邱式鴻2006-07-252018-07-062006-07-252018-07-062003-10-31http://ntur.lib.ntu.edu.tw//handle/246246/10244蛋白凝集可能是引起光散射及水 晶體白內障形成的主因，由於 α-水晶 體蛋白具分子保護者活性，可能可以 保護其它水晶體蛋白，以避免水晶體 蛋白在長期受到紫外線、醣化及氧化 的影響下失去結構的穩定性，凝集產 生沉澱，暗示其與水晶體因老化而形 成白內障有重大關聯。除此之外，αB- 水晶體蛋白在其它器官組織中，尤其 是心臟及骨骼肌，被發現與細胞骨架 (cytoskeleton) 尤其是intermediate filaments 的穩定性有很大的關聯性， 為探討α-水晶體蛋白結構及功能的相 關性，因此我們選殖並表現豬 αB-及 土虱αB-水晶體蛋白，以比較重組αB- 水晶體蛋白的熱穩定性及凝集性質的 差異與蛋白結構上的關聯性。在本計 畫年度中，由於α-水晶體蛋白C 末端 的延伸區被認為與α-水晶體蛋白對水 溶液溶解度有決定性的影響（1,2），而 且C 端的變異在生物體系統中則常見 於α-水晶體蛋白的重大破壞性的改變 （3）；而這些改變很可能導致保護性 功能的喪失，甚至可能引發白內障的 形成，因此我們利用定點突變法進行 豬 αB-水晶體蛋白位於C 末端的延伸 區胺基酸的突變。結果發現，去除尾 端具高保守性的兩個 Lysine 殘基後 在高溫下仍具有蛋白保護活性，除此 之外，利用圓二色偏光譜儀、動態光 散射光譜儀來探討蛋白質的立體結構 與聚集特性。另外，土虱由於其生活 環境之故，被認為其眼睛在退化中， 比較氨基酸序列的結果顯示在αB-水 晶體蛋白的N 端有三個高保守性的氨 基酸缺失(圖十一)，由於αB-水晶體蛋 白的N 端與其形成聚合體有關，而且 土虱αB-水晶體蛋白的聚合體分子量 約在400,000 Da，約為豬αB-水晶體蛋 白的2/3，由此建構土虱多三個氨基酸 與豬少三個氨基酸的αB-水晶體蛋白 突變株，藉以了解這三個氨基酸對αB- 水晶體蛋白聚合體分子量的影響。再 其次，土虱αB-水晶體蛋白的熱穩定性 研究中發現，其半凝集溫度比豬高14.5 ℃，在蛋白保護活性研究中發現，土 虱 αB-水晶體蛋白的蛋白保護活性比 豬的來源高或相似，可能是因為魚類 接觸外來蛋白質變性物質的機會比哺 乳類高的緣故。α-Crystallin, a major protein of all vertebrate lenses, consisting of two subunits αA and αB, forms polymeric aggregates with an average molecular mass of about 800 kDa. In this study, we have employed various physical methods to study aggregate sizes and conformational properties of porcine α-crystallin and homomultimeric aggregates of its purified αA, αB subunits and cloned recombinant αB subunit. From far- and near-UV CD spectra, native α-, αA-, αB- and recombinant αB-crystallins from porcine lenses all show similar β-sheet conformation to that from bovine and human lenses. By means of gel-filtration chromatography and dynamic light scattering, we have found that the molecular sizes of all four crystallin aggregates are polydispersedly distributed in the following order of aggregate sizes, i.e., native α > αA > αB ≈ recombinant αB. In contrast, the molecular weights as determined from sedimentation velocity and equilibrium studies appear to indicate that the molecular size of native or recombinant αB-crystallin is somewhat greater than αA-crystallin. To further investigate the structural and functional relationship, we have also compared the chaperone activities of all four α-crystallin aggregates at different temperatures. From the results of chaperone-activity assays, ANS (8-anilo-1-naphthalene sulfonate) binding and thermal stability studies, there appeared to be at least two factors playing major roles in the chaperone-like activity of these lens proteins: one is the hydrophobicity of exposed protein surface and the other is the structural stability associated with each protein. We showed that αA-crystallin is a better chaperone to protect γ-crystallin against UV-irradiation than αB-crystallin, in contrast to the observation that αB is generally a better chaperoning protein than αA for enzyme protective assays at physiological temperatures. We have also analyzed, expressed and characterized the catfish αB-crystallin, which presents itself as a major structural component in eye lenses and as a small heat shock protein in other tissues of most mammals. Catfishes which reside generally in streams, ponds, ditches and reservoirs, always in the dark or underground environment, are good examples of nocturnal scavengers with atrophied eyes. Sequence comparison with homologues of distantly related taxa has revealed conservation of intron splicing sites and coding regions; however the two intron sequences, 5’ and 3’ untranslated regions of catfish αB-crystallin are shorter than those reported for other vertebrates. The deduced amino-acid sequence of catfish αB-crystallin gene was found to lack a segment of three amino acids (SPF, i.e. Ser-Pro-Phe) in the N-terminal region, which were conserved in all of its homologues characterized from other vertebrate classes. The phylogenetic analysis based on protein sequences indicated that the molecular evolution of αB-crystallins of dogfish, zebrafish and catfish followed three independent evolutionary routes, distinct from αB-crystallins of other vertebrate species. The wild type and insertion mutant (+SPF mutant) of catfish αB-crystallins were expressed and characterized. The most striking feature was that the midpoint for aggregation (Ta) of the purified recombinant catfish αB-crystallin was 15°C higher than that of porcine one, whereas Ta of +SPF mutant was slightly lower than that of wild type. The molecular mass of +SPF mutant as determined by analytical gel filtration is higher than that of the wild type (~440 kDa) and similar to porcine one (~600 kDa). The relative order of chaperone activity was found to be as follows: catfish wild type > catfish +SPF mutant > porcine wild type αB-crystallin for enzyme protection assay at 60°C and insulin reduction assay at 37°C. The surface hydrophobicity as determined by fluorescent dye-binding analysis for the wild type αB-crystallins of catfish is similar to that of porcine but higher than that of catfish +SPF mutant. Therefore both aggregate size and surface hydrophobicity of crystallin may play some role in the chaperone-like function of αB-crystallin.application/pdf41092 bytesapplication/pdfzh-TW國立臺灣大學生化科學研究所水晶體蛋白分子保護者抗熱「保護者」活性老年性白內 障熱休克蛋白reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/10244/1/912311B002035.pdf