- 學位論文
原子力顯微鏡在生命科學上的應用
Application of Atomic Force Microscopy on Life science
摘要
近年來，原子力顯微鏡的技術快速的發展，它具有高解析度、活體掃瞄生物樣品、以及可以用來分析蛋白之間的作用力等優點，因此在生命科學上的應用快速的增加，原子力顯微鏡在生命科學上的應用有助於解決生物樣品上的諸多問題，為生命科學提供新的研究方法來解決問題。我們分別將原子力顯微鏡應用在植物的液泡、液泡膜蛋白結構的功能與動態分析以及蛋白之間的作用力分析，並進一步探討其意義。 在成熟的植物細胞中液泡是一個基本且主要的胞器，液泡佔有植物細胞絕大部分的體積；在高等植物的液泡中，H+-ATPase (EC 3.6.1.3)和H+-PPase(EC 3.6.1.1)是兩個主要的質子傳送幫浦並且位在相同的液泡膜上，其功能皆為在液泡膜上產生質子梯度以提供能量給予液泡中離子與代謝物質的運送。然而，這兩個質子幫浦在植物液泡膜上的特異性目前仍不清楚，在這個研究中我們以白化綠豆的下胚軸細胞的液泡為主要的研究對象，利用不同的滲透壓的調控下觀察這兩個質子幫浦功能上的差異性，在高濃度的蔗糖或山梨醇的處理之下，可以在光散射顯微鏡中觀察到液泡的體積有所變化，同時我們也觀察酵素活性，質子傳送能力，以及偶合反應發現這兩個酵素的反應會呈現不同的抑制結果，然而這些抑制趨勢在純化態的H+-ATPase和H+-PPase並沒有觀察到，因此我們推測這些酵素抑制的結果可能是來自於細胞膜結構的變化，植物液泡膜的結構是影響液泡膜上姪子幫浦的功能的一個重要因素，我們進一步利用酵素動力學的方法來分析不同濃度的蔗糖和山梨醇的處理下KM和Vmax的變化情形，我們發現高濃度的蔗糖和山梨醇的處理Vmax值會有明顯的變化，而KM值變化並不明顯，這結果說明了細胞膜結構的改變會改變H+-ATPase和H+-PPase的水解反應而不是改變H+-ATPase和H+-PPase和受質結合的親合性；利用原子力顯微鏡(AFM)觀察液泡在不同濃度的蔗糖和山梨醇地情況下體積有明顯的變化亦可證明我們所推測的液泡膜的結構是影響液泡膜上質子幫浦的功能，最後我們提出一個植物液泡膜在不同滲透壓下的模型來解釋細胞膜收縮以及皺縮的情況下質子幫浦功能間相互的調控情形。 進一步我們將焦點放在液泡的細胞膜上，液泡膜上存在著兩種質子幫浦，分別是液泡無機焦磷酸水解酶(V-PPase)以及核苷三磷酸水解酶(V-ATPase)， V-PPase利用水解焦磷酸產生能量來驅動質子傳遞造成細胞膜內外的質子梯度，而V-PPase 水解及質子傳遞的活性可被許多的離子所調控，相對高濃度的K+ 可以促進V-PPase的活性而F-、Na+、Ca2+ 以及過多的PPi 則會抑制它的活性，在本研究中我們利用酵母菌表達系統表達帶有六個組胺酸尾飾的V-PPase 接著利用n-dodecyl-β-D- maltoside (DDM) 當作介面活性劑來將V-PPase由細胞膜中溶解出來，再利用Ni2+-NTA 親合性層析管柱將V-PPase 純化出來得到一個分子量大約是73kDa 的純化蛋白質，組胺酸尾飾V-PPase 的水解活性大約是86.4 ± 7.4 μmol PPi/mg.h 比在細胞膜上的還要高約6.5倍； 比較酵母菌表達的組胺酸尾飾V-PPase的活性只有直接由綠豆中純化出來的59%。進一步的分析組胺酸尾飾V-PPase 的特性，發現純化的V-PPase 和位在細胞膜上的有相似的特性。再則，利用光譜分析純化的組胺酸尾飾V-PPase，發現影響V-PPase 活性的這些離子多會造成V-PPase 結構上的改變(特別是二級結構的改變)；這個結果證實影響V-PPase 的離子是經由改變其構型(二級結構) 來影響其活性。 原子力顯微鏡應用在蛋白質結構上有顯著的發展，特別是對於細胞膜蛋白來說，原子力顯微鏡更是一個強而有力的工具，我們就以無機焦磷酸水解酶作為模式蛋白，利用原子力顯微鏡來觀察其構造，特別是我們將無機焦磷酸水解酶利用脂質金屬偶合懸掛法將無機焦磷酸水解酶排列成2D晶體，我們以原子力顯微鏡掃描無機焦磷酸水解酶的2D晶體，再利用原子力顯微鏡之探針在緩衝溶液下進行蛋白質之解構力學分析，可以得到H+-PPase力學圖譜。主要得到七個主要的波峰。另外也紀錄當加入受質或受質類似物的力學圖譜的差異。同時也利用光吸收值、螢光吸收值以及圓形旋轉雙色光光譜來確認當加入受質或受質類似物時，蛋白質結構上的變化情形，並與力學圖譜做一比較。 我們接著利用原子力顯微鏡探討鉤端螺旋體的治病機制，腎小管發炎是由鉤端螺旋體感染引起的相關腎臟疾病，在致病菌性鉤端螺旋體細胞外膜上的脂蛋白，其分子量為32 kDa (簡稱LipL32)，可能是引起腎小管發炎的因子，此蛋白質並不存在非致病性鉤端螺旋體中；之前的研究推測LipL32可能與腎小管細胞膜上的細胞外間質ECM (extracellular matrix)的辨識與結合作用，引起腎小管的發炎，然而確切的引發發炎機制目前仍不清楚。在本次的研究中，我們利用AFM測量探針和腎小管細胞(HK2 cell)之間的作用力，將純化的LipL32修飾在AFM探針上，再將探針在腎小管細胞HK2上做力曲線分析。結果顯示LipL32可以和HK2細胞膜上的蛋白質作用，進一步利用抗體中和的方法來確定這些分子之間的作用力。實驗結果顯示，與LipL32作用的蛋白為細胞膜上的類鐸受體Toll-like receptor 2 (TLR2)作用，但不會和TLR4作用，結合在探針上的LipL32和細胞膜上的 TLR2之間的作用力為59.5 ± 8.7 pico-newton (pN)，這個作用力大小則落在單一分子對之間的作用力，接著再進一步確認LipL32和TLR2之間的作用力，我們利用本身不表現TLR2的細胞株 (HEK293 cell)，比較表達與不表達TLR2的細胞對LipL32的作用力，我們發現作用力會有基本的背景值提升到60.4 ± 11.5 pN，這個作用力與在HK2細胞上所量測的值相近，更證實了LipL32和TLR2之間的作用關係；此外，我們量測LipL32引發發炎反應，利用測量CXCL8/IL-8 mRNA的表現來偵測LipL32引發發炎反應的能力，我們發現當LipL32帶有N端的訊息胜肽會促進細胞產生這些發炎反應因子，總結來說我們發現LipL32 protein會和細胞膜上的TLR2受體作用，但不會和TLR4作用，以及我們提出了LipL32的N端訊息胜肽可能是LipL32引發發炎反應的重要因子，進而提出其感染及致病的機制。
並列摘要
The developments of atomic force microscopy (AFM) are growing up in recently years. The advantages of AFM, such as high resolution at subnano level, application on living cell, and the employment of protein-protein interaction, which make the AFM easily and quickly ultilized on life science. Application of AFM on biological sample could provide new methodology for life science. In this thesis, we applied AFM to plant vacuole, membrane protein investigation and protein-protein interactiona and further provided new insights into these areas. The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. Higher plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, both pumps respond to stress differentially in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. Albeit, no significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is accordingly proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts. In the second part, we took vacuolar proton-translocating pyrophosphatase (V-PPase) as the model system for membrane protein study. V-PPase generates a proton electrochemical gradient across the membrane by hydrolyzing pyrophosphate for maintenance of acidic condition of vacuoles and translocation of secondary metabolites, ions, and even toxics. The enzymatic activity of V-PPase could be stimulated by relatively high concentration of K+, but inhibited by F-, Na+, Ca2+ and excess PPi. In this study, we used the yeast expression system to express hexa-histidine tagged mung bean V-PPase and employed detergent n-dodecyl 刍-D-maltoside (DDM) to solubilize the protein from microsomal membrane, followed by a Ni2+-nitrilotriacetate (Ni2+-NTA) affinity column to yield a highly purified enzyme. The specific activity of purified His-tagged V-PPase was approximately 86.4 ± 7.4 μmol PPi /mg.h, at least 6.5 fold purification compared to that on the vesicle membrane. The specific activity of His-tagged purified V-PPase were approximately 59% compare to the mung bean innate one. Further characterization indicates that the His-tagged V-PPase thus obtained resembles primarily those on membrane in most enzymatic features. The spectroscopic analyses including circular dichroism spectroscopy on His-tagged V-PPase revealed variations in conformational change induced by ions, as those inhibitors Na+, Ca2+, and F-, of this proton translocase. These results confirm the effect of ions are exerted concomitantly with the conformational (secondary structural) changes. The AFM technique uses a tiny stylus on a cantilever that is dragged across the lipid layer surface, and the deflections recorded are used to map the surface topology. Furthermore, the tiny stylus can be used as a fishing pole to fish the membrane protein out of lipid bilayer for determining the force barriers of TMs-TMs of TMs lipid interaction. For this purpose, we reconstituted V-PPase into lipid bilayer in the same orientation to minimize unexpected results when it is poured off the membrane by AFM stylus. The force extension curves thus reflect the mechanical stability of TMDs and interactes with vicinitic TMDs or lipid bilayers. In the presence of PPi and its analog, IDP, the force extension curves revealed significantly changes occurred on the putative PPi binding sites. The conformational changes were also confirmed using other spectroscopes such as Circular Dichroism (CD). Taken together, we proposed a working model for the PPi hydrolysis showing an interaction between domains upon binding of substrate and its analogs. In the third part, we used AFM to investigate the protein-protein interaction, especially the pathogenic protein and host cell interaction. We took Leptospira outer membrane lipoprotein as the model system to explore the protein-protein interaction mechanism. Leptopirosis is a renal disease caused by pathogenic Leptospira that primarily infects the renal proximal tubules, consequently resulting in severe tubular injuries and malfunctions. The protein extracted from outer membrane of this pathogenic strain contains a major component of a 32-kD lipoprotein (LipL32), which is absent in the counter membrane of nonpathogenic strains and is implicated as a crucial factor for host cell infection. Previous studies showed that LipL32 induced inflammatory responses as well as interacted with extracellular matrix (ECM) of the host cell. However, the exact relationship between LipL32 mediated inflammatory responses and ECM binding is still unknown. In this study, atomic force microscope (AFM) with its tip modified by purified LipL32 was used to determine the interaction between LipL32 and cell surface receptors. Furthermore, an antibody neutralization technique was employed to identify Toll-like receptor 2 (TLR2) but not TLR4 as the major target of LipL32 attack. The interaction force between LipL32 and TLR2 was measured as approximately 59.5 ± 8.7 pico-newton (pN), concurring with the theoretical value for a single pair molecular interaction. Moreover, transformation of TLRs deficient cell line with human TLR2 brought the interaction force from basal level to approximately 60.4 ± 11.5 pN, confirming unambiguously TLR2 as counter receptor for LipL32. The stimulation of CXCL8/IL-8 expression by full-length LipL32 protein as compared to that without N-terminal signal peptide domain suggests a significant role of the signal peptide of the protein in the inflammatory responses. This study provides direct evidence that LipL32 binds to TLR2, but not TLR4, on cell surface and a possible virulent mechanism for Leptospirosis is accordingly proposed.