Different contributions of the three CXXC motifs of human protein-disulfide isomerase-related protein to isomerase activity and oxidative refolding.
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.
Horibe
Tomohisa
T
Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.
Gomi
Mitsuhiro
M
Iguchi
Daisuke
D
Ito
Hideaki
H
Kitamura
Yukiko
Y
Masuoka
Toshio
T
Tsujimoto
Ikuo
I
Kimura
Taiji
T
Kikuchi
Masakazu
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D018076:DNA, Complementary; D018832:Molecular Chaperones; D011506:Proteins; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; C526506:PDIA5 protein, human; D019704:Protein Disulfide-Isomerases