甘藷凝集素ipomoelin四級結構與多種醣類之熱力學分析顯示其多變的結合特性

Quaternary structural and energetic analysis of ipomoelin in complex with various carbohydrates reveals its versatile binding properties

張維倢(Wei-Chieh Chang)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10458

摘要

Ipomoelin (IPO)為甘藷受到傷害時所誘導產生之蛋白，屬於mannose-specific jacalin-related lectin (mJRL)蛋白質次家族成員之一。先前蛋白質結構解析顯示IPO的四級結構為四聚體的形式，而單元體的部分是由14個平行的β摺板構成稜柱狀(β-prism)的結構，N端的2個β摺板位於四個單元體結合的介面中，進而推測其參與在四聚體的結合過程。透過膠體過濾色層分析(gel filtration chromatography)，結果顯示IPO在中性溶液下為四聚體，而缺少N端2個β摺板(N-terminal truncated IPO, ∆Nt IPO)則為單元體。另一方面，為了解IPO醣結合專一性，利用等溫滴定微量熱法(isothermal titration calorimetry, ITC)分析IPO與各種差向異構體(epimer)單醣甲基化甘露醣(methyl α-D-mannopyranoside, Me-Man)、甲基化葡萄醣(methyl α-D-glucopyranoside, Me-Glc)、甲基化半乳醣(methyl α-D-galactopyranoside, Me-Gal)，以及不含甲基形式之甘露醣(mannose, Man)、葡萄醣(glucose, Glc)、半乳醣(galactose, Gal)交互作用的情形，結果顯示IPO具有多重專一性(polyspecificity)的結合特性，其中又以甲基化單醣之結合能力高於不含甲基之單醣，進一步利用醣結合區定位點突變(site-directed mutagenesis)確認IPO Trp-142是穩定甲基化單醣結合的重要胺基酸。除此之外，利用ITC實驗進一步分析∆Nt IPO醣結合專一性，由結果顯示∆Nt IPO 與Me-Man、Me-Glc的結合能力增加，然而，與Me-Gal則沒有交互作用的產生。從前人結構的解析可觀察到，單元體IPO可藉由N端的部份與另一單元體醣結合區環狀區域(loop)產生交互作用，推論IPO可透過此種作用力維持可容納具有軸向(axial) O4 Gal之醣結合區，因此在N端缺乏的情況下此作用力便被消除，使IPO回復到Man/Glc專一性結合的情形。綜合以上推論，IPO N端參與四聚體的形成並扮演調控醣類多重專一性結合的角色。另外，利用ITC實驗分析IPO的金屬結合能力，結果顯示IPO會傾向與重金屬鈷離子(Co2+)、鋅離子(Zn2+)、鎘離子(Cd2+)、汞離子(Hg2+)有交互作用的產生。

關鍵字

凝集素 ； 膠體過濾色層分析 ； 等溫滴定微量熱法 ； 差向異構體 ； 甲基化甘露醣 ； 甲基化葡萄醣 ； 甲基化半乳醣 ； 多重專一性

並列摘要

Ipomoelin (IPO), a wound-inducible protein, is regarded as a defense protein from sweet potato (Ipomoea batatas cv. Tainung 57) and belongs to mannose-specific jacalin-related lectin (mJRL) family. Previous structural analysis demonstrated that the quanternary structure of IPO indicated a tetrameric association. In this study, gel-filtration experiments confirmed that the IPO forms as a tetramer and 2 N-terminal extended β-sheets play a role in tetrameric formation. Subsequently, isothermal titration calorimetry (ITC) studies on the interaction of methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), methyl α-D-galactopyranoside (Me-Gal) and those without methyl groups have been carried out. The results indicated that IPO behaves as a polyspecific lectin. Besides, the binding of methylated carbohydrates to IPO showed better affinities than those without methyl groups. By using site-directed mutagenesis, the residue Trp-142 of IPO could be confirmed as a key residue for stabilizing the binding of methylated carbohydrates. On the other hand, ITC studies on the interaction of methylated carbohydrates with N-terminal truncated IPO (∆Nt IPO) have also been carried out. The binding affinity of Me-Man, Me-Glc showed higher affinities from those to the IPO, and it showed no interaction between Me-Gal and ∆Nt IPO. Based on the previous structural analysis, the interaction of N-terminus of IPO with loop β3-β4 in carbohydrate-binding pocket could be observed. The loss of this interaction may account for the inaccessibility of Me-Gal at this relative smaller binding site. As the results of the quaternary structure and energetic analysis, the N terminus of IPO plays dual roles in the formation of tetramers and the regulation of carbohydrate-binding polyspecificity. Furthermore, ITC studies showed that IPO tend to form complexes with heavy metal ions, e.g. Co2+, Zn2+, Cd2+, and Hg2+.

並列關鍵字

lectin ； gel filtration chromatography ； isothermal titration calorimetry ； epimer ； methyl α-D-glucopyranoside ； methyl α-D-galactopyranoside ； methyl α-D-mannopyranoside ； polyspecificity

參考文獻

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