人類嗜伊紅血球核醣核酸水解酶與氨基葡聚糖交互作用之分析

Characterization of glycosaminoglycan-binding activities of human eosinophil ribonucleases

陳依文(I-Wen Chen)

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(2006年)

摘要

人類嗜伊紅血球在氣喘所帶來的組織傷害中扮演一個很重要的角色，其作用主要來自於大量釋放顆粒蛋白的，包括嗜伊紅血球陽離子蛋白(eosinophil cationic protein, ECP) 和嗜伊紅血球神經毒素(eosinophil derived neurontoxin, EDN)。嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素皆屬於人類核糖核酸水解酶A家族的一員均已被推測為氣喘病與過敏性鼻炎相關的重要因子之一，並在臨床檢驗作為測定患者過敏嚴重度的生物標誌。此外二者尚具有抑制數種細胞生長的能力，例如人類上皮腫瘤細胞以及人類前骨髓白血病細胞。但是究竟嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素是藉由何種細胞受器進到胞內仍然未知。
細胞表面的氨基葡聚糖蛋白可充當多種不同蛋白質連結至細胞的引導區，氨基葡聚糖根據其結構與構成的成分不同，可分布在多種不同物種組織細胞的表面上。其中，硫酸乙醯肝素可與多種不同的分子結合，例如生長因子，病毒，細菌以及多種不同蛋白質。嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素已被證實可與肝素結合，因此推測它們也可以與硫酸乙醯肝素結合。在本研究中利用免疫螢光技術，發現硫酸乙醯肝素在嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素的進入細胞過程，扮演很重要的角色。此外亦利用表面電漿共振技術測量了嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素與硫酸乙醯肝素之間反應的強度以及相關特性。研究顯示當嗜伊紅血球陽離子蛋白的立體結構受到高濃度的尿素或是高溫破壞時，它與硫酸乙醯肝素之間的反應會明顯降低。由此可知，嗜伊紅血球陽離子蛋白與硫酸乙醯肝素的結合與其立體結構相關。而當反應環境的酸鹼值降低至接近細胞內小體的微酸環境時，嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素與硫酸乙醯肝素的反應也會明顯減弱。綜合以上所述，本論文證實嗜伊紅血球陽離子蛋白和嗜伊紅血球神經毒素與細胞表面的結合以及進入細胞需要藉由細胞表面的硫酸乙醯肝素產生分子間的交互作用才能發生。

關鍵字

嗜伊紅血球陽離子蛋白 ； 嗜伊紅血球神經毒素 ； 肝素 ； 硫酸乙醯肝素 ； 人類嗜伊紅血球核醣核酸水解酶

並列摘要

The eosinophil appears to be the primary leukocyte responsible for tissue damage in bronchial asthma, which occurs when the granule proteins, including eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN), are released into the extracellular space. ECP and EDN belong to the ribonuclease A superfamily. Both have been suggested as factors in allergic respiratory diseases, and thus used as clinical biomarkers for detecting the severity of asthma. Interestingly, ECP and EDN showed cytotoxicity toward several cell lines, including HL60 and A431 cells. The cytotoxicity has been correlated with the internalization of the RNases, but the cellular receptor for ECP or EDN has not been identified yet.
Cell surface glycosaminoglycan (GAG) proteoglycans act as docking sites for the binding of several different proteins to surface of eukaryotic cells. GAGs are presented almost ubiquitously on cell surface but vary with respect to their composition and quantity among different group of species, cell types, tissues, and cellular development stages. Heparan sulfate (HS), a ubiquitously expressed GAG, bind to a wide variety of extracellular ligands, including growth factors, virus, and bacteria. ECP and EDN known as heparin binding proteins may interact with the HS on the cell surface. Here, we demonstrate that HS is essential in the cellular uptake of ECP and EDN by immunofluorescence study. In addition, surface plasmon resonance instrument (SPR) was used to investigate the binding affinities and kinetics between ECP/EDN and heparan sulfate. The interaction between ECP and HS was obviously reduced while ECP was denatured with urea or boiling, indicating that ECP binding to HS in a 3 D structure dependent manner. The ECP/EDN-HS interactions are decreased at acidic pH, suggesting that ECP and EDN may dissociate from HS under acidic pH within endosomes. Taken together, these findings suggest that heparan sulfate proteoglycan serves as the cellular endocytic receptor for ECP and EDN.

並列關鍵字

ECP ； EDN ； Heparin ； Heparan sulfate ； eosinophil RNase

參考文獻

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