甘藷塊根Sporamin蛋白質定點突變對胰蛋白脢抑制因數活性之影響

Abstract

Sporamin為甘藷塊根中含量最豐富的儲藏性蛋白質，我們將所篩選出的sporamin cDNA構築於表現載體pGEX2T，並轉殖至大腸桿菌(Escherichia coli) XL1-Blue中表現，證實其具有胰蛋白脢抑制因數(Trypsin inhibitor)活性。經由初步分析胺基酸一級排列(Sequence alignment)、二級結構推演(Secondary structure prediction)，並比對資料庫(Protein data bank, PDB)的結果，模擬sporamin的三級結構應與Erythrina caffra所含的ETI結構相近，因而推測出與活性有關的胺基酸位置所在，進一步以定點突變(Site-directed mutagenesis)的方式，分析突變對於sporamin活性的影響。發現Asp70Val、Glu72Arg及Ser73lle突變株均喪失胰蛋白脢抑制因數活性，而Ala 69Ser則仍維持活性，乃確定甘藷塊根sporamin的活性區域為72Arg-73Ser；此結果與先前所模擬出的三級結構相符。此外，由於sporamin的結構中含有兩對雙硫鍵，乃設計將45Cys置換為Leu以打破雙硫鍵，結果發現此突變株喪失了胰蛋白脢抑制因數活性。所以，維持sporamin結構的完整性，對於胰蛋白脢抑制因數活性的存在是有必要的。確定sporamin之活性區所在後，回歸三級結構的預測，便可重新定出sporamin完整的蛋白質三級結構圖。

In our earlier studies, we screened four sporamin cDNA clones from sweet potato tubers and demonstrated their trypsin inhibitory (TI) activities as well as insect resistance. However, we could not identify the precise TI activity domain of our clones by amino acid sequence alignment with other TIs or three-dimensional (3D) modeling. In this study, we identified amino acid residues that were important for the sporamin TI activity by site-directed mutagenesis. Modification on three amino acid residues, namely Asp70Val, Glu72Arg, and Ser7311e, resulted in a complete loss of TI activity. In contrast, modification of Ala69Ser retained the TI activity. Therefore, we concluded that the TI activity domain of the sweet potato sporamin resided in 69Asp-73Ser. Results using 3D modeling also supported our conclusion. We also identified two disulfide bonds within sporamin. When one of the cysteine residues, Cys45, was mutated to leucine, the TI activity was lost. This indicated that disulfide bonding and the integrity of the structure of sporamin were important factors contributing to the TI activity.