趨化因子 (chemokine) 是被分泌的微小細胞因子 (cytokine) ，參與許多生物功能，例如：細胞聚集、淋巴細胞穿透以及血管新生。CXCL4L1是近期被發現在血小板中的新穎趨化因子，為第四型血小板因子 (CXCL4) 的同源蛋白。與CXCL4相比，CXCL4L1的C端區域含有三個突變胺基酸。CXCL4L1可以形成四聚體，突變胺基酸使CXCL4L1在稀釋濃度狀況下能分離為單體。CXCL4L被觀察到具有卓越的抗血管新生功能。先前研究指出單體結構與活化CXCR3A接受體之間有相關。因此，為了得知抗血管新生的作用，需要探討CXCL4L1單體的結構特性。研究中藉由降低pH值低於3.2維持CXCL4L1的單體結構，利用核磁共振擴散排序光譜 (DOSY) 、圓二色光譜 (CD) 以及ANS結合分析法，證實CXCL4L1為類似熔球 (molten globule-like) 型態。此結構特性主要與C端區域的伸縮性有關。設計出人造的CXCL4L1-E28K單體，推測此突變體具有相似的熔球型態。因此，我們推測CXCL4L1-E28K能活化下游CXCR3A接受體，以期具有潛力的臨床用途。

Chemokines are small secreted cytokines, participating in many biological functions, such as cells recruitment, lymphoid trafficking and angiogenesis. A novel chemokine, CXCL4L1, was recently identified in platelet as a homo-logue of platelet factor 4, CXCL4. Comparing with CXCL4, CXCL4L1 con-tains three mutations in its C-terminal region. CXCL4L1 can form tetramer and the mutations make CXCL4L1 to dissociate into monomer under a dilut-ed concentration. CXCL4L1 has been noticed to have predominant function of anti-angiogenesis. Previous study showed the correlation of the mono-meric formation and the activation of the receptor, CXCR3A. Therefore, to characterize the structural property of CXCL4L1 monomer is essential for re-alizing the anti-angiogenic effect. In the study, we kept CXCL4L1 in the monomeric formation by reducing pH value below 3.2. Using NMR diffusion ordered spectroscopy (DOSY), circular dichroism (CD) and ANS binding as-say, the molten globule-like structure of CXCL4L1 was observed. The struc-tural property is mainly related with the flexibility of its C-terminal region. An artificial CXCL4L1-E28K monomer has been designed. The molten glob-ule-like structure is also detected in the mutant. CXCL4L1-E28K contains similar structure property. Therefore, we suspect that the mutant might be capable to active the downstream receptor, CXCR3A and contain a potential in clinical usage.

Contents I
Abstract III
中文摘要 IV
Abbreviations V
Introduction 1
Chemokines 1
The interaction between chemokine and G-protein coupled receptor (GPCR) 2
Chemokine and receptor model 2
The interaction between chemokines and GAG 3
Leukocyte recruitment to the site of inflammation 4
The role of chemokines in angiogenesis and cancer 4
CXCL4 (Platelet factor 4, PF4) 5
CXCL4L1 (Platelet factor 4 variant 1, PF4V1) 6
The downstream receptor of CXCL4 and CXCL4L1: CXCR3 7
CXCL4L1 tetramer dissociation 8
Motivation of this study 18
Experimental protocols 19
The expression of recombinant of CXCL4L1 19
The preparation of pellet protein 19
Refolding of CXCL4L1 20
Purification of refolded CXCL4L1 from pellet 20
8-Anilinonaphthalene-1-sulfonic acid (ANS) binding 21
Circular dichroism spectroscopy 21
Diffusion NMR Measurements 22
NMR experiments 22
Results 24
Improved protein purification for CXCL4L1 24
Identification of CXCL4L1 eluted from different concentration in NaCl gradient 25
NMR condition optimization for CXCL4L1 26
3D backbone assignment of CXCL4L1 monomer 27
CXCL4L1 mutant: CXCL4L1-E28K 29
Identification of CXCL4L1-E28K eluted from Heparin column 30
The monomer state of CXCL4L1 and its mutant confirmed by NMR 31
Comparison of CXCL4L1 monomer and CXCL4L1-E28K 31
The study of secondary structure of CXCL4L1 and its mutant by circular dichroism spectrum 32
The C-terminal helix packing difference between CXCL4L1 and its mutant 33
NMR condition optimization for CXCL4L1-E28K 33
Different conditions of CXCL4L1 and CXCL4L1-E28K samples on SDS-PAGE 34
Discussion 50
The improved purification and optimized NMR condition for CXCL4L1 50
Molten globule-like structure of CXCL4L1 monomer in acid condition 51
Importance of CXCL4L1-E28K for the research of CXCL4L1 monomer in physiological condition 51
SDS-PAGE analysis of CXCL4L1-E28K 52
Hydrophobicity surface of CXCL4 and CXCL4L1 53
Summary and future works 54
References 56

1. Behm B, Babilas P, Landthaler M, Schreml S. Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venere-ol. 2012 Jul; 26(7):812-20.
2. Kim CH. Chemokine-chemokine receptor network in immune cell traf-ficking. Curr Drug Targets Immune Endocr Metabol Disord. 2004 Dec; 4(4):343-61.
3. Gear AR, Camerini D. Platelet chemokines and chemokine receptors: linking hemostasis, inflammation, and host defense. Microcircula-tion. 2003 Jun; 10(3-4):335-50.
4. Dimberg A. Chemokines in angiogenesis. Curr Top Microbiol Immu-nol. 2010; 341: 59-80.
5. Miller MC, Mayo KH. Chemokines from a Structural Perspective. Int J Mol Sci. 2017 Oct 2; 18(10) pii: E2088.
6. Salanga CL, Handel TM. Chemokine oligomerization and interactions with receptors and glycosaminoglycans: The role of structural dynamics in function. Exp Cell Res. 2011 Mar 10; 317(5):590-601.
7. Chew AL, Tan WY, Khoo BY. Potential combinatorial effects of recombi-nant atypical chemokine receptors in breast cancer cell invasion: A re-search perspective (Review). Biomed Rep. 2013 Mar; 1(2):185-192.
8. Fernandez EJ, Lolis E. Structure, function, and inhibition of chemokines. Annu Rev Pharmacol Toxicol. 2002; 42: 469-99.
9. Patel J, Channon KM, McNeill E. The downstream regulation of chemo-kine receptor signaling: implications for atherosclerosis. Mediators In-flamm. 2013; 2013:459520.
10. Lei Sun and Richard D Ye. Role of G protein-coupled receptors in in-flammation. Acta Pharmacol Sin. 2012 Mar; 33(3): 342–350.
11. Balkwill FR. The chemokine system and cancer. J Pathol. 2012 Jan; 226(2):148-57.
12. Allen SJ, Crown SE, Handel TM. Chemokine: receptor structure, interac-tions, and antagonism. Annu Rev Immunol. 2007; 25: 787-820.
13. Kufareva I. Chemokines and their receptors: insights from molecular modeling and crystallography. Curr Opin Pharmacol. 2016 Oct; 30: 27-37.
14. Satoh A, Toida T, Yoshida K, Kojima K, Matsumoto I. New role of gly-cosaminoglycans on the plasma membrane proposed by their interaction with phosphatidylcholine. FEBS Lett. 2000 Jul 21; 477(3):249-52.
15. Yanagishita M. Function of proteoglycans in the extracellular matrix. Acta Pathol Jpn. 1993 Jun; 43(6):283-93.
16. Kuschert GS, Coulin F, Power CA, Proudfoot AE, Hubbard RE, Hoogewerf AJ, Wells TN. Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses. Biochemistry. 1999 Sep 28; 38(39):12959-68.
17. Middleton J, Patterson AM, Gardner L, Schmutz C, Ashton BA. Leukocyte extravasation: chemokine transport and presentation by the endothelium. Blood. 2002 Dec 1; 100(12):3853-60.
18. Dyer DP, Migliorini E, Salanga CL, Thakar D, Handel TM, Richter RP. Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization. Open Biol. 2017 Jan; 7(1). pii: 160286.
19. Li S, Pettersson US, Hoorelbeke B, Kolaczkowska E, Schelfhout K, Martens E, Kubes P, Van Damme J, Phillipson M, Opdenakker G. In-terference with glycosaminoglycan-chemokine interactions with a probe to alter leukocyte recruitment and inflammation in vivo. PLoS One. 2014 Aug 5; 9(8):e104107.
20. Proudfoot AEI, Johnson Z, Bonvin P, Handel TM. Glycosaminoglycan Interactions with Chemokines Add Complexity to a Complex System. Pharmaceuticals (Basel). 2017 Aug 9; 10(3). pii: E70.
21. Bianchi E, Molteni R, Pardi R, Dubini G.Microfluidics for in vitro biomi-metic shear stress-dependent leukocyte adhesion assays. J Bio-mech. 2013 Jan 18; 46(2):276-83.
22. Nagarsheth N, Wicha MS, Zou W.Chemokines in the cancer microenvi-ronment and their relevance in cancer immunotherapy. Nat Rev Immu-nol. 2017 Sep; 17(9):559-572.
23. Belperio JA, Keane MP, Arenberg DA, Addison CL, Ehlert JE, Burdick MD, Strieter RM. CXC chemokines in angiogenesis. J Leukoc Biol. 2000 Jul;68(1):1-8.
24. Bakogiannis C, Sachse M, Stamatelopoulos K, Stellos K. Plate-let-derived chemokines in inflammation and atherosclerosis. Cyto-kine. 2017 Dec 1. pii: S1043-4666(17)30270-3.
25. Sandset PM. CXCL4-platelet factor 4, heparin-induced thrombocytopenia and cancer. Thromb Res. 2012 Apr; 129 Suppl 1:S97-100.
26. Zhang X, Chen L, Bancroft DP, Lai CK, Maione TE. Crystal structure of recombinant human platelet factor 4. Biochemistry. 1994 Jul 12; 33(27):8361-6.
27. Mayo KH, Roongta V, Ilyina E, Milius R, Barker S, Quinlan C, La Rosa G, Daly TJ. NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer. Biochemistry. 1995 Sep 12; 34(36):11399-409.
28. Struyf S, Burdick MD, Proost P, Van Damme J, Strieter RM. Platelets re-lease CXCL4L1, a nonallelic variant of the chemokine platelet fac-tor-4/CXCL4 and potent inhibitor of angiogenesis. Circ Res. 2004 Oct 29; 95(9):855-7.
29. Vandercappellen J, Van Damme J, Struyf S. The role of the CXC chemo-kines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer. Cytokine Growth Factor Rev. 2011 Feb; 22(1):1-18.
30. Struyf S, Salogni L, Burdick MD, Vandercappellen J, Gouwy M, Noppen S, Proost P, Opdenakker G, Parmentier M, Gerard C, Sozzani S, Strieter RM, Van Damme J. Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3. Blood. 2011 Jan 13; 117(2):480-8.
31. Vandercappellen J, Liekens S, Bronckaers A, Noppen S, Ronsse I, Dillen C, Belleri M, Mitola S, Proost P, Presta M, Struyf S, Van Damme J. The COOH-terminal peptide of platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly inhibits angiogenesis and suppresses B16 melanoma growth in vivo. Mol Cancer Res. 2010 Mar; 8(3):322-34.
32. Kuo JH, Chen YP, Liu JS, Dubrac A, Quemener C, Prats H, Bikfalvi A, Wu WG, Sue SC. Alterna-tive C-terminal helix orientation alters chemokine function: structure of the anti-angiogenic chemokine, CXCL4L1. J Biol Chem. 2013 May 10; 288(19):13522-33.
33. Flier J, Boorsma DM, van Beek PJ, Nieboer C, Stoof TJ, Willemze R, Tensen CP. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J Pathol. 2001 Aug; 194(4):398-405.
34. Lacotte S, Brun S, Muller S, Dumortier H. CXCR3, inflammation, and autoimmune diseases. Ann N Y Acad Sci. 2009 Sep; 1173: 310-7.
35. Groom JR, Luster AD. CXCR3 in T cell function. Exp Cell Res. 2011 Mar 10; 317(5):620-31.
36. Mueller A, Meiser A, McDonagh EM, Fox JM, Petit SJ, Xanthou G, Williams TJ, Pease JE. CXCL4-induced migration of activated T lym-phocytes is mediated by the chemokine receptor CXCR3. J Leukoc Bi-ol. 2008 Apr; 83(4):875-82.
37. Vandercappellen J, Van Damme J, Struyf S. The role of the CXC chemo-kines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer. Cytokine Growth Factor Rev. 2011 Feb; 22(1):1-18.
38. Ludeman JP, Stone MJ. The structural role of receptor tyrosine sulfation in chemokine recognition. Br J Pharmacol. 2014 Mar; 171(5):1167-79.
39. Bikfalvi A, Moenner M, Javerzat S, North S, Hagedorn M. Inhibition of angiogenesis and the angiogenesis/invasion shift. Biochem Soc Trans. 2011 Dec;39(6):1560-4
40. Bonacchi A, Romagnani P, Romanelli RG, Efsen E, Annunziato F, Lasagni L, Francalanci M, Serio M, Laffi G, Pinzani M, Gentilini P, Marra F. Signal transduction by the chemokine receptor CXCR3: acti-vation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem. 2001 Mar 30; 276(13):9945-54.
41. Petrai I, Rombouts K, Lasagni L, Annunziato F, Cosmi L, Romanelli RG, Sagrinati C, Mazzinghi B, Pinzani M, Romagnani S, Romagnani P, Marra F. Activation of p38 (MAPK) mediates the angiostatic effect of the chemokine receptor CXCR3-B. Int J Biochem Cell Biol. 2008; 40(9):1764-74.
42. Chen YP, Wu HL, Boyé K, Pan CY, Chen YC, Pujol N, Lin CW, Chiu LY, Billottet C, Alves ID, Bikfalvi A, Sue SC. Oligomerization State of CXCL4 Chemokines Regulates G Protein-Coupled Receptor Activation. ACS Chem Biol. 2017 Nov 17; 12(11):2767-2778.