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

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. Author summary Schistosomes are formidable parasites that cause the debilitating and life-threatening disease human schistosomiasis. We need to better understand the cellular biology of these parasites to develop novel strategies for their control. Within cells, a process called protein phosphorylation controls many aspects of molecular communication or 'signalling' and is central to cellular function and homeostasis. Here, using complementary strategies, we have performed the first in-depth characterisation and functional annotation of protein phosphorylation events in schistosomes, providing one of the richest phosphoprotein resources for any parasite to date. Using this knowledge, we have developed a novel tool to simultaneously evaluate signalling processes in these worms and highlight sex-biased differences in adult worm protein phosphorylation. Several proteins were found to be more greatly phosphorylated by female worm extracts, suggesting their possible importance to female worm function. This work will help drive new research into the fundamental biology of schistosomes, as well as related parasites, and will support efforts to develop new drug or vaccine-based therapeutics for their control.