臺灣博碩士論文加值系統

CC類型的趨化素CCL5與其受體CCR5之間的結合為兩步驟的機制，兩者作用影響造血幹細胞的下游信號，同時在眾多免疫疾病中扮演重要角色。以前的研究提到，受體CCR5中包含硫酸化酪胺酸殘基的N端，對於與CCL5的第一步結合是必要的，這顯示酪胺酸硫酸化是不可或缺的重要修飾。另一方面，具有多個硫酸根的醣類，肝素，對於CCL5在細胞表面的信號傳遞提供另類的協助。由CCR5、肝素與CCL5的作用中發現，硫酸根基團對於調控CCL5的功能調節可能很重要。因此，為了釐清不同的帶硫酸根分子與CCL5作用上的差異，我們進行了核磁共振滴定實驗來評估硫酸根離子、硫酸化酪胺酸與帶硫酸根的肝素低聚物在CCL5溶液中的影響，藉由觀察化學位移變動，我們發現不同的帶硫酸根分子結合位，表示CCL5與不同的帶硫酸根分子間有結合特異性。這些研究結果提供了硫酸化在不同分子中調控趨化素功能的結構基礎，並對於CCL5與肝素、CCR5間進一步的研究提供相關資訊。

CC-type chemokine CCL5 binds to chemokine receptor CCR5 in a two-step mechanism. This interaction derives the downstream signaling in hematopoietic cells, and the molecule plays a crucial role in several immune system pathologies. Previous study established CCR5 N-terminal fragment containing sulfated tyrosine residues is essential for CCL5 recruitment. The step is defined as the first step of CCR5 binding. In addition, the highly sulfated carbohydrate molecule, heparin, acts an alternative role to assist CCL5 signal delivery on cell surface. Considering the two interactions, the sulfate groups could be important for regulating CCL5 functions. Therefore, we want to clarify the discrepancy among the different sulfated molecules in binding with CCL5. We firstly performed NMR titration experiments to evaluate the effects derived from sulfate ion, sulfo-tyrosine and sulfated heparin oligomers in CCL5 solution. Based on chemical shift perturbations, several sulfate-binding sites are characterized which could specifically target to the different sulfate groups. The result provides the structural basis for understanding how sulfation in different molecules could modulate the chemokine functions. The study could provide information for the further study of the interplay between CCL5, heparin and the protein receptor.

目錄
中文摘要 ................................................................................................................................... 1
Abstract ..................................................................................................................................... 2
中英對照 ................................................................................................................................... 3
縮寫 ........................................................................................................................................... 4
第一章、 前言 ..................................................................................................................... 5
1.1 趨化素 ........................................................................................................................... 5
1.2 趨化素與GPCRs和GAGs之作用 ............................................................................... 6
1.2.1趨化素和GPCRs ......................................................................................................... 6
1.2.2趨化素和GAGs ........................................................................................................... 7
1.3 CCL5 ............................................................................................................................... 8
1.3.1 CCL5與CCR5 ............................................................................................................ 9
1.3.2 CCL5與GAGs .......................................................................................................... 11
1.4 Intein .............................................................................................................................. 12
1.5 研究目的 ....................................................................................................................... 13
第二章、材料與方法 ............................................................................................................. 23
2.1 蛋白表現與純化.......................................................................................................... 23
2.1.1 CCL5-E66S表現與純化............................................................................................ 23
2.1.2 Nt-CCR5表現與純化 ............................................................................................... 24
2.2 核磁共振實驗樣品製備 .............................................................................................. 25
2.3 核磁共振15N-1H二維光譜滴定實驗 ......................................................................... 25
2.3.1 CCL5-E66S 與硫酸根 .............................................................................................. 25
2.3.2 CCL5-E66S 與硫酸化酪胺酸 (Sulfotyrosine) ......................................................... 25
2.3.3 CCL5-E66S 與肝素 (Heparin) 四糖 .......................................................................... 26
2.3.4 CCL5-E66S 與Nt-CCR5 ........................................................................................... 26
第三章、結果 ......................................................................................................................... 27
3.1 CCL5-E66S的純化 ...................................................................................................... 27
II
3.2 CCL5-E66S與硫酸根的1H-15N二維光譜滴定實驗 .................................................. 27
3.3 CCL5-E66S與硫酸化酪胺酸的1H-15N二維光譜滴定實驗 ...................................... 28
3.4 CCL5-E66S與肝素四醣的1H-15N二維光譜滴定實驗 .............................................. 29
3.5 CCL5-E66S與Nt-CCR5的1H-15N二維光譜滴定實驗 ............................................. 30
第四章、討論 ......................................................................................................................... 47
4.1 CCL5-E66S與各種帶硫酸根分子之作用 ................................................................... 47
4.1.1 CCL5與硫酸根之作用探討...................................................................................... 47
4.1.2 CCL5與硫酸化酪胺酸之作用探討 .......................................................................... 47
4.1.3 CCL5與GAGs之作用探討...................................................................................... 49
4.2 建立表現硫酸化Nt-CCR5的平台 .............................................................................. 50
參考文獻 ................................................................................................................................. 56

1. Dayanidhi Raman, T.S.-D.a.A.R., Chemokines in health and disease. Experimental cell research, 2011. 317(5): p. 575–589.
2. Amanda E. I. Proudfoot, T.M.H., Zoe Johnson, Elaine K. Lau, Patricia LiWang et al., Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. PNAS, 2003. 100(4): p. 1885-1890.
3. Philip M. Murphy, M.B., Israel F. Charo, Caroline A. Hébert, Richard Horuk et al., International Union of Pharmacology. XXII. Nomenclature for Chemokine Receptors. Pharmacological Reviews, 2000. 52(1): p. 145-176.
4. Lolis, E.J.F.a.E., Structure, Function, and Inhibition of Chemokines. Annual Review of Pharmacology and Toxicology, 2002. 42: p. 469-499.
5. Kazunobu Tachibana, S.H., Hisashi Iizasa, Hisahiro Yoshida, Kenji Kawabata et al., The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature, 1998. 393(6685): p. 591-594.
6. Jessica L. Williams, D.W.H.a.R.S.K., Chemokines in the balance: maintenance of homeostasis and protection at CNS barriers. Frontiers in Cellular Neuroscience, 2014. 8: p. 154-165.
7. Ruiz, E.J., F. Oeztuerk-Winder, and J.J. Ventura, A paracrine network regulates the cross-talk between human lung stem cells and the stroma. Nat Commun, 2014. 5: p. 3175.
8. Cheng, W.J. and G.J. Chen, Chemokines and Chemokine Receptors in Multiple Sclerosis. Mediators of Inflammation, 2014. 2014.
9. Keqiang Chen, Z.B., Peng Tang, Wanghua Gong, Teizo Yoshimura and Ji Ming Wang, Chemokines in homeostasis and diseases. Cellular and Molecular Immunology, 2018. 15(4): p. 324-334.
10. Hoogewerf, A.J., et al., Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry, 1997. 36(44): p. 13570-13578.
11. Campanella, G.S.V., et al., Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity. Journal of Immunology, 2006. 177(10): p. 6991-6998.
12. Ellyard, J.I., et al., Eotaxin selectively binds heparin - An interaction that protects eotaxin from proteolysis and potentiates chemotactic activity in vivo. Journal of Biological Chemistry, 2007. 282(20): p. 15238-15247.
13. Steinke, L.C.B.a.J.W., Cytokines and chemokines. Journal of Allergy and Clinical Immunology, 2003. 111: p. 460-475.
14. Rajagopalan, L. and K. Rajarathnam, Structural basis of chemokine receptor function - A model for binding affinity and ligand selectivity. Bioscience Reports, 2006. 26(5): p. 325-339.
15. Crump, M.P., et al., Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. Embo Journal, 1997. 16(23): p. 6996-7007.
16. Qin, L., et al., Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine. Science, 2015. 347(6226): p. 1117-1122.
17. Ludeman, J.P. and M.J. Stone, The structural role of receptor tyrosine sulfation in chemokine recognition. British Journal of Pharmacology, 2014. 171(5): p. 1167-1179.
18. Kessler, N., et al., Allovalency observed by transferred NOE: interactions of sulfated tyrosine residues in the N-terminal segment of CCR5 with the CCL5 chemokine. Febs Journal, 2021. 288(5): p. 1648-1663.
19. Millard, C.J., et al., Structural Basis of Receptor Sulfotyrosine Recognition by a CC Chemokine: The N-Terminal Region of CCR3 Bound to CCL11/Eotaxin-1. Structure, 2014. 22(11): p. 1571-1581.
20. Christopher T. Veldkamp, C.S., Francis C. Peterson, Thomas P. Sakmar, and Brian F. Volkman, Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1α (SDF-1α/CXCL12). Journal of Molecular Biology, 2006. 359(5): p. 1400-1409.
21. Handel, T.M., et al., Regulation of protein function by glycosaminoglycans - as exemplified by chemokines. Annual Review of Biochemistry, 2005. 74: p. 385-410.
22. Proudfoot, A.E.I., et al., Glycosaminoglycan Interactions with Chemokines Add Complexity to a Complex System. Pharmaceuticals, 2017. 10(3): p. 70-94.
23. Thompson, S., et al., Regulation of Chemokine Function: The Roles of GAG-Binding and Post-Translational Nitration. International Journal of Molecular Sciences, 2017. 18(8): p. 1692-1708.
24. Proudfoot, A.E.I., et al., The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity. Journal of Biological Chemistry, 2001. 276(14): p. 10620-10626.
25. Lau, E.K., et al., Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo. J. Biol. Chem., 2004. 279(21): p. 22294-305.
26. Kuschert, G.S.V., et al., Identification of a glycosaminoglycan binding surface on human interleukin-8. Biochemistry, 1998. 37(32): p. 11193-11201.
27. Crijns, H., V. Vanheule, and P. Proost, Targeting Chemokine-Glycosaminoglycan Interactions to Inhibit Inflammation. Frontiers in Immunology, 2020. 11.
28. Salanga, C.L., et al., Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable It to Function as a Non-oligomerizing Chemokine. Journal of Biological Chemistry, 2014. 289(21): p. 14896-14912.
29. Schall, T.J., et al., A Human T-Cell-Specific Molecule Is a Member of a New Gene Family. Journal of Immunology, 1988. 141(3): p. 1018-1025.
30. Aldinucci, D. and A. Colombatti, The Inflammatory Chemokine CCL5 and Cancer Progression. Mediators of Inflammation, 2014. 2014.
31. Krensky, A.M. and Y.T. Ahn, Mechanisms of disease: regulation of RANTES (CCL5) in renal disease. Nat Clin Pract Nephrol, 2007. 3(3): p. 164-170.
32. Arrildt, K.T., S.B. Joseph, and R. Swanstrom, The HIV-1 Env Protein: A Coat of Many Colors. Current Hiv/Aids Reports, 2012. 9(1): p. 52-63.
33. Eric de Silva, M.P.H.S., HIV and the CCR5-Δ32 resistance allele. FEMS Microbiology Letters 2004. 241(1): p. 1-12.
34. Wilken, J., et al., Total chemical synthesis and high-resolution crystal structure of the potent anti-HIV protein AOP-RANTES. Chem Biol, 1999. 6(1): p. 43-51.
35. Mosier, D.E., et al., Highly potent RANTES analogues either prevent CCR5-using human immunodeficiency virus type 1 infection in vivo or rapidly select for CXCR4-using variants. Journal of Virology, 1999. 73(5): p. 3544-3550.
36. Kawamura, T., et al., PSC-RANTES blocks R5 human immunodeficiency virus infection of Langerhans cells isolated from individuals with a variety of CCR5 diplotypes. J Virol, 2004. 78(14): p. 7602-7609.
37. Jin, H., et al., Structural and functional studies of the potent anti-HIV chemokine variant P2-RANTES. Proteins, 2010. 78(2): p. 295-308.
38. Gaertner, H., et al., Highly potent, fully recombinant anti-HIV chemokines: reengineering a low-cost microbicide. PNAS, 2008. 105(46): p. 17706-17711.
39. Zheng, Y., et al., Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV. Immunity., 2017. 46(6): p. 1005-1017.
40. Isaikina, P., et al., Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist. Sci Adv, 2021. 7(25).
41. Zhang, H., et al., Structural basis for chemokine recognition and receptor activation of chemokine receptor CCR5. Nat Commun, 2021. 12(1): p. 4151-4162.
42. Maeda, S., et al., Development of an antibody fragment that stabilizes GPCR/G-protein complexes. Nat. Commun., 2018. 9.
43. Duma, L., et al., Recognition of RANTES by extracellular parts of the CCR5 receptor. J Mol Biol, 2007. 365(4): p. 1063-1075.
44. Abayev, M., et al., The solution structure of monomeric CCL5 in complex with a doubly sulfated N-terminal segment of CCR5. FEBS J, 2018. 285(11): p. 1988-2003.
45. Cardin, A.D. and H.J.R. Weintraub, Molecular Modeling of Protein-Glycosaminoglycan Interactions. Arteriosclerosis, 1989. 9(1): p. 21-32.
46. Shaw, J.P., et al., The X-ray structure of RANTES: heparin-derived disaccharides allows the rational design of chemokine inhibitors. Structure, 2004. 12(11): p. 2081-2093.
47. Martin, L., et al., Structural and functional analysis of the RANTES-glycosaminoglycans interactions. Biochemistry, 2001. 40(21): p. 6303-6318.
48. Vives, R.R., et al., A kinetics and modeling study of RANTES(9-68) binding to heparin reveals a mechanism of cooperative oligomerization. Biochemistry, 2002. 41(50): p. 14779-14789.
49. Liang, W.G., et al., Structural basis for oligomerization and glycosaminoglycan binding of CCL5 and CCL3. PNAS, 2016. 113(18): p. 5000-5005.
50. Deshauer, C., et al., Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands. Structure, 2015. 23(6): p. 1066-1077.
51. Singh, A., et al., The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH. Journal of Biological Chemistry, 2015. 290(25): p. 15421-15436.
52. Perler, F.B., et al., Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res, 1994. 22(7): p. 1125-1127.
53. Shih, C.K., et al., A Dominant Trifluoperazine Resistance Gene from Saccharomyces-Cerevisiae Has Homology with F0f1 Atp Synthase and Confers Calcium-Sensitive Growth. Molecular and Cellular Biology, 1988. 8(8): p. 3094-3103.
54. Mills, K.V., M.A. Johnson, and F.B. Perler, Protein Splicing: How Inteins Escape from Precursor Proteins. Journal of Biological Chemistry, 2014. 289(21): p. 14498-14505.
55. Aranko, A.S. and H. Iwai, The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins. Molecules, 2021. 26(19): p. 5948-5960.
56. Wood, D.W., et al., A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol, 1999. 17(9): p. 889-892.
57. Oeemig, J.S., et al., Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site-specific chemical modification. Febs Letters, 2009. 583(9): p. 1451-1456.
58. Balkwill, F., Cancer and the chemokine network. Nat Rev Cancer, 2004. 4(7): p. 540-550.
59. Delaglio, F., et al., NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR, 1995. 6(3): p. 277-293.
60. Lee, W., M. Tonelli, and J.L. Markley, NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics, 2015. 31(8): p. 1325-1327.
61. Prem Raj B. Joseph, K.M.P., Krishna Mohan Sepuru, and Krishna Rajarathnam, Characterizing protein-glycosaminoglycan interactions using solution NMR Spectroscopy. Methods Mol Biol., 2015: p. 325–333.
62. Joseph, P.R., et al., Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions. Biochem J., 2015. 472(1): p. 121-133.
63. Ziarek, J.J., et al., Sulfotyrosine Recognition as Marker for Druggable Sites in the Extracellular Space. International Journal of Molecular Sciences, 2011. 12(6): p. 3740-3756.
64. Schwessinger, B., et al., A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection. Integr Biol (Camb), 2016. 8(4): p. 542-545.