Study On Immunoglobulin - Binding Proteome Of Schistosoma Japonicum

Schistosomiasis, also known as bilharzias, is an acute and chronic parasitic disease caused by blood flukes of the genus Schistosoma. It is the second most devastating parasitic disease after malaria according to worldwide rates of both morbidity and mortality. In 2013, at least 261 million people and a large number of animals were infected in 78 countries, and an estimated 700 million people were at risk (WHO, January 2015). Schistosomiasis japonica is caused by parasitization with Schistosoma japonicum and is the only zoonotic schistosomiasis; therefore, it the most difficult to control among the five schistosome species that infect humans. Currently, praziquantel is the only drug that is effective for the treatment of the disease, and a large amount of work is still required to develop a vaccine that is able to control the disease effectively. The surface of the schistosome tegument represents the most accessible interface for a host immune attack and therefore merits investigation for vaccine candidates. Nevertheless, vaccines based on the membrane components or associated proteins have been extensively studied, little success has been achieved.The fact that adult parasites can thrive within the mesenteric or vesicular veins of their hosts for decades indicates that the worms employ a variety of strategies to escape identification by the immune system. It has been hypothesized that schistosomal parasites evade host immune expulsion through surface masking, molecular mimicry, and the active modulation of host immune responses, but the mechanisms of immune evasion were still not well defined.Our laboratory had found that, Sjc23, a member of the tetraspanin (TSP) family of S. japonicum, mediated the acquisition of human IgG via the interaction of a nine amino acids motif with the Fc domain of the IgG molecule, in a similar way as that between paramyosin (SCIP-1) and IgG; the induced antibodies were predominantly the IgG2a type, which has been shown to be inefficient in complement fixation and to exhibit fewer cytophilic properties in ADCC (antibody-dependent cell-mediated cytotoxicity). To further understand the interaction of the schistosomal parasites with human immune system, we perform a proteomic analysis with a purpose to identify the parasite-derived proteins that interact with host immunoglobulins.In this study, we took the advantage of high-throughput protein identification following affinity purification and successfully demonstrated that multiple surface-exposed proteins of S. japonicum exhibited an affinity to a variety of host non-immune immunoglobulins. Of the 437 schistosomal proteins identified,243 bound to human IgG,210 to human IgM, and 134 to human IgE; most of the proteins showed affinity to both IgG and IgM, while fewer showed affinity to IgE. Furthermore, most of the proteins could be categorized based on sequence features, including EF-hand motif, immunoglobulin motif, Ca2+ binding motif, annexin superfamily, immunophilin, and complement binding motif, suggesting common properties of immunoglobulin binding among the proteins identified.To confirm and further characterize the interactions of the schistosomal proteins with the host immunoglobulins, ten proteins were selected, and his-tagged recombinant proteins were generated. The binding of these proteins with host immunoglobulins was confirmed using both an ELISA assay and a pull-down approach. Compared with ELISA assays, pull-down assays require higher affinity between the ligand and receptor due to the high stringency of particle precipitation and the associated washing steps. All of the selected proteins showed a high affinity for the IgGs of both humans and domestic animals and variable but reduced affinity for IgM, IgE and IgA. Previous studies on the antigen paramyosin of S. mansoni and Sjc23 suggested that these proteins only adhere to the Fc domain of Ig; in agreement with this result, we found that all of the tested recombinant proteins bound the Fc domain except Sj31, which showed a slight affinity for the Fab domain. The data collectively demonstrated that schistosomal parasites escape host recognition by binding to the Fc domain of immunoglobulins. One consequence of this Fc domain binding is that the reaction of complement activation and fixation is paralyzed due to the reduced availability of the paratope-epitope interaction of the Fab domain with the antigen, resulting in the unavailability of the complement binding domain. Furthermore, the complement receptor protein identified in the parasite (C1qBP) exhibited higher affinity for human IgG than for complement C1q, strongly suggesting that the parasite utilized non-immune Ig to block complement activation.To further confirm the importance of these proteins for interactions with the host immune system, we investigated their expression during various developmental stages of the parasite using qRT-PCR. The results showed that they were mainly expressed during the stages associated with the infection of their definitive hosts.In summary, our findings propose that human immunoglobulins can interact with multiple S. japonicum-derived ligands and consequently have profound inhibitory effects on anti-schistosome immune responses. Our data are, at the firs time, to provide a global view of the interaction between the schistosomal parasite and human non-immune immunoglobulins, suggesting a molecular basis for the surface masking and evasion of immune recognition after invasion.