Deep phosphoproteome analysis of Schistosoma mansoni leads development of a kinomic array that highlights sex-biased differences in adult worm protein phosphorylation

Abstract

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a 'footprint' of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.

Effect of Praziquantel on Schistosoma mekongi Proteome and Phosphoproteome

“…Recently, drugs acting on phosphorylation and protein kinases have been proposed as novel targets for schistosomiasis treatment [25,26,[55][56][57]. Kinomic array of S. mansoni revealed a number of kinases those are vital for Schistosoma biology [31] in which Src kinase, Cyclin-dependent kinase (CDK), PKC were predicted from bioinformatic tool as the critical kinases for anthelminthic effects of PZQ (Table S3). Our study indicates that kinases of critical pathways in the parasite and Src kinase are important kinases for PZQ response ( Figure S1, Table S3).…”

“…Anti-phosphoserine was used to determine phosphorylation levels and tissue localization in the parasites. In Schistosoma parasite, the main phosphorylated residues are serine (67.8%), threonine (20.1%) and tyrosine (12.1%) [31]. Phosphorylation of tegument, muscle and internal cells of the worms were markedly reduced after PZQ treatment, as indicated by the black spots in Figure 3.…”

Protein Kinases: Potential Drug Targets Against Schistosoma japonicum

“…Therefore, applying Src kinase inhibitors could be a novel approach to treat Mekong schistosomiasis. In another study on S. mansoni , the phosphoproteome of S. mansoni was first comprehensively analyzed based on a novel peptide kinomic array, which has characterized 12936 phosphorylation sites in 3176 proteins ( Hirst et al., 2020 ). The phosphorylation sites identified from S. mansoni cover a wider area than these identified from S. japonicum .…”

“…The phosphorylation sites identified from S. mansoni cover a wider area than these identified from S. japonicum . It was also shown that many PKs such as CaMKII, PKA and CK1/2 are of great importance to schistosome function ( Hirst et al., 2020 ). Interestingly, the distribution of phosphorylations among serine, threonine, and tyrosine is 68%:20%:12% in S. mansoni , and a greater proportion (3 to 4-fold) of phosphotyrosine is determined compared to other organisms ( Rigbolt et al., 2011 ; Lasonder et al., 2012 ).…”

“…In the future, we can construct a corresponding kinase map to explore the interesting targets according to this genome information. The phosphorylation sites may affect the binding effect of the drugs and the targets ( Hirst et al., 2020 ), and widespread phosphorylation data can support the development of kinase drugs. The application of CRISPR technology can allows us to modify the genes of interest, and then functionally analyze the key targets.…”