Structural and functional analysis of Ascaris suum cytochrome b in the nematode methemoglobin reductase system

目的：通过将新型nadh -高铁血红蛋白还原酶系统中的细胞色素b_5编码基因和晶体结构与哺乳动物宿主和线虫细胞色素b_5的编码基因和晶体结构进行比较，研究新型nadh -高铁血红蛋白还原酶系统中的细胞色素b_5。更具体地说，1)解析a.a suum细胞色素b_5在寄生适应过程中获得的结构特性；2)通过在线虫中表达a.a suum细胞色素b_5前体，分析其生物合成方式，即a.a suum细胞色素b_5前置序列的作用。1) A. suum细胞色素b_5的晶体结构分辨率为1.8 A，与有氧哺乳动物细胞色素b_5（红细胞型，可溶性）的结构不同。猪血红蛋白与细胞色素b_5的对接模型强烈提示它们是生理反应的伙伴。免疫组化和免疫印迹研究表明，泡草细胞色素b_5存在于皮下和肠周液中，为一种秘书蛋白。我们提出了一个工作假设，细胞色素b_5是在适应宿主肠腔低氧张力过程中特化的蛋白质。为了验证这一假设，我们从已完成线虫基因组计划的线虫基因数据库中调查了细胞色素b_5种。线虫细胞色素_b5共发现4种，但均不具有序列。对这4种植物的亲水性分析表明，其中2种为可溶性蛋白，其中1种与A. suum细胞色素b_5同源性最高。然而，两者均未检测到表达序列标签。2)由于转染效率低，目前实验条件尚不完善。3)利用Biacore软件，以Ascaris suum和人细胞色素b_5为配体进行亲和力分析，结果表明后者与肠周血红蛋白的亲和力较前者低。这些结果支持了上述工作假设。少

Objectives : This research project was undertaken to elucidate structural properties of Ascaris suum cytochrome b_5, the component of novel NADH-methemoglobin reductase system , by comparing the coding gene and crystal structure with those of cytochromes b_5 from mammalian host and free-living nematode C. elegans. More specifically, 1) To resolve structural properties of A. suum cytochrome b_5 , which have been gained in the course of parasitic adaptation, 2) To analyze its mode of biosynthesis, i. e. the role of presequence of A. suum cytochrome b_5 by expressing the precursor cytochrome b_5 in C. elegans nematode. Achievements 1) The crystal structure of A. suum cytochrome b_5 was resolved at 1.8 A resolution demonstrating the structure different from that of aerobic mammalian cytochrome b_5(erythrocyte type, soluble). Docking models of A. suum hemoglobin and cytochrome b_5 strongly suggest that they are physiological reaction partners. Immunohistochemical and immunoblotting studies … More showed that A. suum cytochrome b_5 was localized in both the hypodermis and perienteric fluid , and that the cytochrome was a secretary protein. We proposed a working hypothesis that the cytochrome b_5 was a protein specialized during adaptation to low-oxygen tension of host intestinal lumen. To test this hypothesis, cytochrome b_5 species were surveyed from gene data bases of C. elegans, of which genome project has been completed. Four species of C. elegans cytochrome _b5 were found although none of them possesses presequence at all. Hydropathy analysis of the four species suggested that two of them were soluble proteins and one of the two exhibited highest homology with A. suum cytochrome b_5. However, none of the two was detected as expressed sequence tag. 2) Experimental conditions are currently examined because of low efficiency of transfection. 3) Affinity analysis using Biacore, employing Ascaris suum and human cytochromes b_5 as ligand, showed that the latter had less affinity with perienteric hemoglobin than the former. These results supported the working hypothesis described above. Less