| 摘要: | 當砷化物進入細胞內，在細胞內的累積作用是造成毒性的重要原因。本研究藉由p-aminophenylarsine oxide (PAO)親和管柱及蛋白質體學技術鑑定抗砷細胞 (SA7, 源自中國倉鼠卵巢細胞CHOA)的砷化物結合蛋白，以利系統性研究砷化物作用的標的，並了解蛋白質序列上cysteine數目及位置與砷結合之機制及對蛋白質特性的影響。結果顯示表現量差異超過兩倍以上的蛋白中，至少有11個蛋白在SA7細胞中表現量是增加的，而有8個蛋白表現量是降低的；除先前已鑑定為砷化物結合蛋白的galectin-1 (hamster GAL1, haGAL1)及peroxiredoxin 1 (Prdx1)外，本研究也發現在胺基酸序列上只含有一個cysteine的heat-shock protein 27 (HSP27)與完全不具有cysteine的reticulocalbin-3 (RCN3)皆會與砷產生結合作用，而與Prdx1同屬peroxiredoxin家族的peroxiredoxin 6 (Prdx6)，雖亦具有一個cysteine，但不具有與砷結合的能力。進一步以蛋白質點突變技術 (site-directed mutagenesis)，探討cysteine在蛋白質中與砷結合所扮演的角色，結果顯示GAL1-Cys16、GAL1-Cys88點突變、所有GAL1-cysteine雙重點突變組及HSP27-CA突變組會顯著降低haGAL1和HSP27對砷化物的結合能力；GAL1-Cys60突變組則較wild type GAL1 (haGAL1WT)及其他突變組與砷有較強的結合能力。圓二色光譜儀 (circular dichroism, CD)的結果也顯示亞砷酸鈉的存在會明顯改變haGAL1WT及HSP27-CA的熱穩定性及熔解溫度 (melting temperature, Tm)。以上結果說明特殊位置的cysteine在影響GAL1與砷的結合力上扮演重要的角色。當單一突變GAL1序列上具-OH基的Thr90、Thr97及帶負電胺基酸的Glu86、Glu105或Glu134後，反而會明顯增加GAL1與砷的結合力；此外，當存在亞砷酸鈉時，會造成這些非cysteine胺基酸突變組的圓二色光譜圖明顯的位移。這些結果也顯示當haGAL1與砷產生結合後，其二級結構也可受特殊非cysteine胺基酸的影響。並利用核磁共振 (nuclear magnetic resonance, NMR)技術探討亞砷酸鈉結合對人類GAL1 (hGAL1)序列中胺基酸的化學位移，顯示隨著亞砷酸鈉濃度的增加，不但會使光譜的訊號減弱，並增加某些胺基酸的位移；此結果顯示，隨著亞砷酸鈉與hGAL1蛋白的結合濃度增加，可能牽動更多胺基酸產生變化。目前已利用坐式蒸氣擴散法 (sitting drop vapor diffusion)培養出hGAL1的晶體，並可觀察到蛋白質的繞射點，進一步利用X-ray晶體繞射法 (X-ray crystallography)分析蛋白質的結構，以釐清hGAL1蛋白質與亞砷酸鈉產生結合的機制。依據本研究我們瞭解在GAL1及HSP27蛋白與砷化物結合上，特定的cysteine扮演了非常重要的角色，與砷結合會改變蛋白質結構，但在RCN3及Prdx6中，與砷的作用力可能受其他因子影響。Arsenic is toxic to the cells in the living organisms. The present dissertation was designed to characterize arsenic-binding proteins, which were isolated from the arsenic-resistant Chinese hamster ovary cell line SA7 by the methods of a p-aminophenylarsine oxide (PAO)-agarose affinity column and proteomic techniques. The study was also to understand how arsenic-binding proteins interact with arsenic based on their number and position of cysteine residues. The results showed that there were 11 up-regulated and 8 down-regulated proteins in SA7 cells. The arsenic-binding proteins eluted from the PAO-agarose column included heat shock protein 27 (HSP27, with one cysteine residue), reticulocalbin-3 (RCN3, with no cysteine residue), and hamster galectin-1 (haGAL1, with six cysteine residues) while peroxiredoxin 6 (Prdx6) containing one cysteine residue was not eluted from this column. Site-directed mutagenesis of these arsenic-binding proteins at the cysteine residue to alanine further indicated that GAL1-C16A, GAL1-C88A, all double mutants of haGAL1, and HSP27-CA mutant exhibited much less binding capacity for sodium arsenite when compared to the wild-type GAL1 (haGAL1WT) and HSP27 proteins. In contrast, the GAL1-C60A mutant had greater binding capacity than the haGAL1WT and other haGAL1 mutants. Circular dichroism (CD) analysis also showed that the presence of sodium arsenite caused significant changes in the thermal stability and the melting temperature of haGAL1WT and HSP27-CA mutants. These observations suggest that the particular positions of cysteine residue affect the binding capacity of haGAL1 for arsenic. Interestingly, the single mutation in amino acid position of either Thr90, Thr97, Glu86, Glu105, or Glu134 was formed to significantly increase the binding capacity for haGAL1 with arsenite. Furthermore, these non-cysteine amino acid mutants displayed a significiently shift of CD curve in the presence of sodium arsenite. This suggests that the secondary structure of haGAL1 responsible for binding to arsenic is also dependent on the particular non-cysteine amino acid residues. Moreover, nuclear magnetic resonance (NMR) analysis showed that sodium arsenite induced chemical shift of amino acids in human GAL1 (hGAL1) in a dose-dependent manner and increasing concentrations of arsenite decreased the NMR signal and increased structural movement of amino acids. Finally, the hGAL1 protein was crystallized by the sitting-drop vapour-diffusion method, and the hGAL1 crystal will form the basis for further X-ray crystallographical analysis to delineate the exact mechanism of how the hGAL1 protein binds to sodium arsenite. Taken together, these results of the dissertation provide the insight into our understanding that the particular cysteine residues in GAL1 and HSP27 may play a critical role in the binding of arsenic and then change the structure of these proteins, but that in the case of RCN3 and Prdx6, this interaction may be mediated by other factors. |
