臺灣博碩士論文加值系統

FMS樣酪氨酸激酶3 (FMS-like tyrosine kinase 3，FLT3)，是一種受體型酪氨酸激酶 (Receptor tyrosine kinase，RTK)， 與c-KIT和PDGFR同屬第三型RTK家族，具有RTKIII成員共同的結構：膜外含有五個免疫球蛋白樣結構區（immunoglobulin-like domains, Ig） ，膜內則含有穿膜區 (transmembrane domain)、近膜區 (juxtamenbrane domain)以及激酶區(tyrosine kinase domain)。FLT3配體則具有跨模型與分泌型兩種形態，分泌型可藉由酵素切割與選擇性剪接產生，其結晶結構於2000年解出。當配體與膜外區結合後，受體結構改變發生二聚體化 (dimerization)，活化胞內受體酪胺酸激酶（tyrosine kinase）進行自體磷酸化，激發下游訊息分子磷酸化連鎖反應（phosphorylation cascade）。FLT3與其配體FLT3 ligand (FLT3L) 所參與的訊息傳遞路徑在造血幹細胞 (Hematopoietic stem cell，HSCs)、樹突狀細胞 (Dendritic cell，DCs)、和自然殺手細胞 (Natural killer cell，NK cell) 的增殖和分化中起著重要的調節作用。
FLT3在生理上所扮演的角色已逐漸明朗，但在細胞與結構層面上的研究文獻卻很有限。直到2011年，FLT3L與FLT3-D1-D4的複合體及FLT3L與FLT3-ECD的複合體的結晶結構才由Verstaete等人所解出，解析度分別是4.3A與7.8A。但其結晶結構解析度不高並與先前的同源模型有相當程度出入：第一、在受體與配體的相互作用中，FLT3複合體只靠著FLT3-D3與FLT3L做結合。其他的同源受體如c-KIT/SCF複合體及CSFR-1/CSF-1複合體則是由D2與D3環抱偶極化的配體。第二、在FLT3複合體中無法觀察到受體間的相互作用，在c-KIT/SCF 2:2 複合體中，偶極化的配體與受體結合後，會造成受體的構型改變，受體間D4-D4與D5-D5會有相互作用。有鑑於此，我們將FLT3與抗體的Fc區域做嵌合，來模仿受體間偶極化的相互作用並希望能以此增加FLT3L與FLT3的親和力以幫助解析結晶結構。
本研究利用果蠅細胞表現系統表現FLT3L與其多種受體：FLT3胞外全長(FLT3-ECD)、FLT3胞外全長與Fc嵌合體(FLT3-ECD-Fc)、FLT3胞外第二個Ig區域至第五個Ig區域(FLT3-D2-D5)及FLT3胞外第二個Ig區域至第五個Ig區域與Fc嵌合體(FLT3-D2-D5-Fc)。重組蛋白分泌至培養液後，經濃縮、透析、親和層析及膠體層析後可得到高純度蛋白液，並將獲得的蛋白進行液相層析質譜分析、酵素免疫分析與小角度X光散射分析。
我們成功地表現出上述的蛋白並以液相層析質譜分析作確認，之後以酵素免疫分析偵測FLT3L與多種受體的親和力，結果顯示具有Fc嵌合的FLT3確實有增強親和力的情形。我們利用共同培養的方式，將可表現FLT3-Fc嵌合蛋白的細胞與可表現配體的細胞混合，希望可以在分泌至培養基的過程中形成複合體，再利用protein A親和管柱純化，建立了一個快速與省工的方法以獲得FLT3L和FLT3-Fc嵌合蛋白之複合體，在確認獲得複合體後，我們進行小角度X光散射實驗，並利用 ATSAS 套件中的程式分析。目前已得到FLT3L和FLT3-Fc嵌合蛋白之複合體的表面構造（ab-initio envelope）。為了獲得更細節的資訊，我們建立了的養晶系統，希望以結晶結構直觀地分析FLT3的複合體。

FMS-like tyrosine kinase receptor 3 (FLT3) belongs to the type-III RTK (Receptor Tyrosine Kinase) family to which c-KIT and the PDGFR (Platelet Derived Growth Factor Receptor) also belong. They share the same structure: 1) Extracellular domain with five immunoglobulin-like domains, 2) Transmembrane domain, 3) Juxtamembrane domain, 4) Cytoplasmic domain with tyrosine-kinase domain with a kinase insert loop. FLT3 ligand (FLT3L) is biologically active both as a transmembrane form and as a soluble form. Soluble FLT3L can be generated by proteolytic cleavage of the transmembrane ligands and/or alternative splicing. The crystal structure of FLT3L was published in 2000. The binding of FLT3L with its cognate receptor (FLT3) will induce the ligand-mediated receptor dimerization which will activate intracellular tyrosine kinase of receptors undergoing auto-phosphorylation and propagate signal-transduction cascade. FLT3 and its cytokine ligand (FLT3L) play a critical role for hematopoiesis and the immune system.
Although the physiologic role of the FLT3 ligand-receptor interaction has been characterized, the FLT3 signaling complex has remained unclear at the molecular and structural levels. Until 2011, Verstraete et al. reported the low-resolution crystal structures of FLT3L in complex with FLT3-D1-D4 and FLT3-ECD, which resolution is 4.3A and 7.8A, respectively. Further, the results are not consistent with previous homology models and the structural features of RTKIII complex by the following two findings. First, ligand-receptor interactions of FLT3 complex are very different from its homologous counterparts. For the FLT3 complex, the FLT3 ligand only interacts with the D3 (the third Ig-like domain) of FLT3. For c-KIT/SCF complex and CSFR-1/CSF-1 complex, the dimer ligand is embraced by two receptor molecules through D2 and D3 domains. Second, no homotypic interaction between two receptors has been observed in the FLT3 complex. In the c-KIT/SCF 2:2 complex, the binding with dimer of ligand induces the conformational change of receptor through the interactions between D4-D4 and D5-D5 interfaces. Therefore, we constructed Fc fusion protein in order to improve the ligand binding affinity and aid for crystallization.
In this study, recombinant proteins of FLT3 ligand, FLT3-D2-D5 with/without Fc region and FLT3-ECD with/without Fc region were expressed by Drosophila Schneider 2 (S2) cells for functional and structural studies of ligand-receptor interaction After purification procedures (Ni-affinity and gel filtration chromatography), we used mass spectrometry to characterize recombinant proteins and conducted ELISA-based binding assays. Our data revealed an increased binding affinity for FLT3 with Fc region. For the formation of FLT3L/FLT3-Fc complex, we co-culture by mixing the stable line of FLT3L and FLT3-Fc together in an attempt that both of them can form complex during secretion in culture medium. With this new expression approach, we obtain FLT3L/FLT3-Fc complex with one–step purification procedure using Protein A affinity gel. The preliminary shape of FLT3L/FLT3-Fc complex was determined by SAXS with the programs implant in ATSAS. To acquire the more detail information of FLT3 signaling complex, we had tried crystal growing for further study by X-ray crystallography.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
ABSTRACT v
CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS xiii
Chapter 1 Introduction 1
1.1 Domain structure and features of FLT3 1
1.2 Domain structure and features of FLT3L 1
1.3 The signaling pathway of FLT3 2
1.4 The function of FLT3 3
1.5 FLT3 mutations in haematopoietic malignancies 4
1.5.1 FLT3 internal tandem duplications. 4
1.5.2 FLT3 activation loop mutation. 5
1.6 Current structural knowledge of RTK III receptors interacting with their cognate ligand 5
1.6.1 The structure of c-KIT/SCF complex 6
1.6.2 The structure of CSF-1R/CSF-1 complex 6
1.6.3 The structure of CSF-1R/IL-34 complex 7
1.6.4 The structure of FLT3 complex 7
1.7 FLT3-Fc 8
1.8 Liquid chromatography-mass spectrometry 9
1.9 Small-Angle X-ray Scattering 10
1.10 Aim and purpose 12
Chapter 2 Material & Methods 15
2.1 Materials 15
2.2 Methods 18
2.2.1 Construction of the expression vectors. 18
2.3 Transfection of the plasmid into Schneider 2 cells. 19
2.4 Purification of secreted recombinant protein 20
2.4.1 FLT3L, FLT3-ECD and FLT3-D2-D5 with/without Fc region 20
2.4.2 The complex of FLT3L/ FLT3-Fc 20
2.5 Sample preparation for LC/MS-MS 21
2.6 ELISA-based binding assays 22
2.7 Small-angle X-ray scattering data acquisition and analysis 23
Chapter 3 Results 25
3.1 Construction of expression vectors. 25
3.2 Expression of recombinant proteins in Drosophila Schneider 2 cells 26
3.2.1 Transient expression of recombinant proteins 26
3.2.2 Large-scale expression of recombinant proteins 26
3.3 Purification of recombinant proteins in Drosophila Schneider 2 cells 27
3.3.1 FLT3L 27
3.3.2 FLT3-ECD and FLT3-D2-D5 27
3.3.3 FLT3-ECD and FLT3-D2-D5 with Fc region 28
3.3.4 The complex of FLT3L/FLT3-Fc 28
3.4 Identification of recombinant proteins 29
3.5 ELISA-based Binding assays 30
3.6 SAXS analysis of FLT3L/FLT3-Fc complex 31
3.7 SAXS-based structural modeling of FLT3L/FLT3-Fc complex 32
Chapter 4 Discussions 33
4.1 The formation of complex 33
4.2 Binding affinity 33
REFERENCES 61
APPENDIX 65

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