Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum.
BACKGROUND
Schistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored.


METHODS
We present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays.


RESULTS
SjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system.


CONCLUSIONS
In this study, we first characterized a PDZ domain-containing protein GIPC3 in S. japonicum. The SjGIPC3-PDZ domain is able to bind both type I and II ligand C-terminal motifs. The identification of native ligand will help reveal the potential biological function of SjGIPC3. These data will facilitate the identification of novel drug targets against S. japonicum infections.
National Key Laboratory of Medical Molecular Biology, Dept, of Physiology and Pathophysiology, School of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, P,R, China.
Yi
Y
Mu
Haiming
H
Huang
Shuai
S
Liu
Pengfei
P
Cai
Youhe
Y
Gao
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D019295:Computational Biology; D008024:Ligands; D054731:PDZ Domains; D010455:Peptides / Q000378:metabolism*; D010802:Phylogeny; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D012549:Schistosoma japonicum / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D008024:Ligands; D010455:Peptides; D015800:Protozoan Proteins