甘藷凝集素與不同金屬結合之晶體結構與熱力學分析

Abstract

植物凝集素 (plant lectin)為一種醣結合蛋白質，能和醣類進行專一且可逆的結合，具抗蟲抗菌的能力，是常見植物防禦蛋白質。本實驗的研究對象為甘藷凝集素(Ipomoelin, IPO)，選殖自甘藷台農57號 (Ipomoea batatas cv. Tainung 57)，歸類於波羅蜜相關凝集素 (Jacalin-related lectin, JRL) 家族之一員，對甲基化的單醣具有較強之結合能力。根據先前實驗結果，發現IPO可能與鎘離子結合，推測IPO具有與金屬離子結合的能力，此特性在JRL家族中尚未被報導。 本研究以蛋白質結晶結構及生物物理分析，探究IPO之金屬結合能力。由IPO–MeMan–Cd及IPO–MeMan–Co共結晶後，解出各為1.9 Å的IPO螯合重金屬鎘、鈷的蛋白質四級結構。原態IPO分子為四聚體，第8個氨基酸Histidine附近，皆具有一個金屬結合位置，且在每一個四聚體的中央，具有兩個金屬結合位置，顯示IPO除了具有醣結合特性，亦具有螯合金屬離子的能力；由等溫滴定微熱量法 (isothermal titration calorimetry)，分析IPO與各種二價金屬結合能力，確認一個IPO分子可結合兩個金屬離子，並可與重金屬鎘、鈷、鋅、銅、鎳、二價鐵 (II)、三價鐵 (III)，而不會結合植物必須金屬元素鈣、鎂、錳，且醣與金屬受質之親和性不會互相影響。以差異性掃描螢光法 (Differential scanning fluorimetry, DSF) 分析IPO蛋白質螯合金屬後之變性溫度 (Tm)，其Tm值與未結合受質之wtIPO蛋白質無顯著差異，顯示金屬離子的存在，不會增加IPO結構的穩定性。 本研究結果表明IPO蛋白質能夠與醣分子及金屬離子結合，而結合金屬後的蛋白質是否能強化其抗蟲能力，是進一步要研究的目標。

Lectins, one of the wound-inducible proteins, play important roles in plants defense systems when plants respond to biotic stress and mechanical wounding. Ipomoelin (IPO), belonging to jacalin-related lectin (JRL) family, is a plant defense protein isolated from sweet potato (Ipomoea batatas cv. Tainung 57). Previous studies identified that IPO can specifically bind to various monocarbohydrates with methyl group. Surprisingly, IPO was found that this protein could bind to not only carbohydrates but also cadmium (Cd). In this study, the structures of IPO in complex with MeMan and cobalt (IPO–MeMan–Co), and in complex with MeMan and cadmium (IPO–MeMan–Cd) are resolved at resolution 1.9 Å. Both of IPO–MeMan–Co and IPO–MeMan–Cd form a tetrameric association and there were metal bind sites adjacent to His 8. Moreover, two metals were chelated in the middle of the tetramer. The results of isothermal titration calorimetry (ITC) indicate that IPO could bind to cobalt, cadmium, zinc, nickel, copper, ferrous (II) and ferric (III), but not to essential metals in plants, such as calcium, magnesium and manganese. The melting point (Tm) of IPO in complex with different metals are analyzed by differential scanning fluorimetry (DSF). The data showed that there was no significant change of Tm while IPO bind to metals, and demonstrated that the metal ion would not provide the stabilized function for IPO structure. Based on the results of IPO and metal ion binding assays, it revealed that IPO would contain the capacity to bind to carbohydrates and heavy metal ions. For further applications of IPO in antipest and antipathogen, IPO would have the potentials toward pesticide.