臺灣博碩士論文加值系統

摘要︰

最近α-水晶體蛋白被歸類為小熱休克蛋白家族的一員，在1992年Joseph Horwitz更證明它具有分子保護作用，可保護水晶體中其他蛋白及酵素，免於高溫形成凝集。本研究的目的︰則是要進一步探討α-水晶體蛋白結構狀態與分子保護功能的相關性。

在此一研究工作，首先經由cDNA的選殖，我們得到豬的αB-水晶體蛋白的cDNA序列，共有525個鹽基對，相當於175個氨基酸殘基；以此αB基因組轉殖入大腸桿菌，表現出來的蛋白質，可與αB抗血清交叉反應，並具有分子保護作用。

其次，我們發現四大類脊椎動物中，包括哺乳類的牛、鳥類的鴨、爬蟲類的鱷魚、及魚類的鯊魚等，其α-水晶體蛋白的分子保護功能，恰如其初級結構在演化上的保守性，彼此間的差異很小；另外在保護的對象上，亦無受質的專一性。

我們的研究也顯示︰α-水晶體蛋白的分子保護功能，也可以作用於紫外線、氧化及其他壓力下；但是其保護效能較差。部份原因在於α-水晶體蛋白，在紫外線的輻射下會逐漸受到破壞，且破壞的程度與分子保護作用的喪失，呈正相關性。另外人類老年性白內障所得的α-水晶體蛋白，比由正常年輕水晶體的此種蛋白之保護作用變差，這種隨著年齡而減低的功能，可能與眼睛在紫外線的長期暴露有關；同時也說明了α-水晶體蛋白在維持水晶體透明度的重要角色。

α-水晶體蛋白的分子保護作用機制，在於它可與部份變性的蛋白結合成複合物，使其免於進一步的變性。我們將α-水晶體蛋白與其受質短暫加熱，使部份形成複合物，以模擬體內α-水晶體蛋白與β-或γ-水晶體蛋白的結合情形，結果這樣會比單純的分子保護作用對紫外線或氧化等壓力，提供更大的保護；所以在維持水晶體的透明度上，α-水晶體蛋白與其受質的結合，另有更深一層的生理意義。

由圓偏光二色光譜及其他分析，可知α-水晶體蛋白的二級結構主要為β層狀構造。在30℃以上，α-水晶體蛋白會逐漸產生一個不可逆的三級結構變化，並導致其表面疏水性質的提高；這與其分子保護作用的增強，亦呈正相關，顯示疏水性交互作用，在此分子保護作用上的意義。另外在50-70℃，α-水晶體蛋白也會產生一個大部份可逆的二級結構變化，然而後者與其分子保護作用的相關性不大。

總結我們的研究顯示︰α-水晶體蛋白除了傳統的結構蛋白角色外，在維持水晶體透明上，更有極重要的〝管家〞功能；這種功能主要取決於其結構狀態，及其與受質結合之複合物的形成；這些將可提供我們未來在探討白內障的成因與防治上，一個新的思考方向。

ABSTRACT:

Recently, α-crystallin is regarded as a member of small heat shock proteins. Joseph Horwitz also demonstrated in 1992 that it can act as a molecular chaperone to prevent thermal aggregation of other crystallins and enzymes. The main purpose of this thesis is to explore the relationship between the conformational states of α-crystallin and its chaperone activity.

We have first determined the cDNA sequence of αB-crystallin from porcine lenses by cDNA cloning technique. It contains one complete full-length reading frame of 525 base pairs, covering a protein sequence of 175 amino acids. Further expression of this αB subunit chain in E.coli generated a polypeptide which can cross-react with the antiserum against the native αB-crystallin, and also possesses chaperone activity.

The next, a comparative study was conducted to analyze the chaperone activity of α-crystallin from bovine (mammal), duck (bird), caiman (reptile), and shark (fish), respectively. Our results show that the difference in the chaperone activity of α-crystallins among different species is relatively small, indicative of the evolutionary conservation in function similar to that revealed by their conserved protein structure. α-Crystallins of different species also show no substrate specificity in their chaperone activity.

In addition to its anti-heat shock property, α-crystallin can also act as a chaperone against UV-irradiation, H2O2-oxidation, and other stress. However, in terms of stoichiometry, such protective ability is not so efficient as that under thermal stress. Part of the reason is the photochemical susceptibility of α-crystallin to UV-irradiation. The UV-induced destruction was found to correlate with its loss of chaperone activity. Similarly, there is some age-dependent change in the chaperone activity of α-crystallin obtained from a young and normal lens as compared to an old and cataractous lens. Thus, the gradual loss of chaperone activity of α-crystallin upon aging is probably through an accumulative event of long term exposure to UV light, which may also shed light on human cataract formation.

The mechanism underlying the chaperone activity of α-crystallin involves preferential binding of the partially denatured substrate protein to α-crystallin, and the formation of a stable complex. We have demonstrated that the binding of substrate proteins to α-crystallin by short-term pre-incubation may mimic the in vivo conditions of crystallin association. Under such conditions, the chaperone activity of α-crystallin to inhibit ultraviolet-, or oxidation-induced protein aggregation can be greatly enhanced. Thus, the presence ofα-β and α-γ complex in vivo may be relevant to the process of maintaining lens transparency.

From the result of circular dichroism and other studies, it is generally accepted that the secondary structure of α-crystallin consists predominantly of β-sheets. A minor but detectable perturbation in the tertiary structure of α-crystallin occurs at above 30℃, which correlates with the extent of exposure of its hydrophobic surfaces. Such increase in surface hydrophobicity also correlates with its increased chaperone activity. These results indicate that hydrophobic interaction play a major role in the chaperone action of α-crystallin. Another thermotropic transition in the secondary structure of α-crystallin occurs at a range between 50 and 70℃, which is largely reversible. However, the heat-induced changes in secondary structure shows a relatively little effect on the chaperone activity of α-crystallin.

In summary, α-crystallin is not only a major structural protein of the lens but may also play an important "house-keeping" role as a molecular chaperone. Both the conformational state of α-crystallin and the association complex with its substrate may contribute to a generalized mechanism of its chaperone function. Such results may provide us a new direction in the study of cataractogenesis and its treatment in the future.

封面
目錄
略語表
中文摘要
英文摘要
第一章緒論
第二章文獻回顧
第三章材料與方法
第1節α-水晶體蛋白的製備、及西方墨點法
第2節mRNA的製備、cDNA的選殖與定序
第3節αB-水晶體蛋白的表現與製備
第4節α-水晶體蛋白分子保護作用的測定
第5節高效液態層析
第6節螢光分析
第7節圓偏光二色光譜
第四章豬αB-水晶體蛋白的特性、選殖與表現
第五章脊椎動物四大綱之α-水晶體蛋白分子保護作用的比較
第六章α-水晶體蛋白在紫外線破壞下的分子保護作用
第七章α-水晶體蛋白與其受質結合成複合物的生理意義
第八章α-水晶體蛋白立體結構的變化與分子保護作用的相關性
第九章綜合討論與未來展望
第十章圖表
第十一章參考文獻
附錄：修業期間已發表之文獻

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