CXCL4屬於CXC類型的趨化素，在生理條件下CXCL4的單體會聚集成四聚體的結構，CXCL4除了趨化細胞的功能之外，也會和受體做專一性的結合，在癌症研究中具有抗血管新生的作用。CXCL4L1則為CXCL4的變異體，兩種趨化素間的差異在於C端有三個氨基酸的不同，造成CXCL4L1的C端螺旋結構會向外展開且具有較高的活動性，也因此在生理環境下CXCL4L1比起CXCL4更容易解離形成單體結構以促使CXCL4L1和下游受體產生結合，而具有更好的抗血管新生作用。 CXCL4和CXCL4L1的受體均為CXCR3，根據不同的RNA splicing可以細分為CXCR3A和CXCR3B兩種亞型，CXCR3B的N端比3A多了52個氨基酸。CXCR3受體的N端有兩個經轉譯後修飾的硫酸化酪胺酸，在許多研究中發現帶有硫酸根的受體會促進受體與趨化素的結合反應。根據晶體結構與結合模型發現受體和趨化素是透過一對一結合，因此在本實驗中會將趨化素突變成單體，並且將CXCR3的胞外區域構築於支架蛋白GB1之上，再藉由核磁共振探討CXCL4/CXCL4L1與CXCR3A/B不同胞外區域的結合選擇性與結合能力的差異，並分析滴定不同濃度的受體模擬蛋白造成的光譜變化，以得出結合曲線與解離常數。在滴定實驗中可以發現，CXCR3受體的N端可以促進受體與CXCL4或CXCL4L1之間的結合能力，而其他三個受體的胞外片段則沒有促進結合的現象。此外透過光譜的變化發現CXCL4L1對CXCR3可能具有更好的結合能力。

CXCL4 and CXCL4L1 belong to CXC-type chemokine family, exhibiting multiple activities in biology. The two chemokines have notable biological functions related with anti-angiogenesis and inflammatory cell recruitment. CXCL4L1, acting as a more potent anti-angiogenic ligand, is a homologue of CXCL4, which differs from CXCL4 in three residues in C-terminal helix. The difference introduces structural flexibility in CXCL4L1 and causes different orientation of the C-terminal helix. CXCL4L1 and CXCL4 execute their functions through binding membrane receptors, CXCR3A and CXCR3B. The only difference between two receptors is the existence of a 52-residue at CXCR3B N-terminal end if comparing with CXCR3A. To understand the binding specificity between CXCR3A/CXCR3B and the two chemokine ligands, we prepared artificial CXCL4 and CXCL4L1 mutants favorably adopting monomeric formations. We further developed a model protein system by transplanting the individual CXCR3A/B extracellular elements including N-terminal region and three extracellular loops on a scaffold protein, GB1. Through solution NMR titration experiments, we can observe the CXCL4/CXCL4L1 resonances perturbed by the presence of the extracellular elements. The result, therefore, allow us to determine the binding affinities and characterize the binding sites. In the study, we observed the three extracellular loops contributing significantly and equally in recruiting CXCL4 and CXCL4L1. The derived binding affinities (Kd) showed less distinguishable difference. However, when the CXCR3A N-terminus co-exist with extracellular loop 2 on the scaffold GB1 protein, the affinities were enhanced for three times where the sulfotyrosine residues provides significant binding specificity in anchoring on the chemokine ligands. The result indicates how the N-terminus contributes to the interaction between CXCL4 and CXCR3. We conclude the synergistic effect of CXCR3 N-terminal fragment and the extracellular loops in recognizing of CXCL4 and CXCL4L1.

CONTENT
Abstract I
中文摘要 II
誌謝 III
Abbreviations IV
Chapter 1. Introduction 1
1.1 Chemokine 1
1.1.1 Chemokine category 1
1.1.2 CXCL4 (PF4) 2
1.1.3 CXCL4L1 (PF4V1) 3
1.2 G protein-couple receptor (GPCR) 3
1.2.1 GPCR 3
1.2.2 The chemokine receptor 4
1.2.3 The chemokine receptor-ligand complex 5
1.2.4 The two-site binding model of chemokine and chemokine receptor 5
1.3 C-X-C type chemokine receptor 3 (CXCR3) 6
1.3.1 CXCR3 function and selectivity 6
1.3.2 CXCR3A/B interact with CXCL4/CXCL4L1 7
1.4 The strategy of the study 10
Chapter 2. Materials and methods 11
2.1 Materials 11
2.2 CXCL4 Cloning, expression and purification 11
2.3 CXCL4L1 Cloning, expression and purification 12
2.4 Soluble receptor mimics cloning, expression and purification 13
2.4.1 Substitution of Cys to Ser 14
2.4.2 The purification of GB1 protein 14
2.5 Nuclear magnetic resonance (NMR) spectroscopy 15
Chapter 3. Results 17
3.1 The binding of CXCL4 to the N-terminus of CXCR3 17
3.1.1 The strategy of experiment design 17
3.1.2 Binding of GB1 18
3.1.2 Binding of GB1-ECL2 18
3.1.3 Contribution derived from CXCR3 N-terminal fragments 19
3.1.4 Contribution derived from sulfated CXCR3 N-terminus 20
3.2 Binding of three extracellular loops with CXCL4 21
3.3 The binding of sulfotyrosine and sulfated CXCR3A peptide on CXCL4 22
3.4 The binding of CXCL4L1 to the N-terminus of CXCR3 23
3.4.1 Binding of GB1-ECL2 23
3.4.2 Contribution derived from CXCR3 N-terminal fragments 24
3.4.3 Contribution derived from sulfated CXCR3 N-terminus 25
3.5 Binding of three extracellular loops with CXCL4L1 25
Chapter 4. Discussion 27
Reference 66
Appendix 70

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