Alloush Habib M, López-Ribot José L, Masten Barbara J, Chaffin W LaJean
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Microbiology (Reading). 1997 Feb;143 ( Pt 2):321-330. doi: 10.1099/00221287-143-2-321.
We have used a polyclonal antiserum to cell wall proteins of Candida albicans to isolate several clones from a cDNA lambda gt11 expression library. Affinity-purified antibody prepared to the fusion protein of one clone identified a 40 kDa moiety present in cell wall extracts from both morphologies of the organism. Indirect immunofluorescence demonstrated expression of this moiety at the C. albicans cell surface. Sequencing of a pBluescript II genomic clone identified with the cDNA clone revealed an open reading frame for a 417 amino acid protein. The nucleotide sequence showed significant homology with 3-phosphoglycerate kinase (PGK) genes, with 88%, 77% and 76% nucleotide homology with the PGK genes from Candida maltosa, Saccharomyces cerevisiae and Kluyveromyces lactis, respectively. The deduced amino acid sequence was consistent with this identification of the sequence as PGK1 of C. albicans. This finding was confirmed by a positive immunological response of a commercially available purified PGK from S. cerevisiae with the affinity-purified antibody against the fusion protein of the cDNA clone. The presence of PGK in the cell wall was confirmed by two additional methods. Cell wall protein were biotinylated with a derivative that does not permeate the cell membrane to distinguish extracellular from cytosolic proteins. Biotinylated PGK was detected among the biotinylated proteins obtained following streptavidin affinity chromatography. Immunoelectron microscopy revealed that the protein was present at the outer surface of the cell membrane and cell wall as well as expected in the cytoplasm. Northern blot analysis revealed that the gene transcript was present in C. albicans cells growing under different conditions, including different media, temperatures and morphologies. Most of the enzyme activity was found in the cytosol. Low enzymic activity was detected in intact cells but not in culture filtrates. These observations confirmed that PGK is a bona fide cell wall protein of C. albicans.
我们使用了一种针对白色念珠菌细胞壁蛋白的多克隆抗血清，从一个cDNA λgt11表达文库中分离出了几个克隆。针对一个克隆的融合蛋白制备的亲和纯化抗体，在该生物体两种形态的细胞壁提取物中都鉴定出了一个40 kDa的部分。间接免疫荧光显示该部分在白色念珠菌细胞表面表达。对与cDNA克隆鉴定出的pBluescript II基因组克隆进行测序，揭示了一个编码417个氨基酸蛋白质的开放阅读框。核苷酸序列与3-磷酸甘油酸激酶（PGK）基因显示出显著同源性，分别与麦芽糖念珠菌、酿酒酵母和乳酸克鲁维酵母的PGK基因具有88%、77%和76%的核苷酸同源性。推导的氨基酸序列与将该序列鉴定为白色念珠菌的PGK1一致。市售的来自酿酒酵母的纯化PGK与针对cDNA克隆融合蛋白的亲和纯化抗体产生阳性免疫反应，证实了这一发现。通过另外两种方法证实了PGK存在于细胞壁中。用一种不渗透细胞膜的衍生物对细胞壁蛋白进行生物素化，以区分细胞外蛋白和胞质蛋白。在链霉亲和素亲和层析后获得的生物素化蛋白中检测到了生物素化的PGK。免疫电子显微镜显示该蛋白存在于细胞膜和细胞壁的外表面，以及预期的细胞质中。Northern印迹分析显示该基因转录本存在于在不同条件下生长的白色念珠菌细胞中，包括不同的培养基、温度和形态。大部分酶活性存在于胞质溶胶中。在完整细胞中检测到低酶活性，但在培养滤液中未检测到。这些观察结果证实PGK是白色念珠菌的一种真正的细胞壁蛋白。
