Venus kinase receptors control reproduction in the platyhelminth parasite Schistosoma mansoni

Abstract

The Venus kinase receptor (VKR) is a single transmembrane molecule composed of an intracellular tyrosine kinase domain close to that of insulin receptor and an extracellular Venus Flytrap (VFT) structure similar to the ligand binding domain of many class C G protein coupled receptors. This receptor tyrosine kinase (RTK) was first discovered in the platyhelminth parasite Schistosoma mansoni, then in a large variety of invertebrates. A single vkr gene is found in most genomes, except in S. mansoni in which two genes Smvkr1 and Smvkr2 exist. VKRs form a unique family of RTKs present only in invertebrates and their biological functions are still to be discovered. In this work, we show that SmVKRs are expressed in the reproductive organs of S. mansoni, particularly in the ovaries of female worms. By transcriptional analyses evidence was obtained that both SmVKRs fulfill different roles during oocyte maturation. Suppression of Smvkr expression by RNA interference induced spectacular morphological changes in female worms with a strong disorganization of the ovary, which was dominated by the presence of primary oocytes, and a defect of egg formation. Following expression in Xenopus oocytes, SmVKR1 and SmVKR2 receptors were shown to be activated by distinct ligands which are L-Arginine and calcium ions, respectively. Signalling analysis in Xenopus oocytes revealed the capacity of SmVKRs to activate the PI3K/Akt/p70S6K and Erk MAPK pathways involved in cellular growth and proliferation. Additionally, SmVKR1 induced phosphorylation of JNK (c-Jun N-terminal kinase). Activation of JNK by SmVKR1 was supported by the results of yeast two-hybrid experiments identifying several components of the JNK pathway as specific interacting partners of SmVKR1. In conclusion, these results demonstrate the functions of SmVKR in gametogenesis, and particularly in oogenesis and egg formation. By eliciting signalling pathways potentially involved in oocyte proliferation, growth and migration, these receptors control parasite reproduction and can therefore be considered as potential targets for anti-schistosome therapies.

Figure 1. SmVKR1 and SmVKR2 expression patterns in adult worms.

A: Quantification of Smvkr1 and Smvkr2 transcripts in adult female and male worms by quantitative RT-PCR. Tubulin transcripts were used as internal controls for each condition. For graphical representation, values were expressed as relative fold-difference using the ΔΔCt method and the Smvkr1 ΔCt value in males as reference. Values are expressed as the mean (+/- SEM) of three determinations. B: Localization in sections of paired adult worms of Smvkr1 (a, c, e) and Smvkr2 (b, d, f) transcripts by in situ hybridization. Smvkr1 transcripts were detected in mature oocytes (a) whereas Smvkr2 transcripts were detected in immature oocytes and in the area surrounding the ootype (b). No expression of Smvkr1 (c) and Smvkr2 (d) was detected in the vitellarium. Sense probes of Smvkr1 and Smvkr2 were used respectively as controls in e and f. Abbreviations: io: immature oocytes, mo: mature oocytes, ot: ootype, v: vitellarium. Scale bar: 100µm.

Figure 2. Efficiency of SmVKR1 and SmVKR2 knock-down by RNA interference in adult worm pairs.

Worm couples were electroporated and incubated for 5 days either with dsSmvkr1 (20 µg) or dsSmvkr2 (20 µg) or with both of them (10 µg each) as described in Materials and Methods. Control worms were treated in the same conditions with irrelevant dsLuc RNA (20 µg). Levels of Smvkr1 (A) and Smvkr2 (B) transcripts were determined by quantitative RT-PCR in each worm sample. RNAi treatment with dsSmvkr1 or with dsSmvkr2 results in specific reduction of Smvkr1 and Smvkr2 transcripts respectively. Treatment with both dsSmvkr1 and dsSmvkr2 affects the expression of both Smvkr1 and Smvkr2 genes. For graphical representation, the ΔΔCt method was used to evaluate the relative expression of transcripts in interfered parasites compared to control dsLuc-treated parasites. Statistical analysis was performed using the Student's t-test and values are expressed as mean+/− SEM of three determinations (** p≤0.01, *** p≤0.001).

Figure 3. Morphological analysis of reproductive organs from worms treated with dsSmvkr1 or dsSmvkr2 RNA.

CLSM images of whole-mount preparations of S. mansoni worm couples stained with carmine red. Worms were treated exactly as described in Fig. 2 with dsSmvkr1 (A, B), dsSmvkr2 (C, D), dsSmvkr1 and dsSmvkr2 (E, F) or control dsLuc (G, H) RNA. The morphology of female (left) and male (right) reproductive organs was analyzed. io: immature oocytes, mo: mature oocytes, ot: ootype, sv: sperm vesicle, t: testes. Scale bar: 20 µm.

Figure 4. Ligand determination of SmVKR1 and SmVKR2. Importance of a conserved Ser residue of the VFT domain in amino-acid binding and activation of the receptors.

A) Dose-effect of selected amino acids on the activation of SmVKR1 and potential to induce GVBD in Xenopus oocytes. L-Arg is the most potent activator of SmVKR1 and is active at 1 µM. B) Dose-effect of Ca²⁺ on the capacity of SmVKR2 to induce GVBD. C) Importance of Ser₄₆₆ on the potential of L-Arg to activate SmVKR1. SmVKR1 mutated on its Ser₄₆₆ residue present in the VFT domain requires 10³ fold higher amounts of L-Arg to be activated. The mutation of Ser₄₁₀ in the VFT of SmVKR2 (which can be activated by 1 mM L-Arg in the absence of Ca²⁺) also abolishes its capacity to be activated by L-Arg, confirming the importance of this Ser residue highly conserved in the amino acid binding motif of VFT , . All experiments have been repeated three times and the mean percentages of GVBD are indicated. D) Western blot analysis of membrane extracts of oocytes expressing wild-type and mutated SmVKR proteins confirming the expression of the receptors and indicating their level of phosphorylation (activation) following L-Arg binding. SmVKR1S₄₆₆A is not recognized by anti-phosphotyrosine antibodies in the presence of L-Arg 1 µM and SmVKR2S₄₁₀A is no more phosphorylated with L-Arg even added at 10 mM.

Figure 5. Homo- and hetero-dimerisation of SmVKR. Requirement of Ca²⁺ and/or L-Arg for receptor dimerisation and kinase activation.

cRNAs encoding different tagged versions (Myc or V5) of SmVKR1 and SmVKR2 were injected in Xenopus oocytes. Oocytes were incubated for 5 h in ND96 medium with or without Ca²⁺ and L-Arg, as indicated. Membrane soluble extracts were immunoprecipitated (IP) by anti-V5 or anti-Myc antibodies and analysed by Western Blot (WB) with anti-V5, anti-Myc or anti-phosphotyrosine (pY20) antibodies. L-Arg and Ca²⁺ induce respectively the formation of active homodimers of SmVKR1 and SmVKR2. SmVKR1/SmVKR2 active heterodimers can be formed in the presence of L-Arg and Ca²⁺ (two bands of 170 kDa and 150 kDa corresponding respectively to SmVKR2 and SmVKR1 are labelled by anti-PY antibodies).

Figure 6. Analysis of signaling pathways triggered by SmVKR activation in Xenopus oocytes.

SmVKR1-Myc and SmVKR2-Myc proteins were expressed in Xenopus oocytes for 5 h in ND96 incubation medium with or without their respective ligands (L-Arg 1 µM and Ca²⁺ 1 mM). A-Oocyte lysates were analyzed by Western blot to detect the phosphorylation state of Akt, S6K, ERK2, JNK and p38γ following SmVKR1 and SmVKR2 activation by L-Arg and Ca²⁺ respectively. As a control, SmVKR1-expressing oocytes were stimulated by progesterone (PG), the natural hormonal stimulus for oocyte maturation. Results show that both SmVKR1 and SmVKR2 activate Akt and Erk2 pathways. S6K can be phosphorylated by both receptors, but more importantly by SmVKR2. Only SmVKR1 can trigger JNK activation. The p38γ pathway, is activated in control PG-stimulated oocytes, but not under activation of SmVKR1 or SmVKR2. B-Immunoprecipitation of oocyte extracts by anti-Myc antibodies allowed us to confirm the expression of receptors (WB anti-Myc) and their activation/phosphorylation (WB anti-pY20) in the presence of their respective ligands.

Figure 7. Summary of the major signalling pathways triggered by the activation of SmVKR and potentially regulating fate and differentiation of oocytes in female schistosome organs.

Proteins identified by Y2-H screening (blue) and kinases detected for their activation in Xenopus oocytes (orange) are indicated. Proved and direct interactions between proteins are indicated by full arrows.