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研究生(外文):Shang-Chiung Chen
論文名稱(外文):Cloning and Protein Structures of a Galactose-Binding Protein and a Lipopolysaccharide-Binding Protein from the Hemolymph of Taiwanese Horseshoe Crab, Tachypleus tridentatus
指導教授(外文):Teh-Yung Liu
外文關鍵詞:horseshoe crablectininnate immunity
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經由親和層析管柱,我在鱟血清中分離出兩個新的外源凝集素,並且從肝胰腺中成功的將其基因找出來,分別將它們命名為半乳糖結合蛋白(galactose-binding protein) 和脂多醣結合蛋白 (LPS-binding protein)。半乳糖結合蛋白和脂多醣結合蛋白分別由232和128個胺基酸所構成的,兩者均為醣蛋白。由電泳分析可看出這兩個蛋白在血清中是以聚合體的方式存在,這暗示著這兩個蛋白在免疫系統上扮演的角色,可能是經由其聚合體的結構來將入侵者限制住,進而引導其他的蛋白或酵素將其摧毀。

The immune system of animals has evolved under the pressure imposed by infectious microorganisms. As a result, all multicellular organisms have developed some kind of defense mechanisms that can be triggered by infection to protect the host by destroying the invading microbes and neutralizing the virulence factors. In contrast to the adaptive immune system in having a repertoire of specific antigen receptors and antibodies, the phylogenetically ancient defense mechanism, known as innate immune system, use germline-encoded receptors for recognition of common antigens on the surface of microbial pathogens. This feature distinguishes the innate immune system found in invertebrate from the adaptive immune system of the vertebrate. In this study, horseshoe crab, Tachypleus tridentatus, also known as living fossil, was used as a model to investigate the innate immune system. Because horseshoe crabs have existed for over 500 million years without any significant change in morphology, their defense mechanism must be a quite extraordinary one.
Two proteins with lectin-like activities, the galactose-binding protein (GBP), and the lipopolysaccharide-binding protein (LBP), and the respective genes were isolated and characterized. Each family of multi-genes that are highly homologous, codes for GBP and LBP. GBP binds tightly to Sepharose CL-4B, and was eluted with buffer containing 0.4 M GlcNAc. The deduced amino acid sequence of GBP consisted of 232 amino acids with an N-glycosylation site, Asn-Gly-Ser at residues 74-76 and shared a 65% identity and similar internal repeats of about 20 amino acid motif punctuated with Trp to tachylectin-1. Tachylectin-1 was identified as an agarose and dextran-binding non-glycosylated intracellular protein from the amebocyte granules of Tachypleus tridentatus.
LBP was eluted from the LPS-column in buffer containing 0.4 M GlcNAc and 2 M KCl. The deduced amino acid sequence of LBP consisted of 128 amino acids with an N-glycosylation site Asn-Cys-Thr, at positions 3-5. It shares 80% sequence identity with tachylectin-3, isolated from the amebocyte of Tachypleus tridentatus. While LBP is a glycoprotein with an apparent molecular mass of about 36 kDa under SDS-PAGE, Tachylectin-3 is an intracellular non-glycosylated protein with an apparent molecular mass about 14 kDa. It recognizes the O-antigen of LPS and the blood group A antigen.
The results obtained in this study support the view that pattern recognition molecules such as the GBP and the LBP have the propensity to form intermolecular dimer/oligomer and possibly heteromultimer to entrap the invading pathogens, which could rival the diversity of the immunoglobulins of the adaptive immune system.

中文摘要 2

1. Fearon D.T., Locksley R.M. (1996) The instructive role of innate immunity in acquired immune response. Science 272, 50-54
2. Lemaitre B., Nicolas E., Michaut L., Reichhart J.M., Hoffmann J.A. (1996) The drosoventral regulatory gene cassette Spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-983
3. Carroll M.C., Prodeus A.P. (1998) Linkages of innate and adaptive immunity. Curr. Opin. Immunol. 10, 36-40
4. Taylor P., Botto M., Walport M. (1998) The complement system. Curr Biol 8, R259-61
5. Janeway C.A. Jr. (1989) Cold Spring Harbor Symp. Quant. Biol. 54, 1-13
6. Medzhitov R., Janeway C.A. Jr. (1997) Innate immunity: the virtues of a nonclonal system of recognition. Cell 91, 295-298
7. Drickmer K. (1988) Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem. 263, 9557-9560
8. Friis-Christinansen P., Thiel S., Svehag S., Dessau R., Svendsen P., Andersen O., Laursen S.B., Jensenius J.C. (1990) In vivo and in vitro antibacterial activity of conglutinin, a mammalian plasma lectin. Scand. J. Immunol. 31, 453-460
9. Lu J., Willis A.C., Reid K.B.M. (1992) Purification, characterization and cDNA cloning of human lung surfactant protein D. Biochem. J. 284, 795802
10. Drickmer K. (1992) Engineering galactose-binding activity into a C-type mannose-binding protein. Nature 360, 183-186
11. Avni O., Pur Z., Yefenof E., Baniyash M. (1998) Complement receptor 3 of macrophages is associated with galectin-1-like protein. J. Immunol. 160, 6151-6158
12. Tanaka K., Nakamura T., Ichihara A. (1989) Structural requirements of lipid A for endotoxicity and other biological activities. CRC Crit. Rev. Microbiol. 16, 477-523
13. Gutierrez J.C., Bluethmann H. (1997) Molecules and mechanisms operating in septic shock: lessons from knockout mice. Immunol. Today 18, 329-334
14. Fenton M.J., Golenbock D.T. (1998) LPS-binding proteins and receptors. J. Leukoc. Biol. 64, 25-32
15. Frey E.A., Miller D.S., Jahr T.G., Sundan A., Bazil V. Espevik T., Finlay B.B., Wright S.D. (1992) Soluble CD14 participates in the response of cells to lipopolysaccharide. J. Exp. Med. 176, 1665-1671
16. Pugin J., Schurer-Maly C.C., Leturcq D., Moriarty A., Ulevitch R.J., Tobias P.S. (1993) Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc. Natl. Acad. Sci. USA 90 (7), 2744-27748
17. Iwanaga S. (1993) The limulus clotting reaction. Curr. Opin. Immunol. 5, 74-82
18. Muta T., Iwanaga S. (1996) The role of hemolymph coagulation in innate immunity. Curr. Opin. Immunol. 8, 41-47
19. Iwanaga S., Kawabata S., Muta T. (1998) New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. J. Biochem. 123, 1-15
20. Muta T., Seki N., Takaki Y., Hashimoto R., Oda T., Iwanaga A.,Tokunaga F., Iwanaga S. (1995) Purified Horseshoe Crab Factor G. J. Biol. Chem. 270, 892-897
21. Muta T., Tokunaga F., Nakamura T., Morita T., Iwanaga S., Methods Enzymol. 223, 336-345 (1993)
22. Fortes-Dias, C. L., Minetti, C.A.S., Lin, Y., and Liu, T.-Y., ( 1993) Agglutination Activity of Limulus Polyphemus Coagulogen Following Limittted Proteolysis. Comp. Biochem. Physiol. 105, 79-85.
23. Liu T., Lin Y., Cislo T., Minetti C.A.S.A, Baba J.M.K., Liu T.Y. (1991) Limunectin. A phosphocholine-binding protein from Limulus amebocytes with adhesion-promoting properties. J. Biol. Chem. 266, 14813-14821
24. Enghild J.J., Thogersen I.B., Salvesen G., Fey G.H., Figler N.L., Gonias S.L., Pizzo S.V. (1990) a-macroglobulin from Limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system. Biochemistry 29, 10070-10080
25. Maniatis T., Fritsch E.F., Sambrook J., Molecular Cloning: A Laboratory Manual (1982), P. 280, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY
26. Saito S., Hatada M., Iwanaga S., Kawabata S. (1997) A Newly Identified Horseshoe Crab Lectin with Binding Specificity to O-antigen of Bacterial Lipopolysaccharides. J. Biol. Chem. 272, 30703-30708
27. Mer E.H., Levin J., Holmer R. (1975) Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab. J. Cell. Physiol. 86, 533-42
28. Kapitany R.A., Zebrowski E.J. (1973) A high resolution PAS stain for polyacrylamide gel electrophoresis. Anal. Biochem. 56, 361-369
29. Furguson M.A.J. (1993) Glycobiology. A Practical Approach (Fukuda M. and Kobata A., eds) P. 349-383, IRL Press, Oxford
30. Chiou S.T., Chen Y.W., Chen S.C., Chao C.F, Liu T.Y (2000) Isolation and Characterization of Proteins That Bind to Galactose, Lipopolysaccharide of Escherichia coli, and Protein A of Staphylococcus aureus from the Hemolymph of Tachypleus tridentatus. J. Biol. Chem. 275, 1630-1634
31. Saito T., Kawabata S., Hirata M., Iwanaga S. (1995) A novel type of limulus lectin-L6. Purification, primary structure, and antibacterial activity. J. Biol. Chem. 270, 14493-14499
32. Huh C.G., Aldrich J., Mottahedeh J., Kwon H., Johnson C., Marsh R. (1998) Cloning and characterization of Physarum polycephalum tectonins. Homologues of Limulus lectin L-6. J. Biol. Chem. 273, 6565-6574
33. Nagai T., Kawabata S., Shishikura F., Sugita H. (1999) Purification, Characterization, and Amino Acid Sequence of an Embryonic Lectin in Perivitelline Fluid of the Horseshoe Crab. J. Biol. Chem. 274, 37673-37678
34. Inamori K., Saito T., Iwaki D., Nagira T., Iwanaga S., Arisaka F., Kawabata S. (1999) A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides. J. Biol. Chem. 274, 3272-3278
35. Nguyen N.Y., Suzuki A., Cheng S.M., Zon G., Liu T.Y. (1986) Isolation and characterization of Limulus C-reactive protein genes. J. Biol. Chem. 261, 10450-10455
36. Iwaki D., Osaki T., Mizunoe Y., Wai S.N., Iwanaga S., Kawabata S. (1999) Functional and structural diversities of C-reactive proteins present in horseshoe crab hemolymph plasma. Eur. J. Biochem. 264, 314-326
37. Hoffmann J.A., Reichhart J.M., Hetru C. (1996) Innate immunity in higher insects. Curr. Opin. Immunol. 8, 8-13
38. Medzhitov R., Janeway C.A. Jr. (1998) An ancient system of host defense. Curr. Opin. Immunol. 10,12-15
39. Minetti, C.S.A., Lin,Y., Cislo,T., Liu, T-Y. (1991).Purification and Characterization of an Endotoxin-binding Protein with Protease Inhibitory Activity from Limulus Amebocytes. J. Biol. Chem. 266, 20773-20780.
40. Schumann, R.R., Leong, S.R., Flaggs, G. W., Gray, P. W., Wright, S.D., Mathison, J. C., Tobias, P.S., and Ulevitch, R. J., (1990). Structure and function of lipopolysaccharide binding protein. Science 249, 1429-1431.

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