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鑑定及特性分析

Characterization and Identification of Heparin/Heparan Sulfate Binding Motifs on Human Eosinophil Derived Neurotoxin

張偉唐(Wei-Tang Chang)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2010年)

摘要

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。

關鍵字

人類嗜酸性白血球神經毒蛋白 ； 肝素 ； 硫酸乙醯肝素 ； 螢光輔助醣電泳

並列摘要

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.

並列關鍵字

eosinophil derived neurotoxin ； heparin ； heparan-sulfate ； Fluorescence-assisted carbohydrate electrophoresis

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