以結構為基礎探討人類嗜酸性球核醣核酸水解酶與肝素之結合模式

Structural basis for differential heparin binding modes of human eosinophil ribonucleases

洪達任(Ta-Jen Hung)

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/博士(2013年)

https://doi.org/10.6843/NTHU.2013.00011

摘要

人類嗜酸性白血球陽離子蛋白(eosinophil cationic protein，ECP)及嗜酸性白血球神經毒蛋白(eosinophil derived neurotoxin，EDN)皆由活化的嗜酸性白血球分泌至血液中，並隸屬於人類核醣核酸水解酶A家族(RNase A superfamily)。ECP與EDN具有極高的序列及結構相似性，然而兩者之生物功能卻不盡相同。本研究以分子對接(molecular docking)模擬ECP/EDN與肝素聚己糖之交互作用，發現ECP序列中之Gln40及His64形成鉗狀結構以穩定與其結合的肝素聚己糖，而EDN則無相似的結構。此外Arg105亦被預測對ECP與肝素聚己糖交互作用具有相當貢獻。為了釐清預測的真實性，Gln40、His64以及Arg105等胺基酸透過點突變置換成丙氨酸(Ala)，並運用恆溫滴定熱量計(isothermal titration calorimetry，ITC)測量ECP突變株與肝素間結合力的變化。此外，本研究利用石英振盪微天平(quartz crystal microbalance，QCM)以及磁減量試驗(magnetic reduction assay，MRA)兩種新穎分子親和力偵測技術測量由ECP序列衍生之細胞穿透胜肽(NYRWRCKNQN，CPPecp)以及肝素結合胜肽(YRWRCK，HBPecp)的肝素/糖胺聚糖結合能力。本研究分析ECP與EDN與肝素結合模式的差異，進而探討ECP與EDN之序列、結構、以及功能的相關性。本論文之主要貢獻為發現ECP/EDN與肝素/糖胺聚糖結合模式與結合強度的差異性，及ECP/EDN與細胞間作用相異性的原因，進而瞭解相關免疫疾病的分子機制。

關鍵字

人類嗜酸性白血球核醣核酸酶 ； 嗜酸性白血球陽離子蛋白 ； 嗜酸性白血球神經毒蛋白 ； 肝素 ； 糖胺聚糖 ； 石英振盪微天平 ； 磁減量試驗

並列摘要

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. Here, a novel molecular model for ECP/EDN binding of heparin hexasaccharide, [GlcNS(6S)-IdoA(2S)]3, was predicted. Interestingly, Gln40 and His64 on ECP formed a clamp-like structure to stabilize heparin hexasaccharide in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln40-His64 clamp contributed to ECP-heparin interaction significantly. Besides, binding affinities of a multi-functional peptide (NYRWRCKNQN, CPPecp), containing major heparin binding region of ECP, to heparin derivatives and glycosaminoglycans (GAGs) were determined by quartz crystal microbalance (QCM) and magnetic reduction assay (MRA). Moreover, a peptide containing minimum length to interact with heparin (YRWRCK, HBPecp) was also used. In conclusion, our in silico and in vitro data demonstrate that ECP uses not only major heparin binding region but also other surrounding residues to interact with heparin. Discovery of such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN, as well as differential binding mode, binding affinity, and cellular interaction between ECP and EDN facilitate further understanding of molecular mechanisms of immune diseases.

並列關鍵字

eosinophil ribonuclease ； eosinophil cationic protein ； eosinophil derived neurotoxin ； heparin ； glycosaminoglycans ； quartz crystal microbalance ； magnetic reduction assay

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