin and only porcine IgG was marginally precipitated (lane 1, the band was indicated with an asterisk). Lane 2 and 4 were controls of the corresponding IgGs.

Figure 5. Binding of human IgG domains to Sjc23-LED.

A In the pull-down assay, Fab and Fc fragments of human IgG were incubated with Sjc23-LED conjugated particles (lane 1 and 3) respectively. After washing, the binding was examined by anti-Fab and anti-Fc antibodies. One Fc fragment bound to Sjc23-LED (lane 3). No binding of Fab fragment to Sjc23-LED was detected. Lane 2 and 4 were loading control of Fab and Fc fragment respectively. B In ELISA assay, the plate was coated with Fab and Fc fragment respectively, and recombinant Sjc23-LED diluted from 100 to 3.125 µg/ml. Sjc23-LED only bound to Fc fragment and the binding was dilution-dependent.

Figure 6. Defining the region of Sjc23-LED that bound human IgG.

A Sequences of the synthetic peptides with overlapping regions. The amino acid sequence of Sjc23-LED was at the top and the 7 partially overlapping peptides were listed below. The sequence region with potential binding activity to human IgG was marked with pink color. B Inhibition of human IgG with Sjc23-LED by peptides. Sjc23-LED was incubated with human IgG in the presence of different peptides. Only peptide 3 and 4 showed inhibitory activity. Lane 8 and 9 were no peptide and loading control respectively.

Figure 7. Structural modeling of Sjc23 and SjcTSP-2 molecule.

A Schematic illustration of Sjc23 molecule anchoring in the membrane. Sjc23 molecule is a member of the tetraspanin family with 4 transmembrane regions and three loops. Loop I (aa 35–53) and II (107–183) are extracellular, while loop III is introcellular. B The structural modeling of the large extracellular domain II of Sjc23. This domain has a high similarity (>94% confidence) to the large extracellular domain of human CD81, another member of tetraspanin family. It is consisted of 4 helix regions and 4 low complex regions. The 9 amino acids region that mediated IgG binding was in helix 2 (red color and arrow-headed). C. Structure prediction of a SjcTSP-2 variant. The molecule has more low complex regions compared to other tetraspanins. Helix 2 is shorter than that of Sjc23. D. Structure of the large extracellular domain II of human CD81. E and F are overlapping of Sjc23 and SjcTSP-2 with human CD81.