Horibe Tomohisa, Gomi Mitsuhiro, Iguchi Daisuke, Ito Hideaki, Kitamura Yukiko, Masuoka Toshio, Tsujimoto Ikuo, Kimura Taiji, Kikuchi Masakazu
Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.
J Biol Chem. 2004 Feb 6;279(6):4604-11. doi: 10.1074/jbc.M310922200. Epub 2003 Nov 19.
Human protein-disulfide isomerase (hPDI)-related protein (hPDIR), which we previously cloned from a human placental cDNA library (Hayano, T., and Kikuchi, M. (1995) FEBS Lett. 372, 210-214), and its mutants were expressed in the Escherichia coli pET system and purified by sequential nickel affinity resin chromatography. Three thioredoxin motifs (CXXC) of purified hPDIR were found to contribute to its isomerase activity with a rank order of CGHC > CPHC > CSMC, although both the isomerase and chaperone activities of this protein were lower than those of hPDI. Screening for hPDIR-binding proteins using a T7 phage display system revealed that alpha1-antitrypsin binds to hPDIR. Surface plasmon resonance experiments demonstrated that alpha1-antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5). Interestingly, the rate of oxidative refolding of alpha1-antitrypsin with hPDIR was much higher than with hPDI or hP5. Thus, the substrate specificity of hPDIR differed from that associated with isomerase activity, and the contribution of the CSMC motif to the oxidative refolding of alpha1-antitrypsin was the most definite of the three (CSMC, CGHC, CPHC). Substitution of SM and PH in the CXXC motifs with GH increased isomerase activity and decreased oxidative refolding. In contrast, substitution of GH and PH with SM decreased isomerase activity and increased oxidative refolding. Because CXXC motif mutants lacking isomerase activity retain chaperone activity for the substrate rhodanese, it is clear that, similar to PDI and hP5, the isomerase and chaperone activities of hPDIR are independent. These results suggest that the central dipeptide of the CXXC motif is critical for both redox activity and substrate specificity.
人蛋白质二硫键异构酶（hPDI）相关蛋白（hPDIR），我们之前从人胎盘cDNA文库中克隆得到（早野，T.，和菊池，M.（1995年）《欧洲生物化学学会联合会快报》372，210 - 214），其突变体在大肠杆菌pET系统中表达，并通过镍亲和树脂柱层析依次纯化。纯化后的hPDIR的三个硫氧还蛋白基序（CXXC）被发现对其异构酶活性有贡献，其活性顺序为CGHC > CPHC > CSMC，尽管该蛋白的异构酶和伴侣活性均低于hPDI。使用T7噬菌体展示系统筛选hPDIR结合蛋白发现α1 - 抗胰蛋白酶与hPDIR结合。表面等离子体共振实验表明α1 - 抗胰蛋白酶与hPDIR相互作用，但不与hPDI或人P5（hP5）相互作用。有趣的是，α1 - 抗胰蛋白酶与hPDIR一起进行氧化重折叠的速率远高于与hPDI或hP5一起时。因此，hPDIR的底物特异性不同于与异构酶活性相关的底物特异性，并且CSMC基序对α1 - 抗胰蛋白酶氧化重折叠的贡献在三个基序（CSMC、CGHC、CPHC）中最为明确。CXXC基序中SM和PH被GH取代会增加异构酶活性并降低氧化重折叠。相反，GH和PH被SM取代会降低异构酶活性并增加氧化重折叠。由于缺乏异构酶活性的CXXC基序突变体对底物硫氰酸酶仍保留伴侣活性，显然，与PDI和hP5类似，hPDIR的异构酶和伴侣活性是独立的。这些结果表明CXXC基序的中央二肽对氧化还原活性和底物特异性都至关重要。
