Disulfide bond formation activity of soybean quiescin sulfhydryl oxidase

Multiple enzymatic systems can catalyse protein disulfide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. The enzyme quiescin sulfhydryl oxidase (QSOX) catalyses disulfide bond formation in unfolded proteins via the reduction of oxygen. We found two QSOX homologues in the soybean genome database, Glycine max QSOX (GmQSOX)1 and GmQSOX2, which encode proteins composed of an N-terminal signal peptide, a thioredoxin-like domain, an FAD-binding domain, Erv/ALR, and a transmembrane region near the C terminus. We subsequently cloned two GmQSOX1 cDNAs, GmQSOX1a and GmQSOX1b, which may be generated by alternative splicing. The GmQSOX1a, GmQSOX1b and GmQSOX2 mRNA levels increased during seed storage protein synthesis in the cotyledon, and were also upregulated under conditions causing ER stress. Recombinant GmQSOX1 expressed in Escherichia coli formed disulfide bonds on reduced and denatured RNase A, but did not show any refolding activity. The reduced and denatured RNase A was effectively refolded by recombinant GmQSOX1 in the presence of the soybean protein disulfide isomerase family protein GmPDIL-2 in the absence of glutathione redox buffer, suggesting that GmQSOX1 plays a role in protein folding in the ER.

The nucleotide sequence data for the GmQSOX1a, GmQSOX1b, GmQSOX2a, GmQSOX2b and glycinin AaB1b cDNAs are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB196647, AB195548, XM-006589586, XM-003536592, and AB113349, respectively.