Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa.

Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member of the thioredoxin family in humans, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx open reading frame encodes for a protein of 486 amino acids composed of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx mRNA is only expressed in human testis, specifically in round and elongating spermatids. Immunostaining of human testis sections identified Sptrx protein in spermatids, while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the postmeiotic phase of spermatogenesis, when the sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.

Combinations of protein-disulfide isomerase domains show that there is little correlation between isomerase activity and wild-type growth.

Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold. A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isomerization of disulfides (Darby, N. J., and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins. Domains a and a' along with constructs ab, aa', aba', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised. The only domain order that retains significant isomerase activity has the b' domain coupled to the N terminus of the a' domain. This b'a'c has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35, 1972-1980). Individual a and a' domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1Delta Saccharomyces cerevisiae strain yeast. Thus, most of the PDI structure is dispensable for its essential function in yeast, and high-level isomerase activity appears not required for viability or rapid growth.

Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms.

Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSH-disulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2--31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5' region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions.

Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.

Reactivity of glutaredoxins 1, 2 and 3 from Escherichia coli and protein disulfide isomerase towards glutathionyl-mixed disulfides in ribonuclease A.

We have examined the activity of protein disulfide isomerase (PDI) and glutaredoxin (Grx) 1, 2 and 3 from Escherichia coli to catalyze the cleavage of glutathionylated ribonuclease A (RNase-SG) by 1 mM GSH to yield reduced RNase. Apparent Km values for RNase-SG were similar, 2-10 microM, for Grx 1, 3 and PDI but Grx I and Grx 3 showed 500-fold higher turnover numbers than PDI. The atypical Grx 2 also catalyzed deglutathionylation by GSH, but had higher Km and apparent turnover number values compared to the two classical Grx. Refolding of RNase in a glutathione redox buffer was catalyzed by PDI. However, it could be measured only after a characteristic lag phase that was shortened by all three E. coli Grxs in a concentration-dependent manner. A role of the glutaredoxin mechanism in the endoplasmic reticulum is suggested.

The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide.

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxin-like domain a'. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal alpha helix of domain a', but this is not the only critical region for any of these functions.

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue.

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.

Effect of glutaredoxin and protein disulfide isomerase on the glutathione-dependent folding of ribonuclease A.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase A (rd-RNase A) and mixed disulfide between glutathione and reduced RNase A derivative (GS-RNase A) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ESMS) time-course analysis and recovery of the native catalytic activity. The ESMS analysis permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding of rd-RNase A and GS-RNase A was also performed in the presence of glutaredoxin (Grx) and/or protein disulfide isomerase (PDI). All the analyses indicate a pathway of sequential reactions in the formation of native RNase A. First, the reduced protein reacts with a single glutathione molecule to form a mixed disulfide which then evolves to an intramolecular S-S bond via thiol-disulfide exchange. Only at this stage, the intermediate containing one intramolecular S-S reacts with a further glutathione molecule, reiterating the process. An analogous mechanism occurs in the refolding of GS-RNase A. The structural analysis of the intermediates formed during the refolding of RNase A showed for the first time that Grx is actually able to catalyze both formation and reduction of mixed disulfides involving glutatione. In both refolding processes, starting from either rd-RNase A or GS-RNase A, Grx displays a significant catalysis at the early stages of the process. Addition of PDI led to a net catalysis of the entire process without appearing to alter the refolding pathway. In the presence of both Grx and PDI, the two enzymes showed a synergistic activity either starting from rd-RNase A, as previously reported [Lundström, J., and Holmgren, A. (1995) J. Biol. Chem. 270, 7822-7828], or starting from GS-RNase A. Present data suggest that the synergistic effect can be explained assuming that Grx actually facilitates PDI action by catalyzing formation or reduction of mixed disulfides. The mixed disulfides are then rapidly converted into intramolecular disulfides in the presence of PDI. These steps are repeated sequentially throughout the whole refolding, resulting in an immediate formation of fully oxidized species even at the very beginning of the reaction. Finally, a Grx mutant, C14S Grx, in which one of the active site cysteine residues (Cys14) had been replaced by serine, had a similar effect on the distribution of folding intermediates, compared to the wild-type protein, thus demonstrating that Grx acts by a monothiol mechanism either in the reduction or in the oxidation step.

Glutaredoxin accelerates glutathione-dependent folding of reduced ribonuclease A together with protein disulfide-isomerase.

Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 microM reduced and denatured ribonuclease RNase-(SH)8 in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S2)4, a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 microM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'0 of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO3(2-)), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.

Two resident ER-proteins, CaBP1 and CaBP2, with thioredoxin domains, are substrates for thioredoxin reductase: comparison with protein disulfide isomerase.

Protein disulfide-isomerase (PDI) is the best known representative of a growing family of enzymes with thioredoxin domains. Two such proteins with thioredoxin (Trx) domains, CaBP1 and CaBP2 (ERp72), have previously been isolated from rat liver microsomes. Here we report that they, like PDI are substrates for thioredoxin reductase and will catalyze NADPH-dependent insulin disulfide reduction. The activity of CaBP1 and CaBP2 in this assay was higher than that of PDI but lower than that of E. coli Trx. Furthermore, as isolated the thioredoxin domains of CaBP1 and CaBP2 were in disulfide form as judged by stoichiometric oxidation of 2 and 3 mol of NADPH in CaBP1 and CaBP2, respectively. The redox potential of the active site disulfide/dithiol was estimated from the equilibrium with a mutant E. coli Trx, P34H Trx, with a known redox potential (-235 mV). This showed that CaBP1 and CaBP2, like PDI, have a much higher redox potential than wild type thioredoxin (-270 mV) in agreement with a role in formation of protein disulfide bonds. In conclusion, in vitro CaBP1 and CaBP2 share catalytic properties in thiol disulfide-interchange reactions with PDI. Thus, the well known activity of PDI is not unique in the endoplasmic reticulum and CaBP1 and CaBP2 may be regarded as functional equivalents.