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

目的：本研究旨在通过对猪蛔虫细胞色素b_5的编码基因和晶体结构与哺乳动物宿主细胞色素b_5和自由生活线虫C.优雅1）解析A.猪细胞色素b_5的生物合成途径。e. A. suum cytochrome b_5前体细胞色素b_5在C.线虫1）研究了A.猪细胞色素b_5的分辨率为1.8A，表明其结构不同于需氧哺乳动物细胞色素b_5（红细胞型，可溶性）。的对接模型。suum血红蛋白和细胞色素b_5强烈提示它们是生理反应伙伴。免疫组织化学和免疫印迹研究 ...更多信息 表明A.猪细胞色素b_5定位于皮下组织和肠周液中，为分泌性蛋白。我们提出细胞色素b_5是适应宿主肠腔低氧环境的一种特化蛋白质的假说。为了验证这一假设，我们从C. elegans，其中基因组计划已经完成。4种C. elegans cytochrome b_5的存在，但它们都不具有前序列。亲水性分析表明，其中两个蛋白为可溶性蛋白，其中一个蛋白与A.猪细胞色素b_5。然而，两者均未被检测到为表达序列标签。2)由于转染效率低，目前正在检查实验条件。3)以猪蛔虫和人细胞色素b_5为配体，用Biacore进行亲和力分析，发现后者与肠周血红蛋白的亲和力低于前者。这些结果支持上述工作假设。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