Abstract
Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis.
Highlights
Human clonorchiasis is caused by infection with Clonorchis sinensis (C. sinensis), the Chinese or oriental liver fluke, which is mainly distributed in various eastern parts of Asia, including China, Korea, and Northern Vietnam
The genome and transcriptome data of C. sinensis demonstrate that the genes that are involved in both the glycolytic pathway and Krebs cycle are expressed during the infection [6,7]
BLASTx showed that the deduced amino acid sequence of CsHK, respectively, shared 69%, 68%, 45%, 36%, 35%, 34%, 31%, 44%, 44% and 44% identity with HK from Schistosoma mansoni (S. mansoni, XP_002575647.1, hexokinase), Schistosoma japonicum (S. japonicum, CAX74187.1, hexokinase A), Haemonchus contortus (H. contortus, CAB40412.1, hexokinase), Trypanosoma brucei (T. brucei, CAC69958.1, hexokinase), Trypanosoma cruzi (T. cruzi, AAL93565.1, hexokinase), Toxoplasma gondii (T. gondii, AAL93565.1, hexokinase), Plasmodium falciparum (P. falciparum, AAA29613.1, hexokinase type IV), Homo sapiens (H. sapiens, NP_000153.1, glucokinase isoform 1), Rattus norvegicus (R. norvegicus, NP_036866.1, the N-terminal half of rat hexokinase-1) and Mus musculus (M. musculus, NP_034422.2, glucokinase) (Figure 1A)
Summary
Human clonorchiasis is caused by infection with Clonorchis sinensis (C. sinensis), the Chinese or oriental liver fluke, which is mainly distributed in various eastern parts of Asia, including China, Korea, and Northern Vietnam. 35 million people suffer from infection with C. sinensis worldwide, of whom 15 million are Chinese [1]. Because the available fecal examination, treatment of patients with praziquantel (PZQ) and interrupting transmission provide limited abilities to prevent the infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. The genome and transcriptome data of C. sinensis demonstrate that the genes that are involved in both the glycolytic pathway and Krebs cycle are expressed during the infection [6,7]. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development [10,11]
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