Screening and verification for proteins that interact with leucine aminopeptidase of Taenia pisiformis using a yeast two-hybrid system
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Leucine aminopeptidase of Taenia pisiformis (TpLAP) belonging to the M17 peptidase family has been implicated as a stage-differentially expressed protein in the adult stage of T. pisiformis. In order to further dissect the biological functions of TpLAP in the growth and development of adult worms, TpLAP-interacting partners were investigated. In this study, a yeast two-hybrid (Y2H) cDNA library from adult T. pisiformis was constructed. Using pGBKT7-TpLAP as bait, proteins interacting with TpLAP were screened by Y2H system and positive preys were sequenced and analyzed using the Basic Local Alignment Search Tool (BLAST). Our results showed that six genuine TpLAP-interacting proteins, including LAP, dynein light chain (DLC), SUMO-conjugating enzyme (UBC9), histone-lysine n-methyltransferase, trans-acting transcriptional, and one unknown protein, were identified via Y2H assay. Furthermore, the interaction between TpLAP and UBC9 of T. pisiformis (TpUBC9), an important protein involved in SUMOylation pathway, was further validated by one-to-one Y2H assay, co-immunoprecipitation, and confocal analysis. These findings provide a deeper understanding of the biological functions of TpLAP and offer the first clue that TpLAP may act as a novel SUMOylated substrate, suggesting that the SUMO modification pathway plays an important role in regulation of adult worm growth and development.
KeywordsTaenia pisiformis Yeast two-hybrid Interacting proteins TpLAP TpUBC9 Co-immunoprecipitation
The author thanks Dr. Xuenong Luo and other members of our laboratory for their help in performing experiments, and Dr. Xuepeng Cai for fruitful advice during the course of the work and critical reading of this manuscript. Shanghai OE Biotech Co., Ltd. (Shanghai, P.R. China) is thanked for technical assistance.
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31772726) and the National Key Research and Development Program of China (No. 2017YFD0501303 and No. 2017YFC1601206).
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The author declares that no competing interests exist.
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