Functional analysis of human P5, a protein disulfide isomerase homologue.
Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.
Kikuchi
Masakazu
M
Department of Bioscience & Technology, Faculty of Science & Engineering, Ritsumeikan University, Noji-higashi, Kusatsu, Shiga 525-8577, Japan. kikuchi@se.ritsumei.ac.jp
Doi
Eisuke
E
Tsujimoto
Ikuo
I
Horibe
Tomohisa
T
Tsujimoto
Yoshiyuki
Y
D020816:Amino Acid Motifs; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
D017931:DNA Primers; D018832:Molecular Chaperones; D011506:Proteins; C073041:P5 protein, Mesocricetus auratus; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002950:Citrate (si)-Synthase; D013884:Thiosulfate Sulfurtransferase; D019704:Protein Disulfide-Isomerases