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標題: XpsN蛋白第96-104胺基酸參與自身交互作用之重要性:cysteine 突變基因的構築與分析
Involvement of residues 96-104 of XpsN in its self-interaction examined by cysteine-scanning mutants analysis
作者: 曹琤宜
Tsao, Cheng-Yi
關鍵字: XpsN
出版社: 生命科學系
摘要: 摘要 XpsN為十字花科黑腐病菌第二型分泌成員中的內膜蛋白之一, 藉由蛋白N端的疏水性區域嵌於內膜上,C端朝向細胞間質區。先前 研究中顯示XpsN蛋白除了與自身蛋白結合外,與第二型分泌系統中 的其他成員,包括位於內膜的XpsL-M 蛋白複合體、位於外膜的XpsD 蛋白、及主要類纖毛蛋白XpsG也有交互作用,推測在第二型分泌機 器中扮演樞紐性的角色。利用免疫共沉澱及GST pull down分析XpsN C端刪除突變蛋白的結果顯示,XpsN蛋白位於第97-110氨基酸區域, 在參與其與自身及與XpsD蛋白結合上,扮演不可或缺的角色。為進 一步探討這區域中個別氨基酸的重要性,本研究針對全長XpsN蛋白 第96-104氨基酸進行cysteine scanning mutagenesis,得到突變基因 分析它們互補xpsN缺損株XC1707分泌 -amylase的能力,定量分 析結果顯示,T96C、V97C分泌酵素能力為野生株XC1701能力的60~90%,R98C、L99C及T100C皆低於30%,而G101C及L104C 則完全失去分泌功能。為檢查分泌能力降低是否由於突變為cysteine 引起的,進一步將分泌能力低於30% 的四個位置分別置換成 alanine ,定量分析α-amylase分泌能力的結果顯示,四個突變為alanine的突 變蛋白分泌能力均較突變為cysteine的突變蛋白有不同程度的提高, 其中L104A突變蛋白分泌能力幾乎與野生株XC1701相同。為檢查 突變為cysteine的XpsN突變蛋白在氧化狀態下是否兩兩相鄰而形成 雙硫鍵結,在不加還原劑β-mercaptoethanol的條件下進行SDS-PAGE 及西方墨點實驗,發現T96C、R98C、T100C、L104C有類似雙倍體 的訊號出現。若在細胞破碎前加入雙硫鍵抑制劑iodoacetic acid,仍 有雙倍體訊號,暗示第96、98、100及104四個位置的cysteine可能 位於相鄰兩個XpsN的界面。
Abstract The XpsN pretien is one of the inner membrane proteins of type II secretion apparatus in Xanthomonas campestris pv. campestris. Near its N-terminus, there is a hydrophobic sequence spanning the cytoplasmic membrane once with a C-terminal periplasmic domain. Previous studies indicated that XpsN could associate with itself. In addition, it also interacts with other members of type II secretion apparatus, including the inner membrane protein complex XpsL-M, the outer membrane protein XpsD and the major pseudopilin XpsG. Thus, it was proposed to play a central role in type II secretion apparatus. Results from co-immune precipitation and GST pull down assay suggested that the sequence between the 97th to 110th residues of XpsN is required for its interaction with itself and with XpsD. To confirm significance of this region, I performed functional analysis of XpsN mutants with single amino acid at 96th-104th residues mutated to cysteine. By introducing each into the xpsN mutant strain of X. campestris XC1707 and assaying for their secretion, I found that T96C and V97C mutant proteins kept 60-90% secretion ability. While the secretion exhibited by TR98C, L99C and T100C mutants were redued to lower than 30%, G101C and L104C have entirely lost the ability. I further constructed four alanine substitution mutants at 99th, 100 th, 101st and 104 th residues and analyzed their function. They all possess higher secretion ability than cysteine substitution. The cysteine scanning mutants were furrher analyzed for dimerization by seperation them on SDS-polyacrylamide gel in absence of -mercaptoethanol. T96C, R98C, T100C, L104C mutants appeared to form disulfide-bonded dimmer with or without addition of iodoacetic acid before cell breakage. These obseravations suggest close proximity of the 96th, 98th, 100st and 104st residue of two neighboring XpsN in the secretion apparatus.
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