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Expression profiles of glyceraldehyde-3-phosphate dehydrogenase from Clonorchis sinensis: a glycolytic enzyme with plasminogen binding capacity

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Abstract

Globally, 15–20 million people are infected with Clonorchis sinensis (C. sinensis) which results in clonorchiasis. In China, clonorchiasis is considered to be one of the fastest-growing food-borne parasitic diseases. That more key molecules of C. sinensis are characterized will be helpful to understand biology and pathogenesis of the carcinogenic liver fluke. Glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) from many species have functions other than their catalytic role in glycolysis. In the present study, we analyzed the sequence and structure of GAPDH from C. sinensis (CsGAPDH) by using bioinformatics tools and obtained its recombinant protein by prokaryotic expression system, to learn its expression profiles and molecular property. CsGAPDH could bind to human intrahepatic biliary epithelial cell in vivo and in vitro by the method of immunofluorescence assays. CsGAPDH also disturbed in lumen of biliary tract near to the parasite in the liver of infected rat. Western blotting analysis together with immunofluorescence assay indicated that CsGAPDH was a component of excretory/secretory proteins (CsESPs) and a surface-localized protein of C. sinensis. Quantitative real-time PCR (Q-PCR) and Western blotting demonstrated that CsGAPDHs are expressed at the life stages of adult worm, metacercaria, and egg, but the expression levels were different from each other. Recombinant CsGAPDH (rCsGAPDH) was confirmed to have the capacity to catalyze the conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate which was inhibited by AMP in a dose-dependent manner. In addition, rCsGAPDH was able to interact with human plasminogen in a dose-dependent manner by ELISA. The interaction could be inhibited by lysine. The plasminogen binding capacity of rCsGAPDH along with the distribution of CsGAPDH in vivo and in the liver of C. sinensis-infected rat hinted that surface-localized CsGAPDH might play an important role in host invasion of the worm besides its glycolytic activity. Our work will be a cornerstone for getting more messages about CsGAPDH and its role in biology and parasitism of C. sinensis.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81101270), the National Key Basic Research and Development Project (973 project, No. 2010CB530000), and the National Important Sci-Tech Special Projects (2012ZX10004220).

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Authors and Affiliations

  1. Department of Clinical Laboratory, The Second People’s Hospital of Hefei, Hefei City, China

    Yue Hu

  2. Department of Parasitology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China

    Yue Hu, Lisi Huang, Wenfang Li, Pei Liang, Xiaoyun Wang, Jin Xu, Yan Huang & Xinbing Yu

  3. Department of Infertility and Sexual Medicine, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China

    Erhong Zhang

  4. Department of Clinical Laboratory, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China

    Lisi Huang

  5. Key Laboratory for Tropical Diseases Control, Sun Yat-Sen University, Ministry of Education, Guangzhou, China

    Wenfang Li, Pei Liang, Xiaoyun Wang, Jin Xu, Yan Huang & Xinbing Yu

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Correspondence to Yan Huang.

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Fig. S1

Amino acid sequence alignment of CsGAPDH with GAPDHs from other species. Amino acid sequence of GAPDH from C. sinensis (Cs, GenBank Accession No.: GAA28380.1), H. sapiens (Hs, GenBank Accession No.: AAH83511.1), R. norvegicus (Rn, GenBank Accession No.: NP_058704.1), M. musculus (Mm, GenBank Accession No.: NP_032110.1), C. elegans (Ce, GenBank Accession No.: CAA28504.1), P. falciparum (Pf, GenBank Accession No.: AAD10249.1) and S. japonicum (Sj, GenBank Accession No.: CAX80263.1) were included in the analysis. Identical amino acids in all sequences were marked with bright yellow background. Identical amino acids in most sequences were showed bright blue background. The NAD (P)-binding domain of GAPDH N-terminus (aa5–152) was illustrated in the rose-red rectangles. The asterisks (*) indicated the active site (aa150–157, ASCTTNCL). The catalytic domain of GAPDH C-terminus (aa157–314) was showed by rose-red underline. (GIF 458 kb)

High resolution image (TIFF 1698 kb)

Fig. S2

Inhibition effect of AMP on the enzymatic activity. The enzymatic activity was inhibited by AMP in a dose-dependent manner. AMP caused nearly to 70 % inhibition at 4 mM. The symbols represented mean±S.D of an experiment done in triplicates. (GIF 5 kb)

High resolution image (TIFF 1188 kb)

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Hu, Y., Zhang, E., Huang, L. et al. Expression profiles of glyceraldehyde-3-phosphate dehydrogenase from Clonorchis sinensis: a glycolytic enzyme with plasminogen binding capacity. Parasitol Res 113, 4543–4553 (2014). https://doi.org/10.1007/s00436-014-4144-x

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