Abstract
The endoplasmic reticulum (ER) contains a number of thiol-disulfide oxidoreductases of the protein-disulfide isomerase (PDI) family that catalyze the formation of disulfide bonds in newly synthesized proteins. Here we describe the identification and characterization of a novel member of the human PDI family, TMX3 (thioredoxin-related transmembrane protein 3). The TMX3 gene encodes a protein of 454 amino acid residues that contains a predicted N-terminal signal sequence, a single domain with sequence similarity to thioredoxin and a CGHC active site sequence, a potential transmembrane domain, and a C-terminal KKKD tetrapeptide sequence that matches the classical KKXX-type consensus sequence for ER retrieval of type I transmembrane proteins. Endogenous TMX3 contains endoglycosidase H-sensitive glycans, localizes to the ER by immunofluorescence microscopy, and is present in the membrane fraction after alkaline extraction of the ER luminal content. The TMX3 transcript is found in a variety of tissues and is not up-regulated by the unfolded protein response. Circular dichroism spectroscopy of the recombinantly expressed luminal domain of TMX3 showed features typical of a properly folded protein of the alpha/beta type. The redox potential of recombinant luminal TMX3 was determined to -0.157 V, similar to the values previously found for PDI and ERp57. Interestingly, TMX3 showed oxidase activity, and in human tissue-culture cells the protein was found partially in the oxidized form, potentially suggesting a function of the protein as a dithiol oxidase.
Highlights
Protein-disulfide isomerase (PDI) is the founding member of a family of thiol-disulfide oxidoreductases in the endoplasmic reticulum (ER)
Sequence Characteristics of the TMX3 Protein—Using a consensus sequence for a thioredoxin-like domain as query sequence, we identified a previously uncharacterized open reading frame (RefSeq: NM_019022.3) by BLAST searching at the NCBI Web interface
The single catalytic thioredoxin-like domain in TMX3 is found at the very N terminus of the mature protein (Fig. 1)
Summary
Protein-disulfide isomerase (PDI) is the founding member of a family of thiol-disulfide oxidoreductases in the ER (reviewed in Ref. 2). The protein contains four domains, named a, b, b, and a, all with homology to thioredoxin Like this cytosolic reductase, the catalytic a and a domains of PDI contain tetrapeptide CXXC active site sequences (where X denotes any amino acid), for catalysis of thiol-disulfide exchange reactions. The catalytic a and a domains of PDI contain tetrapeptide CXXC active site sequences (where X denotes any amino acid), for catalysis of thiol-disulfide exchange reactions These reactions proceed through transient mixed disulfide intermediates between enzyme and substrate and lead to the oxidation (formation), reduction (breaking), or isomerization (rearrangement) of substrate cysteines. In the ER, work on the Ero flavoprotein has revealed that it functions as a thiol oxidase for PDI In this pathway, substrate protein oxidation by PDI reduces its active site disulfide. We describe the characterization of a new human protein of the PDI family, TMX3
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