Heterogeneity within Animal Thioredoxin Reductases

Abstract

Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5 sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-Omethyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membranebound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by ϳ30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle-and cell type-specific location of animal thioredoxin reductases.

Mitochondrial Thioredoxin System

“…This band appears to correspond to a potential polypeptide product generated by alternative splicing of TrxR2 transcripts, a phenomenon that was previously reported by Sun et al (57).…”

“…This result suggests that the partial localization of TrxR2 in the cytosol is not restricted to mouse tissues nor related to a neuronal or tumor origin, because HeLa originated from a uterine human cancer and COS-7 is an immortalized African green monkey kidney cell line. Most interesting, the sites of junction of the MTS coding exon of the mammalian TrxR2 gene correspond to those found in the Drosophila thioredoxin reductase TR gene, which produces both a cytoplasmic and a mitochondrial TR isoform in the fly (57,84). The similar production of mitochondrial and cytosolic TrxR2 isoforms in mammalian cells suggests a conserved mode of TrxR2 expression during evolution (84).…”

“…Because our TrxR2 antibody was obtained using a peptide sequence specific to mitochondrial TrxR2 (44) (see "Experimental Procedures"), this suggests that the cytosolic versions of this protein were derived by a mechanism of gene expression, which discarded the mitochondrial targeting signal in the 5Ј terminus of the TrxR2 gene. Based on computer search analysis of nonredundant and expressed sequence tag data bases, Sun et al (57) have recently predicted the possible existence of different forms of human TrxR2 transcripts that differ in their 5Ј sequences and may be formed by alternate exon splicing. Sequence analysis indicated that one of these transcripts encoded a protein that lacked the mitochondrial signal peptides but instead contained amino acid sequences that could have the effect of confining it to the cytosolic compartment.…”

Mitochondrial and Cytoplasmic Thioredoxin Reductase Variants Encoded by a Single Drosophila Gene Are Both Essential for Viability

“…Moreover, a tagged form of TrxR3 was found in mitochondria (19). A more recent report demonstrated that in addition to the products of the three different TrxR genes, both TrxR1 and TrxR3 exhibit extensive heterogeneity due to differential transcript splicing (18). Comparison between mouse, rat, and human revealed that the multiple isoforms are conserved in mammals (18).…”

“…These additional motifs include an N-terminal disulfide active center, NADPH-and FAD-binding domains, and a dimer interface sequence necessary for homodimerization of the enzymes (4,16,17). Furthermore, sequence homology analyses revealed that TrxR1 and TrxR2 are closely related, whereas TrxR3 is the evolutionarily more distant enzyme but which still exhibits more than 50% overall sequence identity (18).…”