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標題: 十字花科黑腐病菌XpsM蛋白之特性分析
Characterization of the XpsM protein of the type II secretion apparatus of xanthomonas campestris pv. campestris
作者: 吳美婷
關鍵字: 十字花科黑腐病菌
出版社: 生物化學研究所
摘要: 摘 要 革蘭氏陰性菌之第二類型胞外蛋白分泌途徑,可分為兩個步驟,先由Sec系統,將胞外蛋白送到胞質週緣區,再由另一群12-14個蛋白所組成的分泌系統,將胞外蛋白從胞質週緣區分泌到胞外。在十字花科黑腐病菌中,此分泌系統是由XpsD-O十二個蛋白組成,本論文探討的對象是此分泌系統的成員之一XpsM蛋白。XpsM蛋白由213個氨基酸組成,分子量約23 kDa,其氨基酸序列中具有兩個cysteine及兩個預測的 leucine-zipper like motif;靠近N端的部分,有一段疏水性氨基酸序列,推測XpsM蛋白可能以此插在細胞內膜上。Sandkvist等人利用分子篩管柱層析證明,EpsM蛋白在大腸菌中單獨表現時會以雙倍體的形式存在;已有文獻指出,L、M蛋白有相當程度的依存關係,當L蛋白不在的時候,M蛋白會變得不穩定,而M蛋白不在的時候,L蛋白也會變得不穩定;L、M、N蛋白經由免疫共沉澱的實驗,也被證明會有兩兩相互作用的關係。本論文實驗主要針對XpsM蛋白的構造與功能,及其在第二類型分泌途徑中與XpsL蛋白之間的關係進行探討。以分子篩管柱層析分析單獨大量表現的XpsM-His蛋白及以質體大量表現XpsM-His的互補株XC1714(pST116)中的XpsM蛋白,推測單獨表現的XpsM蛋白可能以10-12個單體形成多倍體,而在正常的分泌狀態下,XpsM則會以較小的分子形式與XpsL及XpsN蛋白形成complex。在進行XpsM蛋白的西方墨點分析時,可在不含 b-mercaptoethanol 的情況下,看到XpsM蛋白可能以雙硫鍵形成雙倍體,因此選擇XpsM氨基酸序列中cysteine及預測的 leucine-zipper like motif進行定點突變,進一步探討這兩個序列與XpsM蛋白形成雙倍體之間的關聯性;若將兩個cysteine同時突變就看不到XpsM蛋白雙倍體,但這個突變並不影響XpsM蛋白的功能,推測雙硫鍵的形成可能只代表相鄰的兩個XpsM蛋白中的cysteine十分靠近,並非XpsM蛋白行使功能的必要條件。為了探討XpsM蛋白與XpsL蛋白之間的關係,以不同方式構築XpsM突變蛋白,選擇XpsM的融合蛋白L74::PhoA進行免疫共沉澱分析,結果顯示XpsM蛋白可能只要保留N端74個氨基酸,就具備與XpsL及與XpsN形成穩定complex的能力。
Abstract Gram-negative bacteria have developed different pathways for the secretion of extracellular proteins into melieu. In type II secretion pathway, proteins are secreted in two steps. They are first translocated across cytoplasmic membrane via the Sec machinery. In a second step it takes between 12-14 proteins for the proteins to traverse across the outer membrane. These proteins are designated XpsD-O in Xanthomonas campestris pv. campertris. The xpsM gene, which encodes a protein of 213 amino acid residues with two cysteines and two leucine-zipper like motif, is the subject of this study. The XpsM protein possesses an N-terminal hydrophobic region. It was predicted to be an integral inner membrane protein and was shown to form complex with XpsL and with XpsN. In this study, I performed gel filtration analysis of the XpsM-His protein in the absence (in XC17433) and in the presence (in XC1714) of other Xps proteins that were extracted with b-OG. The XpsM protein in XC17433 appeared as homomultimer of 10-12 subunits. However, it appeared as monomer in the XpsL-XpsM-XpsN complex when expressed from a plasmid- encoded gene in XC1714. SDS-PAGE analysis of XpsM in the absence of b-mercaptoethanol (b-ME) revealed a signal with twice the size of the monomer on the immunoblot, suggesting the involvement of cysteines in dimer formation. When I mutated both cysteines through site-directed mutagenesis, the b-ME sensitive XpsM dimer disappeared. However, it remains functional. This result suggested that disulfide bond formation is probably not essential for normal function of XpsM protein. I further analyzed the XpsM fusion protein L74::PhoA for its interactions with XpsL and with XpsN. Coimmuno- precipitation experiments revealed that XpsM remains capable of forming complex with XpsL and XpsN by keeping its N-terminal seventy-four amino acid residues.
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