title: Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme.
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.
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Tingjin
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Ning
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Hengchang
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Shang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Xuerong
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Chi
authors.initials: C
authors.lastname: Liang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Wenfang
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Mao
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Chuanhuan
authors.initials: C
authors.lastname: Deng
authors.affiliation: The Third Affiliated hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
authors.forename: Lexun
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Zhongdao
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China; Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong, China.
authors.forename: Xinbing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003004:Clonorchis sinensis / Q000201:enzymology*; D017124:Conserved Sequence; D015870:Gene Expression; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011223:Praziquantel / Q000737:chemistry; D020836:Protein Structure, Quaternary; D017422:Sequence Analysis, DNA
chemical_list: D015801:Helminth Proteins; D011223:Praziquantel; D006593:Hexokinase