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.Protein-disulfide isomerase (PDI) 1 catalyzes the oxidation, reduction, and isomerization of protein disulfide bonds (1). It is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding that occurs within the luminal space of the endoplasmic reticulum (ER) (2-4). Recently, many PDI homologs have been identified (5-7), and elucidation of their functions, most of which are unknown, may explain the observed diversity of these proteins. PDI has two distinct regions that contain the CGHC sequence. This sequence is similar to a motif found in thioredoxin, CGPC (also called the TX or CXXC motif). CGHC sequences serve as the active sites in PDI for thiol-disulfide bond exchange reactions (8). PDI and its homologs form a diverse protein superfamily whose members are characterized by two or three CXXC motifs (5).We have identified the cDNA of a novel human PDI (hPDI)-related protein (hPDIR) from a human placental cDNA library (9). The deduced amino acid sequence of hPDIR indicates that it contains three CXXC motifs (Cys-Ser-Met-Cys, Cys-Gly-HisCys, and Cys-Pro-His-Cys) and a putative ER retention signal (Lys-Glu-Glu-Leu) at its carboxyl terminus (9). Based on the number and relative positions of CXXC motifs...