Domain loss events are extensively widespread in S. japonicum, which serves as a notable consequence of the adoption of parasitism . It is estimated that 1,000 protein domains have been abandoned by S. japonicum during long-term evolution, which suggests that the remaining domains are necessary for parasitic growth, development, and maturation. The proteomic information revealed that at least 25 PDZ domains are distributed among 16 S. japonicum proteins, suggesting that the PDZ domain is one of the important modules reserved in the parasite to mediate multiple biological processes. In the present study, we have characterized one of the PDZ domain-containing proteins from S. japonicum, GIPC3, for the first time. The ligand binding properties of the SjGIPC3-PDZ domain were determined by Y2H assays, and its potential ligands were identified.
Screening a highly diverse peptide library provided an opportunity to fish out the potential binding property of SjGIPC3. Whether the SjGIPC3-PDZ domain or full-length SjGIPC3 were used as bait, analogous binding specificities can be deduced from the unique C-terminal tails obtained from both Y2H assays. However, the GH1 and GH2 domains of SjGIPC3 may still affect the binding specificity of the PDZ domain to some degree, because the ligand motifs were more canonical when full-length SjGIPC3 was used as bait. For instance, at P-1, the acidic amino acid Glu was more preferentially selected in the second assay (45.2%) than in the first assay (13.5%). Similarly, at P-3, 16 types of amino acids emerged in the first assay, while only eight kinds of amino acids were presented in the second assay. Also at this position, the occurrence of Glu was 50.0% when screened against the full-length SjGIPC3, twice the occurrence rate as when the SjGIPC3-PDZ domain was used as bait. Thus, the binding specificity screened using full-length SjGIPC3 as bait may be more authentic to its native status.
Previously, GIPCs have been shown to interact specifically with Class I (−X-S/T-X Φ*), II (−Φ X Φ*), and III (−X X-C*) binding motifs . Moreover, Kermit, the GIPC orthologue in Drosophila, and GLUT1CBP each interact with Myosin VI through an internal sequence within the cargo binding domain [43, 44]. In this study, we focused on the C-terminal binding specificity of SjGIPC3 and found that the PDZ domain of SjGIPC3 preferred to bind to Class I and II motifs, consistent with the extensive ligand-binding ability of GIPC orthologues in other species . However, regarding the binding of Class II motifs, the only example presented thus far is that the PDZ domain of synectin binds to sydecan-4 through the “-EFYA*” motif, in which the aromatic amino acid Phe was selected at P-2. In our study, it is worth noting that this site can be occupied by the aliphatic amino acid residues Ile and Leu, in addition to Ser and Thr (Table 1). Moreover, in a previous report, the binding motifs of GIPC orthologues trend toward selecting a Val, Ala, or Cys residue at p0; here, the more hydrophobic amino acids, such as Ile and Leu, were predominantly preferred. These diverse binding characteristics could be utilized for drug development against SjGIPC3.
On average, one PDZ protein is capable of interacting with 17 partners . Although a panel of S. japonicum proteins deposited in the public database bears a C-terminal tail fittting in the consensus binding sequence, the bioinformatic hints at molecular function and localization have prevented them from being ligand candidates of SjGIPC3. Using the Y2H assay, four proteins were determined to be potential ligand candidates for SjGIPC3, which suggests that some other potential ligands may not have been discovered yet, likely restricted by the quality of the proteomic database, as truncated fragments have been extensively deposited in the S. japonicum proteomic database.
To adapt to the complexity of the life cycle, schistosomes are equipped with a complex nervous system to sense environmental signals . Transcriptional analysis has revealed that the expression of SjGIPC3 was dramatically up-regulated after invasion of the host, suggesting that it may play an important role (e.g., signal transduction) in the context of the parasite-host interaction. More importantly, the preferential expression of SjGIPC3 in male rather than female adult worms further hinted that the potential function of the protein was related to the processing of environmental information, as male adult worms interface with the host and female adult worms resided in the gynecophoral canal, more stimulating signal would be received by male adult worms . GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss [48–50]. Previously, Yi and his colleagues suggested that the GIPC orthologue in mouse might associate with extrasynaptic NMDA receptors through binding to the C-terminal tail “ESDV”, and may be involved in the organization and trafficking of this population of receptors . Intriguingly, among the four potential ligands of SjGIPC3 identified in this study, one is the glutamate receptor NMDA, which also bears a class I motif “TTTL”; thus, we cannot rule out the possibility that SjGIPC3 may also have a physiological function in the regulation of NMDA receptor trafficking, similar to its orthologue in mouse.