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J. Biol. Chem., Vol. 279, Issue 6, 4604-4611, February 6, 2004

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Different Contributions of the Three CXXC Motifs of Human Protein-disulfide Isomerase-related Protein to Isomerase Activity and Oxidative Refolding^*

Tomohisa Horibe, Mitsuhiro Gomi, Daisuke Iguchi, Hideaki Ito, Yukiko Kitamura, Toshio Masuoka, Ikuo Tsujimoto, Taiji Kimura, and Masakazu Kikuchi¶

From the Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan

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 ₁-antitrypsin binds to hPDIR. Surface plasmon resonance experiments demonstrated that ₁-antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5). Interestingly, the rate of oxidative refolding of ₁-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 ₁-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.

Received for publication, October 3, 2003 , and in revised form, November 19, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: R&D Company, KOBAYASHI Pharmaceutical Co., Ltd., 30-3 1-Chome, Toyokawa, Ibaraki-Shi, Osaka 567-0057, Japan.

Present address: Fermentation Development Labs., Fujisawa Pharmaceutical Co., Ltd., 156 Nakagawara, Shinkawa-cho, Nishikasugaigun, Aichi 452-0905, Japan.

^¶ To whom correspondence should be addressed. Tel.: 81-77-561-2843; Fax: 81-77-561-2659; E-mail: kikuchi{at}se.ritsumei.ac.jp.

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