Structural and thermodynamics analysis of Ipomoelin with metal ions show the metal-binding ability in Jacalin-related lectin

以結構及熱力學分析甘藷凝集素與金屬離子顯示其金屬結合能力

謝函蓁(Han-Chen Hsieh) ； 鄭貽生(Yi-Sheng Cheng)

《國立臺灣博物館學刊》 71卷4期 (2018/12) Pp. 35-52

https://doi.org/10.6532/JNTM.201812_71(4).03

摘要

Ipomoelin (IPO), a jacalin-related lectin (JRL), is a plant defense protein isolated from sweet potato (Ipomoea batatas cv. Tainung 57). Many kinds of methyl monocarbohydrates can bind to JRL. However, whether JRL can bind to metal ions is unknown. In this study, we demonstrated the metal-binding abilities of IPO by crystal structure and biophysics analysis. We resolved the crystal structures of IPO in complex with methyl α-D-mannopyranoside (Me-Man) and cobalt (IPO-Me-Man-Co) and Me-Man and cadmium (IPO-Me-Man-Cd). The tetrameric structures of IPO-Me- Man-Co and IPO-Me-Man-Cd showed that two metal ions were chelated in the middle of the tetramer, and the binding sites of the metal ion located close to His8 in IPO. Isothermal titration calorimetry revealed that IPO could bind to heavy metal ions, including Co^(2+), Cd^(2+), Zn^(2+), Ni^(2+), Cu^(2+), and Fe^(2+). However, melting points did not differ, which suggests that metal ions did not provide the function for stabilizing IPO tetramers. Moreover, the carbohydrate-binding affinity constant was much higher (~100x) than metal binding in IPO. IPO could be a carbohydrate-binding protein with the capacity to bind several heavy metal ions. The metal-binding ability of IPO might be involved in reducing heavy metal stress in plants.

關鍵字

IPO ； carbohydrate binding protein ； heavy metal binding ； isothermal titration calorimetry ； melting point

並列摘要

甘藷凝集素（Ipomoelin，IPO）是一種植物防禦蛋白，屬於波羅蜜凝集素家族（jacalin-related lectin，JRL），甘藷凝集素基因來自台農57號甘藷（Ipomoea batatas cv. Tainung 57）。在波羅蜜凝集素家族的凝集素蛋白可結合許多種類的甲基單醣。然而，波羅蜜凝集素的蛋白是否可以結合金屬離子仍不清楚。在本研究中，我們以結晶結構及生物物理分析方法闡述IPO的金屬離子結合能力，在結晶結構方面，我們解析IPO、甲基甘露糖及鈷離子（IPO-Me-Man-Co），及IPO、甲基甘露糖及鎘離子（IPO-Me-Man-Cd），在IPO-Me-Man-Co與IPO-Me-Man-Cd四聚體結構中，顯示2個金屬離子可結合在四聚體中央位置，其金屬結合靠近組胺酸8（His 8）的位置。以等溫滴定熱力儀分析重金屬結合特性顯示，IPO可與重金屬離子結合，如鈷Co^(2+)、鎘Cd^(2+)、鋅Zn^(2+)、鎳Ni^(2+)、銅Cu^(2+)及鐵Fe^(2+)。同時，IPO在與這些重金屬離子結合後，其蛋白質熔點並無差異，顯示金屬離子並不提供穩定IPO四聚體的功能。此外，IPO中之糖結合常數較金屬離子結合常數高約100倍，因此本研究推測IPO對重金屬離子結合可能作為植物對抗重金屬逆境的方式。

並列關鍵字

甘藷凝集素 ； 醣結合蛋白 ； 重金屬結合 ； 等溫滴定熱力儀 ； 蛋白質熔點

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