Koser P L, Livi G P, Levy M A, Rosenberg M, Bergsma D J
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Gene. 1990 Dec 15;96(2):189-95. doi: 10.1016/0378-1119(90)90252-m.
A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.
利用人T细胞亲环蛋白（Cyp）cDNA作为杂交探针，从λ噬菌体文库中分离出白色念珠菌的一个cDNA及其基因组对应物。这些克隆含有一个486 bp的开放阅读框，预测编码一个162个氨基酸、约18 kDa的蛋白质，该蛋白质大小与人T细胞Cyp相似，分别与酿酒酵母胞质Cyp有68%和81%的同源性。Northern印迹显示存在一种约800 bp的单一mRNA。然而，基因组Southern印迹表明白色念珠菌中至少存在另一个与Cyp相关的基因。将该cDNA进行工程改造以便在大肠杆菌中表达，产生的重组蛋白与哺乳动物Cyp一样，在体外表现出对环孢素A抑制敏感的肽基脯氨酰顺反异构酶（PPIase）活性。这些结果表明我们克隆的基因编码白色念珠菌的一个Cyp。我们将该基因命名为CYP1（亲环蛋白）。有趣的是，预测的白色念珠菌蛋白仅含有两个半胱氨酸残基，它们与哺乳动物Cyp中保守的四个半胱氨酸中的任何一个都不匹配。这表明PPIase催化机制可能不涉及酶结合的半硫代邻酰胺，如先前报道的猪Cyp那样。
