人類嗜酸性白血球神經毒蛋白(eosinophil derived neurotoxin, EDN)隸屬於人類核醣核酸水解酶A家族(RNase A superfamily)的一員，亦稱為核醣核酸水解酶二 (RNase 2)。EDN係由活化的嗜酸性白血球釋放之分泌性蛋白質，因序列中含12個正電胺基酸，故具有高達8.9的等電點(pI)值。EDN具有抗病毒的能力，血液中EDN含量亦為臨床發炎反應之生物標記。本研究主要探討EDN與人類支氣管上皮細胞(Beas-2B cell)表面的硫酸乙醯肝素(heparan sulfate)或肝素(heparin)之結合作用，並進行EDN序列中肝素結合區域之鑑定及特性分析。正常型及突變型EDN重組蛋白皆利用透過大腸桿菌(E. coli)表現系統生產，並利用酵素免疫分析法(ELISA)及螢光輔助醣電泳(FACE)證實EDN與人類支氣管上皮細胞或肝素分子間的交互作用。本研究利用不同的肝素衍生物進行競爭實驗，發現肝素分子中胺基及氧基之硫酸化作用對於EDN的結合皆相當重要。除此之外，亦於EDN序列中鑑定3個與肝素結合的區域，分別命名為HBR1 (34QRRCKN)、HBR2 (65NKTRKN)、及 HBR3 (113NRDQRRD)。當中HBR1為最重要的肝素結合位，其影響力大於HBR2及HBR3。本研究也驗證EDN進入細胞中之特性，並推測此特性與EDN和細胞表面的硫酸乙醯肝素結合高度相關。本論文針對人類嗜酸性白血球神經毒蛋白之肝素/硫酸乙醯肝素結合區域進行分析及探討，並發現EDN之細胞及生化反應機制，具體貢獻於蛋白質與醣分子交互作用之生物功能研究。

Eosinophil-derived neurotoxin (EDN, also known as RNase2) is secreted by activated eosinophils and is a member of human RNase A superfamily. It contains 12 cationic residues and thus possesses a high pI value of 8.9. EDN is also an antiviral RNase and its level in biological fluid is used as an inflammatory bio-marker. This study focuses on identification and characterization of the essential heparin binding motifs in EDN, and the minimal EDN binding unit in heparin polysaccharides. Recombinant wild-type EDN, and mutant EDNs derived from site-directed mutagenesis were expressed by E. coli expression system. The interaction of EDN to bronchial epithelial cells and heparin has been demonstrated by cell ELISA and FACE, respectively. The results showed direct interaction between recombinant EDN and heparin as well as heparan sulfate (HS). In addition, competitive assays using heparin derivatives indicated that the sulfate groups contributed almost all negative charges for EDN binding, and the crucial role of N-sulfated and O-sulfated groups in heparin in the interaction to EDN was illustrated. The functional heparin binding motif has been identified by site-directed mutagenesis screening. Three heparin binding regions (HBRs) on EDN including HBR1 (34QRRCKN), HBR2 (65NKTRKN), and HBR3 (113NRDQRRD) are identified. HBR1 is the most important motif in governing EDN binding to heparin/HS, for the heparin/HS binding effect of HBR1 is higher than that of HBR2 and HBR3. Furthermore, the penetration of MBP-EDN to Beas-2B cells requires HBR1 and may correlate with the interaction of EDN on cell surface HS. Taken together, we have discovered and characterized functional heparin/HS binding motifs on hEDN.

摘要 I
Abstract II
Acknowledgement III
Tables of contents IV
List of figures VI
List of Tables VIII
List of Appendixes IX
Abbreviation X


Chapter 1 Introduction 1
Chapter 2 Materials and methods 10
2-1 Bacterial strains, vectors, and culture conditions 10
2-2 Cells and cell culture 10
2-3 Antibodies and reagents 11
2-4 Construct 12
2-5 Site-directed mutagenesis and DNA sequencing 12
2-6 In situ PCR for construct checking 14
2-7 Mini-preparation of plasmids 15
2-8 Competent cell preparation and transformation 16
2-9 Large scale expression of recombinant protein 16
2-10 Purification of MBP and recombinant MBP-EDN fusion protein 17
2-11 Isolation and solubilization of EDN-6His inclusion bodies 18
2-12 Purification of recombinant EDN -6His 18
2-13 In vitro folding of EDN-6His 19
2-14 Buffer exchange, protein concentration and quantification 19
2-15 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 20
2-16 Western blotting analysis 21
2-17 RNase activity assay 21
2-18 Cell based enzyme-link immunosorbent assay (ELISA) 22
2-19 GAGs competitor assay and block assay 23
2-20 Fluorescence-assisted carbohydrate electrophoresis (FACE) 24
2-21 EDN uptake assays 24
2-22 Statistical analysis 25
Chapter 3 Results 26
3-1 Expression and purification of soluble MBP, MBP-EDN and mutant MBP-EDN 26
3-2 Expression and purification of wild type and mutant EDN-6His 28
3-3 RNase activities of refold EDN-6His and ECP-6His against yeast tRNA 29
3-4 EDN interacts with Beas-2B cells and heparin 31
3-5 Identification of interaction between recombinant EDN and heparin 32
3-6 Characterization of the interaction between recombinant EDN and heparin derivatives 33
3-7 Identification of heparin binding motifs on EDN 37
3-8 Comparison of the interaction of heparin to EDN and ECP 40
3-9 Uptake of MBP-EDN into Beas-2B cells 41
Discussion 42
Figures 50
Tables 74
Reference 85
Appendix 90

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