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Screening and identification of protein 29 of Echinococcus granulosus interacting molecules
Zailing Shang1, Fei Qiao1, Yaning Li1
1Basic Medical Institute of Ningxia Medical University, Yinchuan, China.
Introduction:
Eg.P29 is a specific protein of E.granulosus metacestod, and has been able to induce high levels of protection in mice and sheep against challenge with protoscolex of E.granulosus. Therefore, Eg.P29 plays a crucial role in the growth and development of the E. granulosus metacestode. However, the function of Eg.P29 remains unknown. During the process life, protein is commonly with other proteins to form a complex network of interactions to play the function. In this study, we identified the interacting molecules of Eg.P29 for foundation to exploring the function of Eg.P29 in E. granulosus metacestode.
Methods:
Some basic information about Eg.P29 through bioinformatics software. A total of 50 molecules interacting with Eg.P29 were screened by three immunoprecipitation-combined liquid chromatography-tandem mass spectrometry intersections. The Eg.P29/pEGFP-C1 vector was transfected into cells to observe the morphological changes. Seven molecules as actin and actin-related proteins were screened using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and the effects of Eg.P29 on them were verified at the mRNA and protein levels. Interacting proteins of Eg.P29 were identified by yeast two-hybrid assay and immunoprecipitation. The localization and distribution of Eg.P29 and Eg.actin on protoscolex were observed by immunohistochemistry.
Results:
Eg.P29 is a soluble cytoplasmic protein containing a BAR structural domain that may play a role in actin polarity and endocytosis. Cells in the Eg.P29 group showed marked morphological changes in the form of rounded, or oval shapes, which were attributed to changes in actin distribution. Seven molecules interacting with Eg.P29 were mainly actin and actin-related proteins (ACTG1, ACTN4, VCL, ARPC1A, LIMA1, FLNB and MYH10), and their mRNA and protein levels were significantly affected by Eg.P29. Yeast two-hybridization experiments showed that VCL did not interact with Eg.P29, whereas the other molecules interacted with Eg.P29. The interaction of LIMA1 and actin with Eg.P29 was verified by co-immunoprecipitation. Additionally, Eg.P29 and Eg.actin had the same histological location on rostellum, and suckers of the protoscolex. Additionally, the seven molecules were all discovered their homologous proteins in E.granulosus and formed a network of interacting proteins.
Discussion:
In this study, we confirmed LIMA1 and actin were Eg.P29-interacting molecules, they were forming a compound regulation system to affect cytoskeleton formation. And we also deduced that Eg.P29 interacts with Eg.actin in E. granulosus, which collaborate to affect the activity and development of protoscolex. These aspects warrant further in-depth study.
