進階搜尋


 
系統識別號 U0026-0812200912114020
論文名稱(中文) 斑馬魚絲胺酸羥基甲基轉移酶之選殖、表現、純化以及特性分析
論文名稱(英文) Cloning, expression,purification and characterization of zebrafish serine hydroxymethyltransferase
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 張文妮
研究生(英文) Wen-Ni Chang
學號 t3693106
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 指導教授-傅子芳
口試委員-謝淑珠
口試委員-蔡振寧
口試委員-黃暉升
中文關鍵字 斑馬魚  葉酸  絲胺酸羥基甲基轉移酶 
英文關鍵字 serine hydroxymethyltransferase  folate  zebrafish 
學科別分類
中文摘要 細胞中的葉酸-單碳循環參與體內許多重要物質的生合成,包括核苷酸、胺基酸、神經傳導物、以及硫化腺嘌呤甲硫胺酸(S-adenosylmethionine)。在此一循環中,絲胺酸羥基甲基轉移酶(serine hydroxymethyltransferase, SHMT, EC 2.1.2.1) 被認為具有催化與調控的功能。絲胺酸羥基甲基轉移酶催化四氫葉酸(tetrahydrofolate)與5,10-甲烯基四氫葉酸(5,10-methylenetetrahydrofolate)及絲胺酸(serine)與甘胺酸(glycine)之間可逆性反應。SHMT的含量被發現在腫瘤細胞中有上升的情形。在大多數的多細胞生物中發現有一種形式以上的絲胺酸羥基甲基轉移酶存在:細胞質型和另一種胞器相關型,通常為粒線體型。許多不同種系生物的絲胺酸羥基甲基轉移酶已被廣泛的研究,但斑馬魚絲胺酸羥基甲基轉移酶卻從未被研究。斑馬魚是近數十年新掘起的一種實驗動物模型。為了評估斑馬魚是否為一個適合做為葉酸相關研究的動物模型,我們必須先了解斑馬魚的葉酸-單碳循環代謝以及其與人類之間的異同。因此我們首先著手於此代謝循環中核心酵素--絲胺酸羥基甲基轉移酶的選殖與研究。在本論文中,斑馬魚細胞質型(zcSHMT)和粒線體型(zmSHMT)的完整編碼序列被選殖並送入表現載體中。進行酵素的表現、純化以及特性和功能分析。同時對此酵素在細胞內表現位點以及組織專一性分佈進行探討。酵素動力學分析結果發現這兩種斑馬魚絲胺酸羥基甲基轉移酶和哺乳類動物的十分相似。而且這兩種絲胺酸羥基甲基轉移酶主要都表現在肝臟和腸胃道。共軛焦顯微鏡觀察結果和細胞免疫染色意外發現,細胞質型絲胺酸羥基甲基轉移酶亦出現在細胞核。意喻著細胞質型絲胺酸羥基甲基轉移酶除了其原本在細胞質代謝中所扮演的角色外,可能還有其他的功能。進一步確認這個結果的實驗正在進行中。



英文摘要 Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) mediates the principle pathway for incorporation of one-carbon units into cells by reversibly converting serine and H4PteGlu to glycine and 5,10-CH2-H4PteGlu which is required for biosynthesis of many important molecules including nucleotides, amino acid, neurotransmitters and S-adenosyl methionine. The level of SHMT is also found to be elevated in tumor cells. In higher organisms more than one SHMT isoforms are often present: a cytosolic isoform and an organelle-associated form, usually mitochondria. SHMT is present in virtually all cell types and has been widely studied in many living systems except in zebrafish, a newly risen and prominent animal model in laboratory. To evaluate the similarity of folate-mediated one-carbon metabolism between zebrafish and human is the prerequisite for using zebrafish as an animal model for folate-related research and diseases. Performing a thorough study on SHMT, the key enzyme of this pathway, is our first step toward this goal. In this study, complete encoding sequences of putative zebrafish cytosolic (zcSHMT) and mitochondrial SHMTs (zmSHMT) were cloned into expression vectors. Enzymes were expressed, purified and characterized for their properties and functions. Intracellular localization and tissue-specific distribution of both isoforms were also analyzed. The kinetic properties of both enzymes suggest strong similarity between these enzymes and their mammalian orthologs. Moreover, both enzymes express predominantly in liver and GI tract. Surprisingly, confocal microscopy reveals that zcSHMT is also present in nuclear, suggesting additional roles of this protein in nuclear beside its metabolic activity in cytosol. Experiments to further confirm the finding are in progress.

論文目次 一、 圖目錄 2
二、 表目錄 3
三、 背景 4 ~ 9
四、 研究材料及方法 10 ~ 20
五、 結果 21 ~ 27
六、 討論 28 ~ 29
七、 參考文獻 30 ~ 36
八、 圖 37 ~ 49
九、 表格 50 ~ 52
十、 附件 53 ~ 58
十一、 自述 59




參考文獻 Agrawal, S., A. Kumar, V. Srivastava and B. N. Mishra, Cloning, expression, activity and folding studies of serine hydroxymethyltransferase: a target enzyme for cancer chemotherapy, J. Mol. Microbiol. Biotechnol., 6(2), 67-75, 2003.
Appling, D. R., Compartmentation of folate-mediated one-carbon metabolism in eukaryotes, FASEB J., 5(12), 2645-2651, 1991.
Bower, C. and F. J. Stanley, Dietary folate as a risk factor for neural-tube defects: evidence from a case-control study in Western Australia, Med. J. Aust., 150(11), 613-619, 1989.
Brody T, Shane B and Stokstad ELR, Folic acid. In:Machlin LJ, ed. Handbook of vitamins., pp. 459-496, Marcel Deller, New York, 1984.
Burns, R. A. and N. Jackson, Time-course studies on the effects of oestradiol administration on the activity of some folate-metabolizing enzymes in chicken liver, Comp Biochem. Physiol B, 71(3), 351-355, 1982.
Chaturvedi, S. and V. Bhakuni, Unusual structural, functional, and stability properties of serine hydroxymethyltransferase from Mycobacterium tuberculosis, J. Biol. Chem., 278(42), 40793-40805, 2003.
Chen, M. S. and L. V. Schirch, Serine transhydroxymethylase. Studies on the role of tetrahydrofolate, J. Biol. Chem., 248(23), 7979-7984, 1973.
Chomczynski, P. and N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162(1), 156-159, 1987.
Chu, E., D. M. Koeller, J. L. Casey, J. C. Drake, B. A. Chabner, P. C. Elwood, S. Zinn and C. J. Allegra, Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase, Proc. Natl. Acad. Sci. U. S. A, 88(20), 8977-8981, 1991.
Chu, E., C. H. Takimoto, D. Voeller, J. L. Grem and C. J. Allegra, Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro, Biochemistry, 32(18), 4756-4760, 1993a.
Chu, E., D. Voeller, D. M. Koeller, J. C. Drake, C. H. Takimoto, G. F. Maley, F. Maley and C. J. Allegra, Identification of an RNA binding site for human thymidylate synthase, Proc. Natl. Acad. Sci. U. S. A, 90(2), 517-521, 1993b.
Cossins EA, Folates in biological materials. In: Blakely RL, Benkovic SJ, eds. Folates and pterins: Chemistry and biochemistry of folate., pp. 1-60, John Wiley & Sons, New York, 1984.
Dasgupta, P. and A. Narayanaswami, Serine transhydroxymethylase activity in vertebrate retina, J. Neurochem., 39(3), 743-746, 1982.
Davis, R. E., Clinical chemistry of folic acid, Adv. Clin. Chem., 25, 233-294, 1986.
di Salvo, M. L., F. S. Delle, B. D. De, F. Bossa and V. Schirch, Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase, Protein Expr. Purif., 13(2), 177-183, 1998.
Don, R. H., P. T. Cox, B. J. Wainwright, K. Baker and J. S. Mattick, 'Touchdown' PCR to circumvent spurious priming during gene amplification, Nucleic Acids Res., 19(14), 4008, 1991.
Finkelstein, J. D., Methionine metabolism in mammals, J. Nutr. Biochem., 1(5), 228-237, 1990.
Fu, T. F., E. S. Boja, M. K. Safo and V. Schirch, Role of proline residues in the folding of serine hydroxymethyltransferase, J. Biol. Chem., 278(33), 31088-31094, 2003.
Gill, S. C. and P. H. von Hippel, Calculation of protein extinction coefficients from amino acid sequence data, Anal. Biochem., 182(2), 319-326, 1989.
Girgis, S., I. M. Nasrallah, J. R. Suh, E. Oppenheim, K. A. Zanetti, M. G. Mastri and P. J. Stover, Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene, Gene, 210(2), 315-324, 1998d.
Girgis, S., I. M. Nasrallah, J. R. Suh, E. Oppenheim, K. A. Zanetti, M. G. Mastri and P. J. Stover, Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene, Gene, 210(2), 315-324, 1998a.
Girgis, S., I. M. Nasrallah, J. R. Suh, E. Oppenheim, K. A. Zanetti, M. G. Mastri and P. J. Stover, Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene, Gene, 210(2), 315-324, 1998b.
Girgis, S., I. M. Nasrallah, J. R. Suh, E. Oppenheim, K. A. Zanetti, M. G. Mastri and P. J. Stover, Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene, Gene, 210(2), 315-324, 1998c.
Girgis, S., J. R. Suh, J. Jolivet and P. J. Stover, 5-Formyltetrahydrofolate regulates homocysteine remethylation in human neuroblastoma, J. Biol. Chem., 272(8), 4729-4734, 1997.
Grunwald, D. J. and J. S. Eisen, Headwaters of the zebrafish -- emergence of a new model vertebrate, Nat. Rev. Genet., 3(9), 717-724, 2002.
Hall, C. A. and R. C. Chu, Serum homocysteine in routine evaluation of potential vitamin B12 and folate deficiency, Eur. J. Haematol., 45(3), 143-149, 1990.
Heil, S. G., N. M. Van der Put, E. T. Waas, H. M. den, F. J. Trijbels and H. J. Blom, Is mutated serine hydroxymethyltransferase (SHMT) involved in the etiology of neural tube defects?, Mol. Genet. Metab, 73(2), 164-172, 2001.
Herbig, K., E. P. Chiang, L. R. Lee, J. Hills, B. Shane and P. J. Stover, Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses, J. Biol. Chem., 277(41), 38381-38389, 2002.
HIBBARD, B. M., THE ROLE OF FOLIC ACID IN PREGNANCY; WITH PARTICULAR REFERENCE TO ANAEMIA, ABRUPTION AND ABORTION, J. Obstet. Gynaecol. Br. Commonw., 71, 529-542, 1964.
J.Sambrook, T.Fritsch and T.Maniatis, Molecular cloning. A labratory Manual, Cold Spring Harbor Labratory, Cold Spring Harbor, NY, 1987.
Kim, Y. I., Folate and carcinogenesis: evidence, mechanisms, and implications, J. Nutr. Biochem., 10(2), 66-88, 1999.
Kimmel, C. B., W. W. Ballard, S. R. Kimmel, B. Ullmann and T. F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn., 203(3), 253-310, 1995.
Kruschwitz, H., S. Ren, S. M. Di and V. Schirch, Expression, purification, and characterization of human cytosolic serine hydroxymethyltransferase, Protein Expr. Purif., 6(4), 411-416, 1995.
Liu, X., B. Reig, I. M. Nasrallah and P. J. Stover, Human cytoplasmic serine hydroxymethyltransferase is an mRNA binding protein, Biochemistry, 39(38), 11523-11531, 2000.
Mason, J. B. and T. Levesque, Folate: effects on carcinogenesis and the potential for cancer chemoprevention, Oncology (Williston. Park), 10(11), 1727-3, 1996.
McNeil, J. B., E. M. McIntosh, B. V. Taylor, F. R. Zhang, S. Tang and A. L. Bognar, Cloning and molecular characterization of three genes, including two genes encoding serine hydroxymethyltransferases, whose inactivation is required to render yeast auxotrophic for glycine, J. Biol. Chem., 269(12), 9155-9165, 1994.
Milunsky, A., H. Jick, S. S. Jick, C. L. Bruell, D. S. MacLaughlin, K. J. Rothman and W. Willett, Multivitamin/folic acid supplementation in early pregnancy reduces the prevalence of neural tube defects, JAMA, 262(20), 2847-2852, 1989.
Miranda, C. L., P. Collodi, X. Zhao, D. W. Barnes and D. R. Buhler, Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio), Arch. Biochem. Biophys., 305(2), 320-327, 1993.
Nakshatri, H., P. Bouillet, P. Bhat-Nakshatri and P. Chambon, Isolation of retinoic acid-repressed genes from P19 embryonal carcinoma cells, Gene, 174(1), 79-84, 1996.
Narkewicz, M. R., S. D. Sauls, S. S. Tjoa, C. Teng and P. V. Fennessey, Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells, Biochem. J., 313 ( Pt 3), 991-996, 1996.
Nikiforov, M. A., S. Chandriani, B. O'Connell, O. Petrenko, I. Kotenko, A. Beavis, J. M. Sedivy and M. D. Cole, A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism, Mol. Cell Biol., 22(16), 5793-5800, 2002.
Pfendner, W. and L. I. Pizer, The metabolism of serine and glycine in mutant lines of Chinese hamster ovary cells, Arch. Biochem. Biophys., 200(2), 503-512, 1980.
Rassin, D. K., J. A. Sturman and G. E. Gaull, Sulfur amino acid metabolism in the developing rhesus monkey brain: subcellular studies of the methylation cycle and cystathionine beta-synthase, J. Neurochem., 36(3), 1263-1271, 1981.
Renwick, S. B., K. Snell and U. Baumann, The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer chemotherapy, Structure., 6(9), 1105-1116, 1998.
Roux, K. H., Optimization and troubleshooting in PCR, PCR Methods Appl., 4(5), S185-S194, 1995.
Sanborn, T. A., R. L. Kowle and H. J. Sallach, Regulation of enzymes of serine and one-carbon metabolism by testosterone in rat prostate, liver, and kidney, Endocrinology, 97(4), 1000-1007, 1975.
Scarsdale, J. N., G. Kazanina, S. Radaev, V. Schirch and H. T. Wright, Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications, Biochemistry, 38(26), 8347-8358, 1999.
Schirch, L., Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate, Arch. Biochem. Biophys., 189(2), 283-290, 1978.
Schirch, L., Serine hydroxymethyltransferase, Adv. Enzymol. Relat Areas Mol. Biol., 53, 83-112, 1982.
Schirch, L. and D. Peterson, Purification and properties of mitochondrial serine hydroxymethyltransferase, J. Biol. Chem., 255(16), 7801-7806, 1980.
Schirch, L. and M. Ropp, Serine transhydroxymethylase. Affinity of tetrahydrofolate compounds for the enzyme and enzyme-glycine complex, Biochemistry, 6(1), 253-257, 1967.
Schirch, L. V., C. M. Tatum, Jr. and S. J. Benkovic, Serine transhydroxymethylase: evidence for a sequential random mechanism, Biochemistry, 16(3), 410-419, 1977.
Schirch, V., S. Hopkins, E. Villar and S. Angelaccio, Serine hydroxymethyltransferase from Escherichia coli: purification and properties, J. Bacteriol., 163(1), 1-7, 1985.
Skibola, C. F., M. T. Smith, A. Hubbard, B. Shane, A. C. Roberts, G. R. Law, S. Rollinson, E. Roman, R. A. Cartwright and G. J. Morgan, Polymorphisms in the thymidylate synthase and serine hydroxymethyltransferase genes and risk of adult acute lymphocytic leukemia, Blood, 99(10), 3786-3791, 2002.
Snell, K., Y. Natsumeda, J. N. Eble, J. L. Glover and G. Weber, Enzymic imbalance in serine metabolism in human colon carcinoma and rat sarcoma, Br. J. Cancer, 57(1), 87-90, 1988.
Stabler, S. P., P. D. Marcell, E. R. Podell, R. H. Allen, D. G. Savage and J. Lindenbaum, Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry, J. Clin. Invest, 81(2), 466-474, 1988.
Steegers-Theunissen, R. P., Folate metabolism and neural tube defects: a review, Eur. J. Obstet. Gynecol. Reprod. Biol., 61(1), 39-48, 1995.
Stover, P. and V. Schirch, 5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase, J. Biol. Chem., 266(3), 1543-1550, 1991c.
Stover, P. and V. Schirch, 5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase, J. Biol. Chem., 266(3), 1543-1550, 1991b.
Stover, P. and V. Schirch, Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate, J. Biol. Chem., 265(24), 14227-14233, 1990.
Stover, P. and V. Schirch, 5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase, J. Biol. Chem., 266(3), 1543-1550, 1991a.
Stover, P. J., Physiology of folate and vitamin B12 in health and disease, Nutr. Rev., 62(6 Pt 2), S3-12, 2004.
Stover, P. J., L. H. Chen, J. R. Suh, D. M. Stover, K. Keyomarsi and B. Shane, Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene, J. Biol. Chem., 272(3), 1842-1848, 1997.
Strong, W. B. and V. Schirch, In vitro conversion of formate to serine: effect of tetrahydropteroylpolyglutamates and serine hydroxymethyltransferase on the rate of 10-formyltetrahydrofolate synthetase, Biochemistry, 28(24), 9430-9439, 1989.
Strong, W. B., S. J. Tendler, R. L. Seither, I. D. Goldman and V. Schirch, Purification and properties of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from L1210 cells, J. Biol. Chem., 265(21), 12149-12155, 1990.
Szebenyi, D. M., F. N. Musayev, M. L. di Salvo, M. K. Safo and V. Schirch, Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism, Biochemistry, 43(22), 6865-6876, 2004.
Ulevitch, R. J. and R. G. Kallen, Purification and characterization of pyridoxal 5'-phosphate dependent serine hydroxymethylase from lamb liver and its action upon beta-phenylserines, Biochemistry, 16(24), 5342-5350, 1977.
V.Schirch, Mechanism of folate-requiring enzymes in one-carbon metabolism, in:M.L. Sinnott(Ed.), Comprehensive Biological Catalysis, pp. 211-252, Acaademic Press, London, 1998.
Wang, Y. M., W. Guo, X. F. Zhang, Y. Li, N. Wang, H. Ge, L. Z. Wei, D. G. Wen and J. H. Zhang, [Correlations between serine hydroxymethyltransferase1 C1420T polymorphisms and susceptibilities to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma], Ai. Zheng., 25(3), 281-286, 2006.
Werler, M. M., S. Shapiro and A. A. Mitchell, Periconceptional folic acid exposure and risk of occurrent neural tube defects, JAMA, 269(10), 1257-1261, 1993.
Wilson, R. D., G. Davies, V. Desilets, G. J. Reid, A. Summers, P. Wyatt and D. Young, The use of folic acid for the prevention of neural tube defects and other congenital anomalies, J. Obstet. Gynaecol. Can., 25(11), 959-973, 2003.
Xu, H. M., G. Y. Zhang, X. D. Ji, L. Cao, L. Shu and Z. C. Hua, Expression of soluble, biologically active recombinant human endostatin in Escherichia coli, Protein Expr. Purif., 41(2), 252-258, 2005.


論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2006-08-30起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2006-08-30起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw