Millership Jason J, Zhu Guan
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA.
Int J Parasitol. 2002 Nov;32(12):1477-85. doi: 10.1016/s0020-7519(02)00135-2.
Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism. Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain. Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C. parvum genome-sequencing project. The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C. parvum. The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein. However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum. Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites. However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites. Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays. The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties. Studies indicate that CpRPA1B binds ssDNA of >or=5 nucleotides (dT), while CpRPA1 only binds ssDNA >or=20 nucleotides (dT). This study indicates that C. parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts.
复制蛋白A是一种单链DNA结合蛋白，在真核生物DNA代谢中具有多种作用。通常，真核生物复制蛋白A是一种稳定的异源三聚体复合物，由70 kDa（RPA1）、32 kDa（RPA2）和14 kDa（RPA3）的三个亚基组成。我们之前已经从小隐孢子虫中克隆并鉴定了一个RPA1亚基，它与其他真核生物复制蛋白A 1蛋白具有高度同源性，但缺少一个N端结构域。在此，我们从正在进行的小隐孢子虫基因组测序项目中鉴定出了第二个复制蛋白A 1（称为CpRPA1B）。推导的CpRPA1B蛋白序列与CpRPA1的序列同一性仅为16%，这表明小隐孢子虫中存在两种不同类型的RPA1亚基。CpRPA1B基因预测的一个75.5 kDa的肽段，其大小与高等真核生物的相似，但与53.9 kDa的N端短型CpRPA1蛋白不同。然而，蛋白质印迹分析表明，尽管整个CpRPA1B开放阅读框可能被翻译，但该蛋白可能会通过翻译后修饰被切割，类似于在恶性疟原虫中观察到的复制蛋白A 1基因产物的情况。间接免疫荧光研究表明，这两种蛋白在子孢子中均呈弥散分布模式。然而，观察到CpRPA1定位于含有顶复合器的前部区域，而CpRPA1B定位于子孢子细胞核内或其周围的中部区域。为了进行功能测定，表达了CpRPA1和CpRPA1B的全长开放阅读框。CpRPA1和CpRPA1B重组蛋白在大肠杆菌中作为麦芽糖结合蛋白融合蛋白表达，并对整个融合蛋白的DNA结合特性进行了测定。研究表明，CpRPA1B结合长度≥5个核苷酸（dT）的单链DNA，而CpRPA1仅结合长度≥20个核苷酸（dT）的单链DNA。这项研究表明，小隐孢子虫拥有两种不同类型的复制蛋白A大亚基（复制蛋白A 1），两者均与其宿主有显著差异。
