行政院國家科學委員會專題研究計畫 成果報告
利用基因重組方法探討大白鼠 alphaA-水晶體蛋白之功能與 結構(Ⅱ)
計畫類別： 個別型計畫
計畫編號： NSC92-2113-M-006-020-
執行期間： 92 年 08 月 01 日至 93 年 07 月 31 日 執行單位： 國立成功大學化學系（所）
計畫主持人： 黃福永
報告類型： 精簡報告
處理方式： 本計畫可公開查詢
中 華 民 國 93 年 11 月 1 日
行政院國家科學委員會專題研究計畫成果報告
*********************************************************************
利用基因重組方法探討大白鼠 α-水晶體蛋白之功能與結構(II)
Functional and Structural Studies of Mutant Rat Lens α-Crystallins (II)
*********************************************************************
計劃類別：個別型計畫
計畫編號：NSC92-2113-M-006-020
執行期間：92 年 8 月 1 日 至 93 年 7 月 31 日
個別型計畫：計畫主持人：黃福永
處理方式：▓已送發表
執行單位：國立成功大學化學系 中華民國 93 年 10 月 25 日
1
In the project of “Functional and Structural Studies of Mutant Rat Lens alpha-Crystallin (II)”, we have constructed the αins-crystallin, αA-crystallin and mutant D69R and D69W αA-crystallin genes, and obtained the pure crstallins. The chaperone-like activity of these three crystallins against the DTT-induced insulin B chian aggregation has been measured. In addition to this functional and structural studies of wild type and mutant α-crystallins, we also completed the studies in the purification and characterization of rice coleoptile protease inhibitors and also collaborated with Dr. T.–H. Wu of Department of Clinical Pharmacy, Taipei Medical University and completed a project: In vitro and in vivo studies of Astaxanthin, a cartenoid with powerful biological antioxidant, in the prevent of the development of selenite-induced lens cataract and the damage of hydroxyl radical induced α-crystallins damage. The results of the above three projects have been basiclly completed and summarized. Followings are the paper title and the abstract contents.
Paper # 1:
Title: Structural and Chaperone-like activity Studies of Mutant D69R and D69W Rat Lens αA-crystallins
Abstract: Wild type αins-crystallin, αA-crystallin and mutant D69R and D69W αA-crystallin genes had been cloned and expressed and the purified crystallins have been obtained. The chaperone-like activities of these four crystallins have been performed against DTT-induced insulin B chain aggregation. It was found that in the molar ratio of insulin B chain to α-crystallin, the αA-crystallin and mutant D69R αA-crystallin showed have the similar activity as that of α-crystallin, while the αins-crystallin and mutant D69R αA-crystallin showed much less activities (about 55% lower) compared with that of αA-crystallin and mutant D69R αA-crystallin.
The αins-crystallin has extra 23 amino acids than αA-crystallin, which is inserted into the 23th and 24th amino acids of αA-crystallin. This indicates the extra amino acids
may affect the overall hydrophobic region as to cause the decrease of activity. The mutant D69R showed the same activity as that of wild type αA-crystallin, while D69W did not. These results implied that the polarity of the 69th amino acid plays an important role in maintaining the chaperone-like activity. As for whether the size of amino acid will affect the chaperone-like activity, the mutant R69L is under exploration.
Paper #2
Title: Oxidative Growth Retardation of Rice Coleoptiles Up-regulate the Expression of Bowman-Birk Inhibitors When Germinating in Submergence
Abstract: One of the rice Bowman-Birk inhibitor proteins, BBIrc1, from fast elongating coleoptiles has been purified to its homogeneous and identified by partial N-terminal sequence, LC-MS and ESI-MS as a 133 amino acid polypeptide expressed from a splicing gene of BBIrc3. This protease inhibitor displayed a competitive inhibition toward trypsin with a dissociation constant (Ki) of 4.0 x 10-7 M; while it displayed an noncompetitive inhibition toward α-chymotrypsin with a Ki of 9.3 x 10-6 M. And it bonded to trypsin and α-chymotrypsin in a molar ratio of 2:1 and 1:1. The result of the anti-sera arose against this protein presented that the expression pattern of BBIs proteins correlated to the developmental stage in terms of morphological changes. This anti-serum recognized two species of BBI proteins with molecular size around 28 K and 16 K in the elongating coleoptiles. The expression of 16K species proteins was in a nearly constant quantity under both aerobic and hypoxia conditions;
however, the 28 K proteins expression were greatly up-regulated when the fast elongating coleoptiles were transferred from hypoxia into aerobic condition. This expression pattern matched to that of the elongation retardation of coleoptiles and
3
indicated that the more BBI of 28 K expressed the less coleoptiles elongated. Both species of BBI proteins could be detected in the coleoptile organ even after the cell death. This study implies that the BBI proteins might play multiple biological functions inside the rice coleoptiles.
Paper #3
Title: The in vitro and in vivo Studies of Astaxathin against the Oxidative stress-induced Alpha-crystallin damage and cataractogenesis
Abstract: Cataract is the major cause of blindness; the most common form is the age-related senile cataract. Environmental toxins or the foreign insults and the nutritional status have shown greatly link to the development of cataracts. The formation of hydroxyl radicals and the accumulation of calcium ion in lens have also been recognized as two major causes resulting in the lens protein modifications, which in turn leads to the development of cataract. These two mechanisms also account for the selenite-induced cataractogenesis in rat pups. Astaxanthin(ASTX), a carotenoid with antioxidant properties, exists in most plants, algae, and in marine seafood as well. Astaxanthin is also available as a food supplement. Currently much attention has been focused on its biological activities. The anti-cataractogenesis activities of ASTX on lenses have been studied in vitro and in vivo, respectively. In vitro study showed that antioxidative actions of ASTX towards lens proteins were
demonstrated by measuring tryptophan oxidations using fluorescence at 290 nm. The declines in tryptophan fluorescence intensity of crystallins, reflecting the destruction of these residues, were observed while incubated with iron and hydrogen peroxide in a concentration and time-dependent manner. Among crystallins, loss of βHigh-crystallin were the most significant following radical insults and α- and γ-crystallins were partially degraded. Inhibitory of ASTX on calcium-induced turbidity was measured daily at 405 nm and SDS-PAGE then assessed proteolysis of
lens crystallins. ASTX (1 mM) was equivalent to 10 mM glutathione in preventing crystallin aggregations. Appreciable in vitro protection of ASTX against lens protein fragmentations induced by metal-mediated free radicals occurred. ASTX, similar to calpain inhibitor E64, also inhibits crystallins proteolysis by calcium-activated calpain in a dose-dependent manner. ASTX (1 mg/kg) also provides anti-cataractogenesis (75%) in a selenite-induced cataract animal model. ASTX possesses the observed in vivo protection of ASTX against experimental cataract development by virtue of its
antioxidant properties and capacities of endogenous reduced glutathione reservations in lenses. These data are consistent with the hypothesis that supplementation of ASTX may be beneficial for oxidative stress-related disease prevention. A promising protective action of ASTX for prophylaxis against cataract is demonstrated in this study.
