臺灣博碩士論文加值系統

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。

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.

Abstract (in Chinese)…………………………………………………………………...I
Abstract (in English)………………………………………………………………….IV
Abbreviations..………………………………………………………………………..XI
List of Figures……………………………………………………………………….XIV
List of Tables……………………………………………………...…………………XVI
Chapter 1. Differential response of vacuolar proton pumps to osmotica 1
1.1. Introduction 1
1.2. Materials and methods 3
1.2.1. Plant materials 3
1.2.2. Membrane preparation and partial purification of V-ATPase and V-PPase 3
1.2.3. Enzyme assay and protein determination 3
1.2.4. Measurement of H+ translocation 5
1.2.5. Atomic force microscopy (AFM) 5
1.2.6. Light scattering measurement 6
1.2.7. SDS-PAGE and immunoblotting analysis 6
1.2.8. Control over K+, Na+, and Ca2+ contamination 6
1.2.9. Chemicals 7
1.3. Results 7
1.3.1. Vacuolar membrane from etiolated hypocotyls of mung bean seedlings 7
1.3.2. Enzymatic activities of V-PPase and V-ATPase under high osmotic pressure 8
1.3.3. Proton translocation of V-ATPase and V-PPase under high osmotic pressure 10
1.3.4. Immunoblotting analysis of vacuolar membranes upon high osmotic treatment 12
1.3.5. AFM image and light scattering of tonoplast under osmotic pressure 12
1.4. Discussion 14
Chapter 2. Purification, characterization, and spectral analyses of histidine-tagged vacuolar H+-pyrophosphatase expressed in yeast 18
2.1. Introduction 18
2.2. Materials and Methods 20
2.2.1. Chemicals 20
2.2.2. Construction of His-tagged plasmid 20
2.2.3. Heterologous expression of V-PPase in yeast 21
2.2.4. Purification of His-tagged V-PPase 22
2.2.5. Enzyme assay and protein determination 23
2.2.6. Measurement of proton translocation 23
2.2.7. SDS-PAGE and immunoblotting analysis 24
2.2.8. Spectral measurements 25
2.2.9. Ion contamination in reaction media 25
2.2.10. V-PPase sequence alignment and membrane topology prediction 25
2.2.11. V-PPase two-dimensional crystallization 26
2.2.12. AFM observation and force-distance curve 26
2.3. Results and Discussions 27
2.3.1. Expression and purification of mung bean V-PPase in yeast 27
2.3.2. Kinetic parameters and optimal pH of purified His-tagged V-PPase 29
2.3.3. Ion effects on His-tagged V-PPase 30
2.3.4. Spectroscopic properties of purified His-tagged V-PPase 32
2.3.5. Sequence alignment of Sc. V-PPase and Vr.V-PPase 33
2.3.6. The dimmer structure of V-PPase in two-dimensional crystal 34
2.3.7. The force-distance curves of V-PPase two-dimensional crystal 35
2.4. Conclusions and outlook 36
Chapter 3. Leptospiral outer membrane lipoprotein LipL32 binding on Toll-like Receptor 2 of renal cell as determined by atomic force microscope 37
3.1. Introduction 37
3.2. Materials and Methods 40
3.2.1. Reagents 40
3.2.2. Expression and purification of LipL32 40
3.2.3. Size exclusion chromatography, SDS-PAGE, and western blot analysis 41
3.2.4. Cell culture and transient transfection 41
3.2.5. AFM tip functionalization 42
3.2.6. AFM observation 42
3.2.7. RNA extraction and RT-PCR 43
3.2.8. AFM force-distance analysis 44
3.3. Results 44
3.3.1. LipL32 over-expression, purification, and characterization 44
3.3.2. Analysis of force-distance curves on cell surface 45
3.3.3. Identification of cell surface receptors 48
3.3.4. Dynamic force spectroscopy 50
3.4. Discussion 51
3.4.1. LipL32 interacts with TLR2 51
3.4.2. The LipL32 binding domain to TLR2 52
References………………………………………………………….……………..……55

Akira, S., Takeda, K., and Kaisho, T. (2001). Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2, 675-680.
Alexopoulou, L., Thomas, V., Schnare, M., Lobet, Y., Anguita, J., Schoen, R.T., Medzhitov, R., Fikrig, E., and Flavell, R.A. (2002). Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med 8, 878-884.
Allen, S., Chen, X., Davies, J., Davies, M.C., Dawkes, A.C., Edwards, J.C., Roberts, C.J., Sefton, J., Tendler, S.J., and Williams, P.M. (1997). Detection of antigen-antibody binding events with the atomic force microscope. Biochemistry 36, 7457-7463.
Anders, H.J., Banas, B., and Schlondorff, D. (2004). Signaling danger: Toll-like receptors and their potential roles in kidney disease. J Am Soc Nephrol 15, 854-867.
Baltscheffsky, M., Schultz, A., and Baltscheffsky, H. (1999). H+-PPases: a tightly membrane-bound family. FEBS Lett 457, 527-533.
Baltscheffsky, M.a.B., H. (1993). Molecular mechanisms in bioenergetics (Elsevier, Amsterdam).
Barbosa, A.S., Abreu, P.A., Neves, F.O., Atzingen, M.V., Watanabe, M.M., Vieira, M.L., Morais, Z.M., Vasconcellos, S.A., and Nascimento, A.L. (2006). A newly identified leptospiral adhesin mediates attachment to laminin. Infect Immun 74, 6356-6364.
Baykov, A.A., Dubnova, E.B., Bakuleva, N.P., Evtushenko, O.A., Zhen, R.G., and Rea, P.A. (1993). Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme. FEBS Lett 327, 199-202.
Belogurov, G.A., and Lahti, R. (2002). A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans. J Biol Chem 277, 49651-49654.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.
Britten, C.J., Turner, J.C., and Rea, P.A. (1989). Identification and purification of substrate binding subunit of higher plant H+ translocating inorganic pyrophosphatase. FEBS Lett 256, 200-206.
Bulach, D.M., Zuerner, R.L., Wilson, P., Seemann, T., McGrath, A., Cullen, P.A., Davis, J., Johnson, M., Kuczek, E., Alt, D.P., et al. (2006). Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. Proc Natl Acad Sci USA 103, 14560-14565.
Bush, D.R. (1993). Proton-coupled sugar and amino acid transporters in plants. Annu Rev Plant Physiol Plant Molec Biol 44, 513-542.
Butt, H.J., and Jaschke, M. (1995). Calculation of thermal noise in atomic force microscopy. Nanotechnology 6, 1-7.
Carystinos, G.D., Macdonald, H.R., Monroy, A.F., Dhindsa, R.S., and Poole, R.J. (1995). Vacuolar H+-translocating pyrophosphatase is induced by anoxia and chilling in seedlings of rice (Oryza sativa L.). Plant Physiol 108, 641-649.
Cooperman, B.S., Baykov, A.A., and Lahti, R. (1992). Evolutionary conservation of the active site of soluble inorganic pyrophosphatase. Trends Biochem Sci 17, 262-266.
Davies, J.M. (1997). Vacuolar energization: pumps, shunts and stress. J Exp Bot 48, 633-641.
de Souza, E.F., and Teschke, O. (2003). Liposome Stability Verification by Atomic Force Microscopy. Review of Advances in Material Sciences 5, 34-40.
Drozdowicz, Y.M., and Rea, P.A. (2001). Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6, 206-211.
Dschida, W.J.A., and Bowman, B.J. (1995). The vacuolar ATPase: sulfite stabilization and the mechanism of nitrate inactivation. J Biol Chem 270, 1557-1563.
Elston, T., Wang, H.Y., and Oster, G. (1998). Energy transduction in ATP synthase. Nature 391, 510-513.
Espenel, C., Giocondi, M.C., Seantier, B., Dosset, P., Milhiet, P.E., and Le Grimellec, C. (2008). Temperature-dependent imaging of living cells by AFM. Ultramicroscopy 108, 1174-1180.
Farr, R.W. (1995). Leptospirosis. Clin Infect Dis 21, 1-6.
Fiske, C.H., and Subbarow, Y. (1925). The colorimetric determination of phosphorous. J Biol Chem 66, 378-400.
Gans, P.J., Lyu, P.C., Manning, M.C., Woody, R.W., and Kallenbach, N.R. (1991). The helix-coil transition in heterogeneous peptides with specific side-chain interactions: theory and comparison with CD spectral data. Biopolymers 31, 1605-1614.
Gerold, G., Ajaj, K.A., Bienert, M., Laws, H.J., Zychlinsky, A., and de Diego, J.L. (2008). A Toll-like receptor 2-integrin beta3 complex senses bacterial lipopeptides via vitronectin. Nat Immunol 9, 761-768.
Gordon-Weeks, R., Steele, S.H., and Leigh, R.A. (1996). The role of magnesium, pyrophosphate, and their complexes as substrates and activators of the vacuolar H+-pumping inorganic pyrophosphatase (Studies using ligand protection from covalent inhibitors). Plant Physiol 111, 195-202.
Griffith, C.J., Rea, P.A., Blumwald, E., and Poole, R.J. (1986). Mechanism of stimulation and inhibition of tonoplast H+-ATPase of Beta vulgaris by chloride and nitrate. Plant Physiol 81, 120-125.
Haake, D.A., Chao, G., Zuerner, R.L., Barnett, J.K., Barnett, D., Mazel, M., Matsunaga, J., Levett, P.N., and Bolin, C.A. (2000). The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun 68, 2276-2285.
Hauk, P., Guzzo, C.R., Roman Ramos, H., Ho, P.L., and Farah, C.S. (2009). Structure and calcium-binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp. J Mol Biol 390, 722-736.
Hauk, P., Macedo, F., Romero, E.C., Vasconcellos, S.A., de Morais, Z.M., Barbosa, A.S., and Ho, P.L. (2008). In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin. Infect Immun 76, 2642-2650.
Hedrich, R., and Schroeder, J.I. (1989). The physiology of ion channels and electrogenic pumps in higher plants. Annu Rev Plant Physiol Plant Molec Biol 40, 539-569.
Hirschfeld, M., Kirschning, C.J., Schwandner, R., Wesche, H., Weis, J.H., Wooten, R.M., and Weis, J.J. (1999). Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll-like receptor 2. J Immunol 163, 2382-2386.
Hsiao, Y.Y., Van, R.C., Hung, S.H., Lin, H.H., and Pan, R.L. (2004). Roles of histidine residues in plant vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1608, 190-199.
Hsu, S.H., Hsiao, Y.Y., Liu, P.F., Lin, S.M., Luo, Y.Y., and Pan, R.L. (2009). Purification, characterization, and spectral analyses of histidine-tagged vacuolar H+-pyrophosphatase expressed in yeast. Botanical Studies 50, 291-301.
Hung, C.C., Chang, C.T., Tian, Y.C., Wu, M.S., Yu, C.C., Pan, M.J., Vandewalle, A., and Yang, C.W. (2006). Leptospiral membrane proteins stimulate pro-inflammatory chemokines secretion by renal tubule epithelial cells through Toll-like receptor 2 and p38 mitogen activated protein kinase. Nephrol Dial Transplant 21, 898-910.
Hung, S.H., Chiu, S.J., Lin, L.Y., and Pan, R.L. (1995). Vacuolar H+-pyrophosphatase cDNA from etiolated mung bean seedlings. Plant Physiol 109, 1125-1127.
Jin, M.S., Kim, S.E., Heo, J.Y., Lee, M.E., Kim, H.M., Paik, S.G., Lee, H., and Lee, J.O. (2007). Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130, 1071-1082.
Kaiser, G., and Heber, U. (1984). Sucrose transport into vacuoles isolated from barley mesophyll protoplasts. Planta 161, 562-568.
Kane, P.M., Yamashiro, C.T., and Stevens, T.H. (1989). Biochemical characterization of the yeast vacuolar H+-ATPase. J Biol Chem 264, 19236-19244.
Kienberger, F., Kada, G., Mueller, H., and Hinterdorfer, P. (2005). Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability. J Mol Biol 347, 597-606.
Kim, E.J., Zhen, R.G., and Rea, P.A. (1995). Site-directed mutagenesis of vacuolar H+-pyrophosphatase. Necessity of Cys634 for inhibition by maleimides but not catalysis. J Biol Chem 270, 2630-2635.
Kim, H., Arakawa, H., Hatae, N., Sugimoto, Y., Matsumoto, O., Osada, T., Ichikawa, A., and Ikai, A. (2006). Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM. Ultramicroscopy 106, 652-662.
Kuo, S.Y., Chien, L.F., Hsiao, Y.Y., Van, R.C., Yan, K.H., Liu, P.F., Mao, S.J., and Pan, R.L. (2005). Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings. J Plant Physiol 162, 129-138.
López-Marqués, R.L., Pérez-Castiñeira, J.R., Buch-Pedersen, M.J., Marco, S., Rigaud, J.L., Palmgren, M.G., and Serrano, A. (2005). Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins. Biochim Biophys Acta 1716, 69-76.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Lalonde, S., Boles, E., Hellmann, H., Barker, L., Patrick, J.W., Frommer, W.B., and Ward, J.M. (1999). The dual function of sugar carriers: transport and sugar sensing. Plant Cell 11, 707-726.
Lanfermeijer, F.C., Venema, K., and Palmgren, M.G. (1998). Purification of a histidine-tagged plant plasma membrane H+-ATPase expressed in yeast. Protein Expr Purif 12, 29-37.
Larson, E., Howlett, B., and Jagendorf, A. (1986). Artificial reductant enhancement of the Lowry method for protein determination. Anal Biochem 155, 243-248.
Lee, S.H., Kim, K.A., Park, Y.G., Seong, I.W., Kim, M.J., and Lee, Y.J. (2000). Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai. Gene 254, 19-28.
Levett, P.N. (2001). Leptospirosis. Clin Microbiol Rev 14, 296-326.
Levy, D., Mosser, G., Lambert, O., Moeck, G.S., Bald, D., and Rigaud, J.L. (1999). Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins. J Struct Biol 127, 44-52.
Lien, E., Sellati, T.J., Yoshimura, A., Flo, T.H., Rawadi, G., Finberg, R.W., Carroll, J.D., Espevik, T., Ingalls, R.R., Radolf, J.D., et al. (1999). Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 274, 33419-33425.
Lin, H.H., Pan, Y.J., Hsu, S.H., Van, R.C., Hsiao, Y.Y., Chen, J.H., and Pan, R.L. (2005). Deletion mutation analysis on C-terminal domain of plant vacuolar H+-pyrophosphatase. Arch Biochem Biophys 442, 206-213.
Liu, W., Kamensky, Y., Kakkar, R., Foley, E., Kulmacz, R.J., and Palmer, G. (2005). Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems. Protein Expr Purif 40, 429-439.
Madl, J., Rhode, S., Stangl, H., Stockinger, H., Hinterdorfer, P., Schutz, G.J., and Kada, G. (2006). A combined optical and atomic force microscope for live cell investigations. Ultramicroscopy 106, 645-651.
Maeshima, M. (1991). H+-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases. Eur J Biochem 196, 11-17.
Maeshima, M. (2000). Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465, 37-51.
Maeshima, M. (2001). Tonoplast transporters: Organization and function. Annu Rev Plant Physiol Plant Molec Biol 52, 469-497.
Maeshima, M., and Yoshida, S. (1989). Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J Biol Chem 264, 20068-20073.
Matsunaga, J., Barocchi, M.A., Croda, J., Young, T.A., Sanchez, Y., Siqueira, I., Bolin, C.A., Reis, M.G., Riley, L.W., Haake, D.A., et al. (2003). Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol Microbiol 49, 929-945.
Matsuuraendo, C., Maeshima, M., and Yoshida, S. (1990). Subunit composition of vacuolar membrane H+-ATPase from mung bean. Eur J Biochem 187, 745-751.
Matsuuraendo, C., Maeshima, M., and Yoshida, S. (1992). Mechanism for the decline of V-ATPase activity in mung bean hypocotyls during chilling. Plant Physiol 100, 718-722.
Maurel, C., Tacnet, F., Guclu, J., Guern, J., and Ripoche, P. (1997). Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity. Proc Natl Acad Sci U S A 94, 7103-7108.
McIntosh, M.T., and Vaidya, A.B. (2002). Vacuolar type H+-pumping pyrophosphatases of parasitic protozoa. Int J Parasitol 32, 1-14.
McRae, S.R., Christopher, J.T., Smith, J.A.C., and Holtum, J.A.M. (2002). Sucrose transport across the vacuolar membrane of Ananas comosus. Funct Plant Biol 29, 717-724.
Mimura, H., Nakanishi, Y., Hirono, M., and Maeshima, M. (2004). Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis. J Biol Chem 279, 35106-35112.
Morillon, R., and Lassalles, J.P. (1999). Osmotic water permeability of isolated vacuoles. Planta 210, 80-84.
Moriyama, Y., and Nelson, N. (1989). Cold inactivation of vacuolar H+-ATPase. J Biol Chem 264, 3577-3582.
Morrison, W.I., Wright, N.G., and Cornwell, H.J. (1976). An immunopathologic study of interstitial nephritis associated with experimental canine adenovirus infection. J Pathol 120, 221-228.
Muller, D.J., Helenius, J., Alsteens, D., and Dufrene, Y.F. (2009). Force probing surfaces of living cells to molecular resolution. Nat Chem Biol 5, 383-390.
Murray, G.L., Srikram, A., Hoke, D.E., Wunder, E.A., Jr., Henry, R., Lo, M., Zhang, K., Sermswan, R.W., Ko, A.I., and Adler, B. (2009). Major surface protein LipL32 is not required for either acute or chronic infection with Leptospira interrogans. Infect Immun 77, 952-958.
Nakanishi, Y., Saijo, T., Wada, Y., and Maeshima, M. (2001). Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase. J Biol Chem 276, 7654-7660.
Nakayasu, T., Kawauchi, K., Hirata, H., and Shimmen, T. (1999). Cycloprodigiosin hydrochloride inhibits acidification of the plant vacuole. Plant Cell Physiol 40, 143-148.
Neuert, G., Albrecht, C., Pamir, E., and Gaub, H.E. (2006). Dynamic force spectroscopy of the digoxigenin-antibody complex. FEBS Lett 580, 505-509.
Nishi, T., and Forgac, M. (2002). The vacuolar H+-ATPases - nature's most versatile proton pumps. Nat Rev Mol Cell Biol 3, 94-103.
Obermeyer, G., Sommer, A., and Bentrup, F.W. (1996). Potassium and voltage dependence of the inorganic pyrophosphatase of intact vacuoles from Chenopodium rubrum. Biochim Biophys Acta 1284, 203-212.
Ozinsky, A., Underhill, D.M., Fontenot, J.D., Hajjar, A.M., Smith, K.D., Wilson, C.B., Schroeder, L., and Aderem, A. (2000). The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc Natl Acad Sci USA 97, 13766-13771.
Palaniappan, R.U., Chang, Y.F., Hassan, F., McDonough, S.P., Pough, M., Barr, S.C., Simpson, K.W., Mohammed, H.O., Shin, S., McDonough, P., et al. (2004). Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J Med Microbiol 53, 975-984.
Palaniappan, R.U., Chang, Y.F., Jusuf, S.S., Artiushin, S., Timoney, J.F., McDonough, S.P., Barr, S.C., Divers, T.J., Simpson, K.W., McDonough, P.L., et al. (2002). Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. Infect Immun 70, 5924-5930.
Palmgren, M.G., Askerlund, P., Fredrikson, K., Widell, S., Sommarin, M., and Larsson, C. (1990). Sealed inside-out and right-side out plasma membrane vesicles. Optimal conditions for formation and separation. Plant Physiol 92, 871-880.
Picardeau, M., Bulach, D.M., Bouchier, C., Zuerner, R.L., Zidane, N., Wilson, P.J., Creno, S., Kuczek, E.S., Bommezzadri, S., Davis, J.C., et al. (2008). Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS One 3, e1607.
Ratajczak, R. (2000). Structure, function and regulation of the plant vacuolar H+-translocating ATPase. Biochim Biophys Acta-Biomembr 1465, 17-36.
Rea, P.A., Kim, Y., Sarafian, V., Poole, R.J., Davies, J.M., and Sanders, D. (1992). Vacuolar H+-translocating pyrophosphatases: a new category of ion translocase. Trends Biochem Sci 17, 348-353.
Rea, P.A., and Poole, R.J. (1993). Vacuolar H+-translocating pyrophosphatase. Annu Rev Plant Physiol Plant Mol Biol 44, 157-180.
Rief, M., Oesterhelt, F., Heymann, B., and Gaub, H.E. (1997). Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy. Science 275, 1295-1297.
Riethmuller, C., Schaffer, T.E., Kienberger, F., Stracke, W., and Oberleithner, H. (2007). Vacuolar structures can be identified by AFM elasticity mapping. Ultramicroscopy 107, 895-901.
Ristow, P., Bourhy, P., da Cruz McBride, F.W., Figueira, C.P., Huerre, M., Ave, P., Girons, I.S., Ko, A.I., and Picardeau, M. (2007). The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog 3, e97.
Ron, A., Singh, R.R., Fishelson, N., Socher, R., Benayahu, D., and Shacham-Diamand, Y. (2008). Site localization of membrane-bound proteins on whole cell level using atomic force microscopy. Biophys Chem 132, 127-138.
Saftner, R.A., Daie, J., and Wyse, R.E. (1983). Sucrose uptake and compartmentation in sugar beet taproot tissue. Plant Physiol 72, 1-6.
Schäffer, T.E., Cleveland, J.P., Ohnesorge, F., Walters, D.A., and Hansma, P.K. (1996). Studies of vibrating atomic force microscope cantilevers in liquid. Journal of Applied Physics 80, 3622-3627.
Schneider, S.W., Larmer, J., Henderson, R.M., and Oberleithner, H. (1998). Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pflugers Arch 435, 362-367.
Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., and Kirschning, C.J. (1999). Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. J Biol Chem 274, 17406-17409.
Shiratake, K., Kanayama, Y., Maeshima, M., and Yamaki, S. (1998). Changes in tonoplast protein and density with the development of pear fruit. Physiol Plant 103, 312-319.
Stevenson, B., Choy, H.A., Pinne, M., Rotondi, M.L., Miller, M.C., Demoll, E., Kraiczy, P., Cooley, A.E., Creamer, T.P., Suchard, M.A., et al. (2007). Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement. PLoS ONE 2, e1188.
Sze, H., Li, X.H., and Palmgren, M.G. (1999). Energization of plant cell membranes by H+-pumping ATPases: Regulation and biosynthesis. Plant Cell 11, 677-689.
Takasu, A., Nakanishi, Y., Yamauchi, T., and Maeshima, M. (1997). Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies. J Biochem (Tokyo) 122, 883-889.
Takeda, K., and Akira, S. (2004). TLR signaling pathways. Semin Immunol 16, 3-9.
Takeda, K., Kaisho, T., and Akira, S. (2003). Toll-like receptors. Annu Rev Immunol 21, 335-376.
Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R.L., and Akira, S. (2002). Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 169, 10-14.
Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.
Tian, Y.C., Chen, Y.C., Hung, C.C., Chang, C.T., Wu, M.S., Phillips, A.O., and Yang, C.W. (2006). Leptospiral outer membrane protein induces extracellular matrix accumulation through a TGF-beta1/Smad-dependent pathway. J Am Soc Nephrol 17, 2792-2798.
Tsai, Y.R., Yang, S.J., Jiang, S.S., Ko, S.J., Hung, S.H., Kuo, S.Y., and Pan, R.L. (1998). High-pressure effects on vacuolar H+-ATPase from etiolated mung bean seedlings. J Protein Chem 17, 161-172.
Tsuboi, N., Yoshikai, Y., Matsuo, S., Kikuchi, T., Iwami, K., Nagai, Y., Takeuchi, O., Akira, S., and Matsuguchi, T. (2002). Roles of Toll-like receptors in C-C chemokine production by renal tubular epithelial cells. J Immunol 169, 2026-2033.
Tung, J.Y., Yang, C.W., Chou, S.W., Lin, C.C., and Sun, Y.J. (2009). Calcium binds to LipL32, a lipoprotein from pathogenic Leptospira, and modulates fibronectin binding. J Biol Chem, (In press) doi:10.1074/jbc.M1109.006320.
Tzeng, C.M., Yang, C.Y., Yang, S.J., Jiang, S.S., Kuo, S.Y., Hung, S.H., Ma, J.T., and Pan, R.L. (1996). Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation. Biochem J 316 ( Pt 1), 143-147.
Uchida, E., Ohsumi, Y., and Anraku, Y. (1985). Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 260, 1090-1095.
Vanheeswijk, M.P.E., and Vanos, C.H. (1986). Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine. J Membr Biol 92, 183-193.
Verma, A., Hellwage, J., Artiushin, S., Zipfel, P.F., Kraiczy, P., Timoney, J.F., and Stevenson, B. (2006). LfhA, a novel factor H-binding protein of Leptospira interrogans. Infect Immun 74, 2659-2666.
Vivian, J.P., Beddoe, T., McAlister, A.D., Wilce, M.C., Zaker-Tabrizi, L., Troy, S., Byres, E., Hoke, D.E., Cullen, P.A., Lo, M., et al. (2009). Crystal structure of LipL32, the most abundant surface protein of pathogenic Leptospira spp. J Mol Biol 387, 1229-1238.
Walker, R.R., and Leigh, R.A. (1981). Mg2+-dependent, cationstimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 153, 150-155.
Wang, M.Y., Lin, Y.H., Chou, W.M., Chung, T.P., and Pan, R.L. (1989). Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings. Plant Physiol 90, 475-481.
Ward, J.M., Kuhn, C., Tegeder, M., and Frommer, W.B. (1998). Sucrose transport in higher plants. In International Review of Cytology - a Survey of Cell Biology, Vol 178 (San Diego, Academic Press Inc), pp. 41-71.
Ward, J.M., and Sze, H. (1992). Proton transport activity of the purified vacuolar H+-ATPase from oats. Direct stimulation by Cl-. Plant Physiol 99, 925-931.
Webb, M.R. (1992). A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89, 4884-4887.
Werts, C., Tapping, R.I., Mathison, J.C., Chuang, T.H., Kravchenko, V., Saint Girons, I., Haake, D.A., Godowski, P.J., Hayashi, F., Ozinsky, A., et al. (2001). Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat Immunol 2, 346-352.
Wu, J.J., Ma, J.T., and Pan, R.L. (1991). Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation. FEBS Lett 283, 57-60.
Yamaki, S. (1987). ATP-promoted sorbitol transport into vacuoles isolated from apple fruit. Plant Cell Physiol 28, 557-564.
Yang, C.W. (2007). Leptospirosis renal disease: understanding the initiation by Toll-like receptors. Kidney Int 72, 918-925.
Yang, C.W., Wu, M.S., Pan, M.J., Hsieh, W.J., Vandewalle, A., and Huang, C.C. (2002). The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells. J Am Soc Nephrol 13, 2037-2045.
Yang, S.J., Jiang, S.S., Hsiao, Y.Y., Van, R.C., Pan, Y.J., and Pan, R.L. (2004). Thermoinactivation analysis of vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1656, 88-95.
Yang, S.J., Jiang, S.S., Kuo, S.Y., Hung, S.H., Tam, M.F., and Pan, R.L. (1999). Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N, N'-dicyclohexylcarbodi-imide. Biochem J 342 Pt 3, 641-646.
Yang, S.J., Jiang, S.S., Tzeng, C.M., Kuo, S.Y., Hung, S.H., and Pan, R.L. (1996). Involvement of tyrosine residue in the inhibition of plant vacuolar H+-pyrophosphatase by tetranitromethane. Biochim Biophys Acta 1294, 89-97.