十字花科黑腐病菌第二類型胞外蛋白分泌系統中XpsN 蛋白與XpsL-XpsM蛋白複合體結合區域之探討

Abstract

摘要 革蘭氏陰性菌之第二類型胞外蛋白分泌途徑須由二段步驟，將胞外蛋白由胞內送至胞外。胞外蛋白在細胞質內合成後具有辨識訊號 (signal peptide)，經由Sec蛋白系統，將胞外蛋白送至胞質週緣區 (periplasm) 後，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送達胞外。十字花科黑腐病菌的分泌系統由XpsD~O十二個蛋白產物所組成，本實驗探討之XpsN蛋白為分泌系統的成員之一。XpsN蛋白由261個氨基酸所組成，其N-端具有一段厭水性序列，XpsN藉此厭水性序列以C-端朝向胞質週緣區的方式嵌入內胞膜，可能與另外兩個內胞膜蛋白XpsL及XpsM形成complex。為分析XpsN蛋白構造與功能的關係，本實驗利用跳躍子 (transposon) TnphoA/in的隨機插入，構築成功11個xpsN::phoA突變株，並進一步利用位在跳躍子兩端的BamHI切割位，刪除大部分的跳躍子序列，留下93個鹼基對，而造成31個氨基酸的插入，獲得另一群xpsN::i31插入突變株。利用XpsN::PhoA突變蛋白，以免疫共沉澱分析XpsN突變蛋白與 XpsL-XpsM complex間的結合區域。結果顯示，與 XpsL-XpsM 的結合區域可能位於 XpsN 的 N-端1-97個氨基酸；且發現XpsN 蛋白本身可能藉由其 N-端110個氨基酸形成 dimer 以上的結構。為排除XpsN::PhoA中PhoA引起的效應，又構築另一群truncated xpsN 突變株。互補實驗結果顯示，XpsNΔ159-261在xpsN基因缺損株中有互補能力，推測 XpsN 蛋白N-端158個氨基酸即具有分泌 α-amylase 的能力。為進一步檢驗97-110氨基酸序列在XpsN與其本身結合中的重要性，構築了XpsNΔ97-110刪除蛋白，在互補測試中並無功能，進行分子篩管柱層析分析時，顯示其仍然形成大的complex，但分子大小較全長XpsN的complex小，其意義仍有待進一步分析。

Abstract The type II secretion pathway in Gram-negative bacteria occurs in two steps. The secreted proteins possessing an N-terminal signal peptide are exported to periplasm through the Sec pathway. In the second step they traverse across the outer membrane via a secretion apparatus composed of 12-14 proteins. In Xanthomonas campestris pv. campestris, there are 12 genes designated as xpsD-O involved in the second step. Among them, the xpsN gene encodes a protein of 261 amino acid residues. Near its N-terminus, there is a hydrophobic sequence spanning the membrane once with a C-terminal periplasmic domain. The XpsN protein probably forms a complex with two other inner membrane proteins XpsL and XpsM. To study the structure-function relationship of XpsN protein, I constructed mutants utilizing random insertion property of the transposon TnphoA/in. I obtained a total of 11 xpsN::phoA mutants. The transposon has a BamHI site on both ends. Upon BamHI cleavage and self-ligation, almost the entire trnasposon can be deleted. The remaining 93 base pairs caused an inframe insertion of 31 amino acid residues. I thus obtained a series of xpsN::i31 mutants. By performing immunoprecipition experiments, I analyzed the XpsN::PhoA fusion proteins for their interaction with the XpsL-XpsM complex. The results suggested that for stable complex formation between XpsN and XpsL-XpsM, the N-terminal 97 amino acid residues are required. Moreover, I also found that the PhoA fusion containing the N-terminal 110 amino acid residues of XpsN could form stable complex with the wild type XpsN protein. In order to exclude the effect caused by the PhoA moiety, I constructed a series of truncated xpsN mutants. Complementation experiments showed that the XpsNΔ159-261 protein is functional, suggesting that the XpsN protein only needs its N-terminal 158 amino acid residues to be functional. In order to confirm the role of the region covering 97-110 amino acid residues of XpsN in its formation of complex with itself, I constructed a gene encoding the XpsNΔ97-110 deletion protein. It is no longer functional. Upon gel filtration chromatography, it appeared as a homomultimeric complex. Its molecular size is smaller than that of the wild type XpsN complex. The exact meaning of this result awaits further experimentations.