烟曲霉蛋白质分泌载体的构建及酸性磷酸酯酶的细胞定位

Analysis of acid-phosphatase localization in Aspergillus fumigatus by a secreted chimeric green fluorescent protein as reporter

【目的】在酵母细胞中蛋白质的糖基磷酸肌醇化(GPI)修饰是将GPI定位于细胞膜或细胞壁的信号。目前已对酵母GPI蛋白的细胞定位信号有一定了解,但对丝状真菌GPI蛋白的定位则了解甚少。AfPhoA是丝状真菌烟曲霉(Aspergillus fumigatus)的酸性磷酸酯酶,是GPI修饰的蛋白。该蛋白首先分离自细胞膜,随后又发现该蛋白与细胞壁结合。分析其C-端序列也未发现已知的定位信号,因此目前还不能确定其细胞定位。【方法】我们以绿色荧光蛋白(GFP)作为报告分子,将AfPhoA的C-端序列与GFP的C-端融合后检测融合GFP的细胞定位【。结果】我们用烟曲霉几丁质酶AfChiB1的启动子和N-端信号肽构建了可在烟曲霉中分泌表达GFP的表达载体pchiGFP。在此基础上将AfPhoA的C-端与GFP融合,融合质粒与pCDA14共转化烟曲霉后筛选到一株转化子。该转化子可表达融合GFP,在诱导和非诱导条件下,融合GFP均主要分布在细胞膜上,随培养时间的延长,融合GFP在细胞壁上也有少量分布;在培养上清液中只能检出约30KD的GFP融合蛋白,而没有完整的GFP融合蛋白,推测为从GPI锚上水解释放的。【结论】我们的研究结果表明,AfPhoA蛋白GPI修饰的作用是使该蛋白定位于细胞膜。本研究不仅初步确定了AfPhoA蛋白GPI修饰的细胞膜定位功能,而且为烟曲霉基因与蛋白质功能的研究建立了一个有效表达系统。

[Objective] In yeast glycosylphosphatidylinositol （GPI） anchoring is a signal directing localization of GPI proteins to the plasma membrane or cell wall. Some of the cis-requirements for the localization of GPI proteins are now understood, however, little is known the signals directing distribution of the GPI proteins in filamentous fungi. Previously, AfPhoA, a GPI-anchored acid phosphatase in filamentous fungus Aspergillus fumigatus, was first isolated from the cell membrane and latter found to be associated with the cell wall. The actual distribution of the AfPhoA remains unclear. Meanwhile, the signature amino acid motif that determines the distribution of GPI protein in yeast is not found in the C-terminal sequence of the AfPhoA. We aimed to elucidate the cell distribution of the AfPhoA. [Methods] The green fluorescent protein （GFP） was used as reporter to track the localization of the AfPhoA. The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP. [Results] We first constructed the expression plasmid pchiGFP, in which the N-terminal signal sequence of the A. fumigatus AfChiB 1 was fused to the N-terminus of the GFP. After transformation, a secreted expression of the GFP was achieved in A. fumigatus. Based on this construct, The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP to construct a chimeric GFP. After the co-transformation of the fusion construct with plasmid pCDA14, a transformant was confirmed to harbor the chimeric GFP in its genome and could express the chimeric GFP. The transformant cultivated with or without chitin induction could express the chimeric GFP mainly attached to the cell membrane, a prolonged cultivation led to a minor distribution of the chimeric GFP in the cell wall. Although a 30KD of GFP fragment, instead of an intact 43.SKDa chimeric GFP, was also detected in the culture supernatant, which might be released by the cleavage between the fusion protein and its GPI anchor. [Conclusion] Our results suggest that GPI anchoring determines the distribution of the AfPhoA in the cell membrane. In addition to our investigation of the GPI anchoring, an expression vector was also constructed, which would be useful for analyses of the function and regulation of the genes and proteins in A. fumigatus.