革蘭氏陰性菌之胞外蛋白分泌系統至少分為四種，Type II是其中常見的一種，稱為一般分泌途徑(General Secretion Pathway, GSP)。該系統之外膜分泌機器主要由十二至十三個基因產物所構成，包括GspC-O。在這些基因蛋白中由於GspGHIJ的NH2-端具有與PilA纖毛的NH2-端相當高的同源性，因此這些蛋白被認為是屬於同一家族，可是GspGHIJ的纖毛結構一直未被鑑定、分離出。在本研究中，我們主要以生化與遺傳方法對十字花科黑腐病菌蛋白質外膜分泌系統的GspGHIJ纖毛結構作一有系統的探討。在過去幾年，我們已成功製備好XpsG蛋白的抗體與xpsG基因 knockout突變菌種。將細胞打破，以抗體偵測XpsG的分布，結果發現XpsG蛋白同時存在於可溶與不可溶部分。以膠質篩濾層析法分析這兩種型式的XpsG蛋白，結果發現酯溶性是dimer型式，而水溶性則是一種higher-order的型式。此higher-order結構在高濃度清潔劑DOC下，會發生解離，成為dimer型式。將解離的dimer型式集中後，進行第二次膠質篩濾層析，此時緩衝液中不含DOC，結果higher-order的型式不再恢復，表示此型式的形成可能需要細胞內其他蛋白質的參與。higher-order的的型式在高溫八十度處理後仍然很穩定，不會解離。在低濃度(0.1%)清潔劑DOC下，此結構在pH8.0不會解離，但在pH10.3下則發生解離。以定位突變法檢查XpsG蛋白五個演化保留的氨基酸Asp，將D70、D103、D121、D128及D139均改變成Glu後，發現只有D103E突變蛋白的功能喪失。以管柱層析法分析D103E水溶性與酯溶性突變蛋白的分子大小，發現突變蛋白與野生蛋白並無差異，由此可知D103E在蛋白中扮演某種特別功能，而此功能與分子結構可能無關。總結，在本研究中XpsG蛋白很可能會形成纖毛類似結構，而水溶性higher-order form的XpsG蛋白很可能就是從膜上斷裂的纖毛類似結構。
The protein translocation machinery in Gram-negative bacteria are classified into three types. The type II pathway or the so-called general secretion pathway (GSP) is the most common one. The outer membrane GSP secretion machinery is composed of twelve to thirteen gene products designated as GspCDEFGHIJKLMNO. The N-termini of GspGHIJ are highly homologous to the N-terminus of PilA pilus, and were considered as the same family. However, the pili structure of GspGHIJ structure had never been identified or isolated. In this study, we used biochemical and genetic methods to systematically investigate the GspGHIJ pili structure in the outer membrane protein secretion system of Xanthomonas campestris. We had prepared anti-XpsG antibody and xpsG knockout mutant strain. XpsG protein was found to present in the soluble and insoluble fraction of lysed cells. Gel filtration chromatography analysis showed that the XpsG from membrane fraction was in the dimer form and from soluble fraction was in a higher order form. In the presence of high concentration of detergent DOC, the higher-order form dissociated to dimers. Second gel filtration analysis of the dissociated dimer in the absence of DOC revealed that the proteins could not revert to the higher-order form, indicating that the formation of the higher order from might require the participation of other factors. The soluble high-order form was stable at 80℃ and did not be dissociated. Under low concentration of DOC, the structure did not dissociate at pH 8 but dissociated at pH10.3. The XpsG-containing, high-order structures observed in this study could be the pilus-like structure proposed for the pilin-like protein family.
The five-conserved aspartate residues of XpsG protein at the 70, 103, 121, 128 and 139 positions were substituted to glutamate by site-directed mutagenesis. By -amylase secretion function assay, only mutant D103E was found that could not compensate the secretion ability of XC1713 and could interfere the secretion ability of XC1701. Using gel filtration chromatography analyses, no difference of elution profiles were found between wild type and mutant XpsG proteins in soluble and membrane forms. These results indicated that D103 of XpsG protein might be play a special function on protein secretion mechanism and this function was not related to the multimer function.
In conclusion of this study, XpsG protein might form pilus-like structure, and the soluble higher-order form of XpsG protein might be cleavaged from membrane pilus-like structure.
