Abstract
We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.
Highlights
The endoplasmic reticulum (ER) is a highly specialized organelle involved in the maturation of extracellular membrane proteins and secreted proteins
To identify novel genes involved in the unfolded protein response in B. mori–derived Bm5 cells, we used a differential screening method [13]
We investigated whether or not bPDI and bERO1 are required for oxidative protein folding in the ER as well as mammals
Summary
The endoplasmic reticulum (ER) is a highly specialized organelle involved in the maturation of extracellular membrane proteins and secreted proteins. Cooperative activity of two proteins, protein disulfide isomerase (PDI) and flavin adenine dinucleotide (FAD)-dependent oxidase ER oxidoreductin 1 (ERO1) formed disulfide bonds, which both have protein oxidation with redox reactions [2,3]. Disulfide bonds formed between cysteines in the active site of PDI are transferred directly to the folding secretory protein. In Saccharomyces cerevisiae, most disulfide bonds are formed by thiol-disulfide transfer mechanisms with the oxidized Pdi1p [8,9]. Disulfide bonds are directly transferred to PDI by ERO1 in both yeast and mammalian cells [8]. We present a novel protein termed bERO1, endoplasmic reticulum oxidoreductin 1 of B. mori, containing two cysteine motifs, N-terminal CAMKYC (Cys-X-X-X-X-Cys motif; active-site cysteine) and C-terminal CVECDKC (Cys-X-X-Cys-X-X-Cys motif; shuttle cysteine). We provide direct evidence of the gene structure, molecular characterization, and connection between bERO1 and bPDI expression in B. mori for the first time
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