Spermatid-Specific Thioredoxin Reductases
The unexpected finding of several spermatid-specific thioredoxins immediately raised the question of whether additional thioredoxin reductases might also exist in t3st1s to maintain the different thioredoxins in their reduced form. The human genome contains only three thioredoxin reductases genes (Table I): TrxR1, TrxR2 and TGR (28, 29, 53, 93, 94) (Figure 3). Of these, TrxR1 and TGR are of particular interest regarding t3st1s-specific thioredoxin activity.
TrxR1, a cytosolic enzyme, is expressed highly in Sertoli cells, spermatocytes and spermatids, cells that express little or no Trx-1 (33, 80). This lack of colocalization suggests that reductase-substrates other than Trx-1 might exist in these cells and the spermatid-specific thioredoxins are obvious candidates. However, using recombinant protein expressed in bacteria only Sptrx-1 (56), but not Sptrx-2 and Txl-2 (81, 82), is a substrate for TrxR1. As aforementioned, the lack of activity of both proteins might be due to the requirement of additional cofactors or interacting proteins not present in the in vitro assay, or to posttranslational modifications which are not are not achieved when the proteins are expressed in bacteria. In order to investigate in more detail the complexity of the different thioredoxin systems in t3st1s, we have initiated a developmental study of Trx-1 and TrxR1 during spermatogenesis.
Thioredoxin glutathione reductase (TGR) deserves special attention in the context of t3st1s and spermatogenesis. TGR was initially identified in human and mouse databases in the course of a genomic screening for novel thioredoxin reductases (94). Evaluation of TGR mRNA expression pattern by northern blot analysis indicated a prominent expression in t3st1s which has been confirmed at the protein level using specific antibodies (94). A subsequent cloning of the full-length mouse cDNA showed that TGR is composed of two defined domains: The N-terminal domain is similar to that of glutaredoxins, although the active site contains only one cysteine residue and the C-terminal domain is typical of thioredoxin reductases, including the SeCys insertion as penultimate residue (Figure 3). Studies using mouse TGR protein confirmed that both domains are enzymatically active, and TGR is able not only to reduce Trx-1 but also GSSG and GSH-linked disulfides in vitro, providing specificity for both the thioredoxin and the glutathione system, and broadening even more the substrate specificity shown by thioredoxin reductases (93). Recent data indicate that TGR is mostly expressed in spermatocytes and elongating spermatids (V. Gladyshev, personal communication), strongly supporting a role as reductant for the spermatid-specific thioredoxins, a possibility that we are currently exploring.
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