Human DCXR - another 'moonlighting protein' involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility?

Biol. Rev. (2014), pp. 000–000. doi: 10.1111/brv.12108

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Human DCXR – another ‘moonlighting protein’ involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility? Bettina Ebert, Michael Kisiela and Edmund Maser∗ Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Brunswiker Str. 10, 24105 Kiel, Germany

ABSTRACT Dicarbonyl/l-xylulose reductase (DCXR; SDR20C1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of 𝛼-dicarbonyl compounds and monosaccharides. Its role in the metabolism of l-xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as ‘sperm surface protein P34H’, it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non-catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS-21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non-primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm–zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry. Key words: dicarbonyl/l-xylulose reductase, DCXR, carbonyl reductase 2, P34H, carbonyl metabolism, AGE precursor, cell adhesion, gamete interaction, phylogenetic relationship. CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Nomenclature and alternative names for human DCXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Structure of human DCXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Cold inactivation of rodent Dcxr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Protein variants of human DCXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a)Does DCXR contain post-translational modifications? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Catalytic activity of human DCXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Human DCXR: metabolism of sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Murine Dcxr: metabolism of dicarbonyls and renal clearance of AGE precursors in mice . . . . . . (a)Dcxr overexpression reduces 3-deoxyglucosone accumulation in mouse kidney . . . . . . . . . . . . . III. DCXR and disease – the multifunctional nature of DCXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) DCXR and pentosuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) DCXR expression in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a)DCXR expression in melanoma: a function in cell adhesion and metastasis? . . . . . . . . . . . . . . . . .

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* Address for correspondence (Tel: +49 431 597 3540; Fax: +49 431 597 3558; E-mail: [email protected]). Biological Reviews (2014) 000–000 © 2014 The Authors. Biological Reviews © 2014 Cambridge Philosophical Society

Bettina Ebert and others

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(3) Idiopathic male infertility: DCXR and its role in fertilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a)DCXR (P34H) is involved in sperm maturation and gamete interaction . . . . . . . . . . . . . . . . . . . . . . (b)DCXR is differentially expressed along the male reproductive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) DCXR on the sperm surface as a predictor of male fertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (d)Vasectomy affects DCXR expression in the human epididymis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (e) Sperm maturation and cell–cell contacts: epididymal principal cells from infertile donors display characteristics of an EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. DHS-21, a DCXR ortholog from c. elegans modulates longevity and reproduction – another multifunctional protein? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) DHS-21 is expressed on spermatids and plays a role in reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) DHS-21 expression increases the lifespan of C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Cbr2, a carbonyl reductase closely related to DCXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Cbr2 from mouse (Mus musculus): ‘mouse lung carbonyl reductase (MLCR, AP27)’ . . . . . . . . . . . (2) Cbr2 from golden hamster (Mesocricetus auratus): ‘hamster sperm protein P26h’ . . . . . . . . . . . . . . . . (a)Catalytic properties of hamster P26h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Cbr2 from cow (Bos taurus): ‘P25b’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Phylogenetic relationship between DCXR and Cbr2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Cbr2 has arisen from a gene duplication event . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION The short-chain dehydrogenase/reductase (SDR) super-family member dicarbonyl/l-xylulose reductase (DCXR) is widely distributed among eukaryotes and prokaryotes (Kisiela et al., 2011) indicating an evolutionarily conserved function. The enzymatic activity of human DCXR (SDR20C1) was discovered in 1970, when the major enzymatic defect in essential pentosuria (characterized by high urinary excretion of l-xylulose) was attributed to a deficiency of l-xylulose reductase (Wang & Van Eys, 1970). More than 30 years later the human enzyme and four of its rodent orthologs were cloned and enzymatically characterized (Nakagawa et al., 2002). Based on the diacetyl reductase activity of the recombinant enzyme and its additional capability to reduce l-xylulose it was concluded that l-xylulose reductase (EC 1.1.1.10) was identical with diacetyl reductase (EC 1.1.1.5). The enzyme was therefore designated ‘dicarbonyl/l-xylulose reductase’ (DCXR). Recombinant human DCXR reduced several aliphatic and aromatic dicarbonyl compounds as well as sugars (pentoses, tetroses) in a NADPH-dependent manner. The rodent DCXR counterparts from rat, mouse, hamster and guinea pig all displayed substantially higher catalytic efficiencies than the human form for almost all compounds tested (Nakagawa et al., 2002). In invertebrates, the only ortholog of DCXR that has been studied to date is that from the nematode Caenorhabditis elegans, DHS-21 (Kisiela et al., 2011; Son le et al., 2011). Despite the high evolutionary distance between mammals and nematodes, DHS-21 shows some remarkable similarities

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with the human protein (e.g. structural features and catalytic properties; see Section IV.1). In addition to its metabolic activity, some apparently non-catalytic functions that might involve protein–protein interactions have been reported for DCXR. This enzyme thus has been termed a ‘moonlighting protein’: proteins that combine two distinct functions in a single polypeptide chain (Jeffery, 2003). First, DCXR (often referred to in this context as P34H) plays a crucial role in the sperm–zona pellucida interaction and hence in the fertilization process (Boue & Sullivan, 1996; Boué, Blais & Sullivan, 1996; Moskovtsev et al., 2007). Second, DCXR is thought to have a role in cell adhesion (Cho-Vega et al., 2007b) and probably in tumour dissemination (Fig. 1). However, further experimental evidence is needed to substantiate these functions. This review attempts to illustrate the multifunctional nature of DCXR to stimulate investigation of non-enzymatic functions in other members of the SDR superfamily. For several SDRs no physiological role has been identified to date based on their catalytic properties; it might be possible that their main functions are non-catalytic. This review therefore emphasizes the non-catalytic properties of DCXR, which we are only just beginning to understand. Moreover, because of inconsistent and confusing use of numerous alternative names for DCXR, other tetrameric enzymes and the closely related carbonyl reductase 2 (Cbr2), we provide an overview of the systematic classification of these proteins. In addition, a phylogenetic analysis illustrates the ancestry of DCXR and Cbr2 and highlights the biochemical and functional relationships between these tetrameric enzymes.

Biological Reviews (2014) 000–000 © 2014 The Authors. Biological Reviews © 2014 Cambridge Philosophical Society

Human DCXR – another ‘moonlighting protein’?

ic funct talyt ion Ca bolism Xylu lo se

O C H3

H3 C

OH

m et a

HO

OH

O

OH

m is ol b

Ca rb o

a et m O yl n

DCXR

a

ct

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es

a

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(?)

n-c a

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t

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t a l yti c f u n c

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Fig. 1. Multifunctional nature of DCXR: catalytic and non-catalytic functions. DCXR mediates the reductive metabolism of carbonyl compounds such as 𝛼-dicarbonyls and l-xylulose. In addition, due to its role in gamete interaction and its possible function in cell-adhesion, DCXR has been referred to as a ‘moonlighting protein’.

(1) Nomenclature and alternative names for human DCXR Since 2009, the systematic name for DCXR is short chain dehydrogenase/reductase family 20C, member 1 (SDR20C1) (Persson et al., 2009); other commonly used names are l-xylulose reductase (XR) and sperm surface protein P34H [P34H; ‘H’ indicating ‘human’ and 34 identifying the protein’s size (34 kDa)]. Less frequent designations for DCXR include kidney dicarbonyl reductase (KIDCR), dicarbonyl reductase (DCR) and, confusingly, human carbonyl reductase 2 (HCR2). Although sharing a considerable degree of sequence similarity, carbonyl reductase 2 (Cbr2) and DCXR are two distinct proteins (see Table 1) and the Cbr2 gene is absent in humans, therefore the name ‘HCR2’ can be misleading. It should be noted that at the amino acid level, DCXR and P34H are identical except for one amino acid substitution in P34H at position 239 (G239R): in the sequence obtained from a human epididymal cDNA library (Legare et al., 1999b), codon GGG (Gly) in DCXR was replaced by AGG (Arg). As numerous single-nucleotide polymorphisms (SNPs) have been identified in the DCXR sequence, it can be assumed that this substitution represents a SNP. However, such a SNP has not been listed in public databases to date. (2) Structure of human DCXR The crystal structure of DCXR was initially resolved in 2002 (El-Kabbani et al., 2002), with further detail

3 provided later including the substrate binding site (Ishikura et al., 2003a) and cofactor binding site (El-Kabbani et al., 2004) as well as residues responsible for binding inhibitors (El-Kabbani et al., 2005). The latter study provided crystallographic evidence that functional DCXR has a tetrameric structure, which was reported previously for mouse DCXR using gel-filtration analysis (Ishikura et al., 2003b). Two monomers are connected via salt bridges formed between Arg203 from one monomer and the carboxyl group of the C-terminal Cys244 from the adjacent monomer. Two such dimers form a homotetramer, with each subunit containing one molecule of NADP+ (El-Kabbani et al., 2005). Further refinement of the crystal structure of human DCXR together with mutation analyses revealed an interesting structural feature that might serve as a post-translational mechanism to regulate the enzyme’s activity (Zhao et al., 2009). In the purified protein, the cysteine residues Cys138 and Cys150, which are located within the active site, normally occur in their reduced form with free SH-groups. But in an oxidizing environment a disulfide bond between those cysteines is created and this modification decreases the catalytic efficiency of DCXR threefold. However, this disulfide bond is unstable and can be reversed by reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol (2ME) and glutathione (GSH). Notably, a decreased content of 2ME in the protein buffer alone was sufficient to allow for oxidation resulting in inactivation of DCXR (Zhao et al., 2009). The strong dependence of DCXR activity on the supplementation of assay buffers with reducing agents might explain the variability in results from different laboratories in the catalytic activities of this enzyme. Incubation with millimolar concentrations of cysteine caused covalent modification of DCXR with cysteine resulting in a 10-fold reduction of catalytic activity. This was prevented by the addition of both NADP(H) and the inhibitor n-butyric acid, suggesting S-cysteinylation of residues within the active site of the enzyme (Zhao et al., 2009). The pronounced inactivation of DCXR in an oxidizing environment might be a mechanism by which the enzyme’s activity can be modulated and thus, similarly to many other proteins (e.g. transcription factors, receptors), DCXR might sense and respond to the cell’s redox state. Although seen as a defence mechanism to reduce oxidative cell damage by detoxification of reactive carbonyls, DCXR has been reported to produce reactive oxygen species (ROS) itself through the redox cycling of 9,10-phenanthrenequinone, a polycyclic aromatic hydrocarbon (PAH) derivative (Matsunaga et al., 2008). From this point of view, the impairment of the enzyme’s catalytic activity in an oxidizing cellular environment could be a mechanism to prevent the generation of ROS by DCXR.



l-Xylulose reductase; DCXR

Q920N9 (83%)

Guinea pig: DCXR_CAVPO

Metabolism of: 𝛼-dicarbonyls, sugars, xylulose;

Metabolism of: 𝛼-dicarbonyls, sugars, xylulose;

—

Unknown

Q9Z245 (85%)

Q29529 (85%)

G3N1E7 (89%)

Guinea pig: H0VPQ5_CAVPO H0VPQ5 (82%)

Golden hamster: Q9Z245_MESAU

Pig: CBR2_PIG Data incomplete∗

Bovine: G3N1E7_BOVIN

Q542P5_MOUSE Q542P5 (100%) Rat: No entry —

P08074 (100%)

—

Unknown

Unknown

Metabolism of: 3-oxosteroids, ketones; activated by arachidonic acid; adipocyte differentiation

—

Function

Metabolism of: 3-oxoandrostane, ketones Carbonyl Carbonyl metabolism reductase (acetone, [NADPH] 2-like acetaldehyde, etc.)

P26h; surface sperm protein P26h

Pig lung carbonyl Carbonyl reductase metabolism; (PLCR) activated by arachidonic acid

P25b (?) P21b (?)

—

MLCR; AP27; NADPHdependent carbonyl reductase 2 (Cbr2)

—

UniProt accession no. (% identity with Alternative CBR2_MOUSE) name(s)

Cbr2

∗ Note: The sequence of CBR2_PIG was deduced from a cDNA (Nakanishi et al., 1993). However, it does not align to the Sus scrofa genome that was later released, most probably due to variations between different inbred strains. Large parts of this genome still lack annotation.

P31h; DCXR; hamster diacetyl reductase

Q91XV4 (86%)

—

Golden hamster: DCXR_MESAU

—

P25b (?) P25b (?)

Q1JP75 (85%) A5PJR3 (85%)

Bovine: DCXR_BOVIN A5PJR3_BOVIN

Pig: No entry

l-Xylulose Metabolism of: reductase; Dcxr 𝛼-dicarbonyls, sugars, xylulose

Q920P0 (84%)

Rat: DCXR_RAT

Human: No entry

Species UniProtKB identifier

Mouse: l-Xylulose Metabolism of: CBR2_MOUSE reductase; Dcxr 3-deoxyglucosone, 𝛼-dicarbonyls, sugars

Q91X52 (84%)

Mouse: DCXR_MOUSE

DCXR, P34H, XR, Metabolism of: HCR2, KIDR, 𝛼-dicarbonyls, DCR xylulose; gamete interaction; cell adhesion (?); marker of epididymal sperm maturation

Function

Q7Z4W1 (100%)

UniProt accession no. (% identity with Alternative DCXR_HUMAN) name(s)

Human: DCXR_HUMAN

Species UniProtKB identifier

DCXR

Table 1. Overview of carbonyl reductase 2 (Cbr2) and dicarbonyl/l-xylulose reductase (DCXR) in different species (only those included in the UniProt database are shown)

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Human DCXR – another ‘moonlighting protein’? (3) Cold inactivation of rodent Dcxr Cold inactivation is a characteristic feature of rodent forms of DCXR that is not observed in the human enzyme (Nakagawa et al., 2002; Ishikura et al., 2003b). At low temperatures, mouse Dcxr was inactivated, with a 50% loss of catalytic activity after only 5 min of incubation at 0∘ C (Ishikura et al., 2003b). Gel-filtration analysis revealed that this reversible process resulted from dissociation of the active tetramer into a dimeric form lacking catalytic activity. From an analysis of a series of mutated enzymes, Ishikura et al. (2003b) demonstrated that the residues Asp238 and Leu242 were predominantly involved in the destabilization of the tetrameric form of mouse Dcxr. Cold inactivation was completely prevented by the double mutation D238E/L242W that corresponds to the human cold-stable form, in which Asp238 is replaced with Glu and Leu242 with Trp. However, it remains unexplained whether this phenomenon serves a physiological purpose in rodents. It is conceivable that cold inactivation represents an evolutionary relict that originally had a function in ancestral species such as amphibians and was lost during the course of evolution in human DCXR. (4) Protein variants of human DCXR The human DCXR gene is encoded on the reverse strand of chromosome 17 (17q25.3). According to the ENSEMBL database (www.ensembl.org) it has 21 transcripts from which 7 have been identified as coding for proteins with lengths ranging from 86 to 244 amino acids. By contrast, the NCBI nucleotide database (www.ncbi.nlm.nih.gov/gene/51181) reports only two protein variants for human DCXR. The longer isoform 1 is encoded by an 860 base pair (bp) mRNA (NM_016286.3) that translates into a protein composed of 244 amino acids (NP_057370.1). The shorter isoform 2 (NM_001195218.1) is the product of an 854 bp mRNA and harbours a deletion of two amino acids (ΔG18, ΔI19) within the cofactor binding region resulting from an alternate in-frame splicing event affecting exon 2 (NP_001182147.1). Because isoleucine 19 is involved in the binding of NADP+ in the active tetrameric enzyme (El-Kabbani et al., 2005), it is more than likely that deletion of this residue would change the catalytic properties of DCXR isoform 2. Note that DCXR isoform 2 is missing from the ENSEMBL database. Since the 1980s, the existence of two DCXR isoforms with different subcellular localization has been known (Lane, 1985). While the major isoform of the enzyme was detected in both mitochondria and the cytosolic fraction of human liver, the minor isoform was absent in mitochondria and only found in the cytosol (Lane & Jenkins, 1985). These isoforms displayed different electrophoretic behaviour and substrate affinities toward l-xylulose. The minor isoform with low substrate affinity toward l-xylulose had a slightly smaller molecular

5 mass than the major isoform, which exhibited a higher substrate affinity towards this sugar. Moreover, the more efficient major isoform was absent in pentosuric individuals, while the minor form was still detectable. Although the smaller size and the low catalytic activity of the minor isoform would support its identity with DCXR isoform 2 that harbours a defective cofactor binding site (NP_001182147.1), due to methodological restrictions that were present in the 1980s this is difficult to verify. (a) Does DCXR contain post-translational modifications? Based on its amino acid sequence, the calculated molecular weight of DCXR is 26 kDa, which is in agreement with the size of the recombinant protein cloned by Nakagawa et al. (2002). Most interestingly, although expression data of the native protein are rare, researchers have found two different sizes for DCXR expressed in vivo. Western blot analyses revealed that DCXR from prostate (Cho-Vega et al., 2007a) had the same apparent size (34 kDa) as DCXR (P34H) isolated from sperm surface (Boué et al., 1994, 1996; Boue & Sullivan, 1996). By contrast, two different studies reported that in liver the DCXR-specific signal migrated at approximately 26 kDa (Liu et al., 2006; Cho-Vega et al., 2007a). When the tissue distribution of DCXR in the human body was first analyzed, different protein sizes were not described (Nakagawa et al., 2002): corresponding immunoreactive bands from liver, kidney and epididymis all had the same apparent size. In the Western blot presented by Nakagawa et al. (2002) no protein standard is shown, therefore the molecular mass can only be inferred from the size of the positive control, a His-tagged recombinant human DCXR (around 30 kDa). The inconsistency among these studies might have methodical causes, such as variation in resolution of the separating sodium dodecyl sulphate (SDS) gel. Interestingly, extraction of spermatozoa-bound DCXR by treatment with membrane-destabilizing detergents resulted in the appearance of two bands in Western blot analysis: the expected 34-kDa band and a second one at 30 kDa (Legare et al., 1999b). The authors explained this as resulting from proteolytic cleavage of DCXR during this process. They also predicted three potential N-glycosylation sites (positions 119–121, 131–132, and 164–166) using their sequence-analysis software. Likewise, using bioinformatics tools available from public proteomic servers such as ExPasy (www.expasy.org), possible modification sites (N-myristolyation, N-glycosylation, and phosphorylation) have been predicted for DCXR as well as one putative transmembrane domain at position 68–89. (Cho-Vega et al., 2007b). Taken together, these observations suggest that DCXR may be modified by post-translational mechanisms, perhaps explaining the observed tissue-dependent differences in molecular weights. It also raises the question of whether sperm-associated DCXR is a phosphatidyl-anchored


6 protein, similar to the sperm surface proteins from the Cbr2 subfamily P26h (golden hamster Cbr2) and P25b (bovine Cbr2) (see Sections V.2 and V.3). II. CATALYTIC ACTIVITY OF HUMAN DCXR Human DCXR combines both 𝛼-dicarbonyl reductase and l-xylulose reductase activities and, based on its catalytic properties, it performs several distinct physiological functions. By the reductive metabolism of carbonyl compounds it serves as clearance/defence mechanism against endogenous or exogenous potentially harmful carbonyls. The recombinant human enzyme (26 kDa) was active with several aliphatic (e.g. diacetyl, 2,3-heptanedione, 3,4-hexanedione) and aromatic (e.g. 1-phenyl-1,2-propanedione) dicarbonyl compounds that are by-products of endogenous metabolic processes (diacetyl) or occur naturally in foodstuffs and are used as flavourings. The enzyme preferably reduced 𝛼-dicarbonyls but was inactive with diketones without an 𝛼-carbonyl group (e.g. 2,5-hexanedione, 2,4-pentanedione) as well as with mono-carbonyls (e.g. acetone, cyclohexanone, pyridine-4-aldehyde) and 3-oxo-androstanes. Moreover, it was inhibited by short-chain fatty acids, particularly by n-butyric acid (Nakagawa et al., 2002) (a constituent of butter flavourings, similar to diacetyl), but also occurring endogenously. A potential defensive role against xenobiotics is evident from the finding that 9,10-phenanthrenequinone, a major component of diesel exhaust particles, was metabolized by DCXR in a human T-lymphoma cell line (Matsunaga et al., 2008). However, this is the only xenobiotic compound to be a known DCXR substrate to date. Although experimentally substantiated only for the mouse ortholog, Dcxr also removes highly reactive 𝛼-dicarbonyls (see Section II.2), precursors of advanced glycation end products (AGEs). By this action, DCXR might prevent covalent modifications of proteins that would otherwise lead to the impairment of cellular functions. Moreover, AGEs have been recognized as sources of persistent low-grade inflammation, as they can bind to specific receptors (receptor for AGE; RAGE) and thereby activate an inflammatory response. Thus, by the removal of AGE precursors DCXR might counteract the emergence of AGE-related inflammation associated with diabetes or cellular ageing. By converting l-xylulose to the organic osmolyte l-xylitol DCXR is believed to be involved in water reabsorption and to protect against osmotic stress, however, direct experimental evidence is still awaited. The reduction of l-xylulose to l-xylitol is a part of the uronate cycle; this alternative pathway of glucose metabolism is known to account for 5% of total daily glucose utilization.

Bettina Ebert and others (1) Human DCXR: metabolism of sugars From all the monosaccharides, including aldotriose d,l-glyceraldehyde, tetroses (e.g. d-erythrose, l-threose), pentoses (e.g. d-ribulose), and hexoses (e.g. d-glucose, d-fructose), tested as possible substrates for human DCXR, only l-xylulose showed considerable substrate affinity with a Michaelis constant (K m ) value of 0.21 mM and a turnover number/Michaelis constant (k cat /K m ) ratio of 3.1 s−1 mM−1 (Nakagawa et al., 2002). Based on this rather poor catalytic activity observed with the recombinant enzyme, a physiological role for DCXR in the metabolism of l-xylulose would seem unexpected. This is surprising because l-xylulose deficiency has long been known to be the cause of pentosuria, a hereditary metabolic condition (see Section III.1). Therefore, it would be interesting to explore whether endogenously expressed DCXR and the recombinant enzyme have different catalytic properties and if this is attributable to post-translational modifications that only occur in vivo. Recombinant enzymes from rat, mouse and guinea pig all exhibited much higher catalytic efficiencies for l-xylulose; guinea pig Dcxr was the most active ortholog with a more than 200-fold higher k cat /K m ratio of 740 s−1 mM−1 (Nakagawa et al., 2002). These interspecific differences indicate that catalytic data obtained from rodent models should not be extrapolated to humans, especially with regard to the conversion of l-xylulose by DCXR. (2) Murine Dcxr: metabolism of dicarbonyls and renal clearance of AGE precursors in mice Reactive 𝛼-dicarbonyls (e.g, 3-deoxyglucosone, methylglyoxal and glyoxal) are formed during cellular metabolic processes by degradation of sugars, lipid peroxidation or ascorbate autoxidation. These compounds can covalently modify proteins and give rise to AGEs such as N𝜖 -(carboxymethyl)-lysine (CML). While normal cellular ageing is accompanied by accumulation of AGEs, glycated proteins are frequently observed in many disease states, most prominently in uncontrolled diabetes as a result of high blood glucose levels. Hyperglycemic conditions occurring in untreated diabetes or resulting from renal insufficiency can lead to the overabundance of carbonyl compounds in the bloodstream, emphasizing the central importance of the kidney in carbonyl metabolism. The initial catalytic characterization of recombinant human DCXR demonstrated that the enzyme accepted the AGE precursor methylglyoxal as a substrate, although with rather low catalytic efficiency (k cat = 0.57 s−1 ) and poor substrate affinity (K m ) in the millimolar range. An even worse substrate was 3-deoxyglucosone, for which human DCXR showed almost no activity. Both compounds also were rather poor substrates for mouse Dcxr (Nakagawa et al.,


Human DCXR – another ‘moonlighting protein’? 2002). Interestingly, double immunostaining in rat renal tubules revealed a co-localization of Dcxr with CML, a predominant AGE found in diabetic patients. This finding raised the speculation that rodent Dcxr might play a role in the metabolism of dicarbonyl precursors, possibly protecting against the formation of AGEs. In a search for carbonyl-metabolizing enzymes that might contribute to the reduction of renal carbonyl stress, the role of Dcxr was investigated using rodent models including mice and rats (Sudo et al., 2005; Asami et al., 2006; Odani et al., 2008). High expression of mouse Dcxr in the brush border epithelium of renal tubules was described previously (Nakagawa et al., 2002) and implied a potential role in the detoxification of AGE-precursors in the kidney. Sudo et al. (2005) generated a Dcxr-overexpressing transgenic (Tg) mouse model and observed a sixfold increase in Dcxr protein content in homozygous (Tg/Tg) animals, which were physiologically normal. Immunostaining revealed elevated Dcxr-expression in Tg/Tg animals not only in the kidney but also throughout the body, while the renal distribution resembled that of endogenous Dcxr being predominantly expressed in the collecting tubules. To investigate further the role of Dcxr in the progression of diabetic nephropathy, Dcxr-overexpressing mice were crossed with the type-2 diabetic mouse model KK-Ay . The generated Tg/Ay hybrid mice showed a threefold higher Dcxr protein expression, reduced body mass, lower blood glucose levels and improvement in renal parameters compared to their wild type/Ay littermates (+/Ay ). Overexpression of Dcxr obviously accelerated the metabolism of l-xylulose and its subsequent renal clearance, thereby contributing to the removal of excess glucose from the blood of Tg/Ay hybrid mice. The authors also suggested that the decreased blood glucose levels might have resulted from the reduced food intake observed in these animals. However, at least with regard to CML, protection against the formation of AGEs by Dcxr overexpression could not be confirmed, as no reduced accumulation of CML in kidney tissue of Tg/Ay mice was observed. This raises the question of whether Dcxr can mediate protection against other AGEs by metabolizing their respective precursors. (a) Dcxr overexpression reduces 3-deoxyglucosone accumulation in mouse kidney Unilateral ureteral obstruction (UUO) is a well-established experimental animal model of renal injury where obstruction of one of the kidneys is simulated by ligation of the ureter. This condition is known to generate oxidative stress leading to the formation of CML and renal fibrosis (Kawada et al., 1999). As expected, rats receiving UUO treatment for 7 days exhibited increased accumulation of CML

7 in the obstructed kidney (Asami et al., 2006). By contrast, Dcxr protein content in both the obstructed and the non-obstructed kidney decreased continuously during the course of the experiment from 12 h to 7 days. This implies that the elevated CML levels in the obstructed rat kidneys were not only a result of the carbonyl stress, but also might be a consequence of the decreased Dcxr protein expression recorded throughout the treatment. This reduced DCXR expression provoked by carbonyl stress might indicate a possible regulatory mechanism for DCXR that is widely unexplored. It remains unknown whether ROS are involved in this down-regulation (in both the obstructed and non-obstructed kidney), as has been described for the incubation of cultured cells with 9,10-phenanthrenequinone (Matsunaga et al., 2008). When the effects of UUO treatment in heterozygous Dcxr transgenic (Tg/+) mice was compared with their wild-type (+/+) counterparts, it emerged that the Dcxr-overexpressing animals accumulated less 3-deoxyglucosone in the obstructed kidney tissue (Asami et al., 2006). A similar tendency was observed for methylglyoxal and glyoxal but differences were not statistically significant. These findings indicate a role of Dcxr in the metabolism of renal carbonyl compounds such as AGE precursors. However, overexpression of Dcxr did not prevent an initial renal fibrosis as evidenced by similar collagen type III mRNA contents in obstructed kidneys of both transgenic and wild-type mice (Asami et al., 2006). Taken together, Dcxr overexpression in mice did not prevent accumulation of CML in kidney tissue but obviously accelerated the clearance of 3-deoxyglucosone in kidneys during obstruction. Given that the native mouse Dcxr resembles the recombinant protein with respect to its catalytic properties, this outcome is rather surprising, because the recombinant enzyme had a low activity with 3-deoxyglucosone in vitro (Nakagawa et al., 2002). Conversely, the rather marginal effect of Dcxr on methylglyoxal accumulation in vivo was to be expected from the poor substrate affinity and catalytic efficiency (K m = 8.1 mM; k cat /K m = 1.4 s−1 mM−1 ) of the recombinant enzyme (Nakagawa et al., 2002). So, although both 3-deoxyglucosone and methylglyoxal are poor substrates in vitro, at least with respect to the clearance of 3-deoxyglucosone, overexpression of mouse Dcxr in vivo was beneficial. As DCXR is seen as a defence mechanism against reactive carbonyls and AGE precursors, overexpression of DCXR instead of down-regulation during the UUO treatment described above would have been expected. If Dcxr establishes a biochemical barrier against AGE precursors that are produced during UUO, the physiological significance of this down-regulation is hard to explain and appears contradictory. Nevertheless, this reduced Dcxr expression might point to a possible regulatory mechanism that remains unexplored for Dcxr.


8 III. DCXR AND DISEASE – THE MULTIFUNCTIONAL NATURE OF DCXR

(1) DCXR and pentosuria Pentosuria is an inherited metabolic condition that is caused by DCXR deficiency. Because impaired reduction of l-xylulose to l-xylitol leads to the accumulation of l-xylulose, elevated levels of this sugar are found in blood and urine of pentosuric individuals. Pentosuria is a completely benign condition that has no impact on the lifespan of affected individuals, but before 1950, due to the lack of clinical tests that could distinguish between urinary glucose and xylulose, individuals with pentosuria were often wrongly diagnosed with diabetes. Although the enzyme deficiency responsible for essential pentosuria was identified in 1970 (Wang & Van Eys, 1970), the molecular background was resolved only very recently (Pierce et al., 2011). As a recessive trait, pentosuria has almost exclusively been diagnosed among ethnic groups with a history of endogamy, such as the Ashkenazi Jewish population (Lane & Jenkins, 1985), within which the frequency of pentosuria is one in 3300 individuals (Pierce et al., 2011). By sequencing the DCXR gene in pentosuric individuals from this ethnic group, two mutated alleles responsible for DCXR deficiency were discovered (Pierce et al., 2011). The DCXR c.583ΔC allele contains a deletion of a single cytosine within exon 7, inserting a premature stop codon that leads to a loss of 44 amino acids in the C-terminus of the translated protein. Because the C-terminal part of DCXR harbours two amino acids (Arg203 and Cys244) critical for the formation of the active tetrameric structure of the enzyme (El-Kabbani et al., 2005), this allele will likely produce non-functional protein. In the second mutant allele, DCXR c.52(+1)G > A, a substitution at the first base pair of intron 1 alters the splice donor site of exon 1. This mutation results in different alternative transcripts, all of which contain an altered 3′ end of exon 1. Notably, the deduced amino acid sequence of one transcript variant carries a deletion of two amino acids within the coenzyme-binding region (K17 and G18) and resembles the sequence of DCXR isoform 2 (NP_001182147.1) that also contains a two amino acid deletion, although on positions G18 and I19 (see Section I.4). Most interestingly, Western blot analysis of DCXR in lymphoblasts from individuals either homozygous for DCXR c.583ΔC or DCXR c.52(+1)G > A, or compound heterozygous for both mutant alleles showed no DCXR-specific signal in either case, indicating that none of the mutant alleles produced stable protein. These findings are in agreement with the elevated xylulose levels (0.9–1.9 mg dl−1 ) in blood plasma samples from individuals homozygous or compound

Bettina Ebert and others heterozygous for the mutant alleles (Pierce et al., 2011). Moreover, homozygosity mapping of the genomic region surrounding DCXR revealed that the DCXR c.52(+1)G > A allele arose more recently than the DCXR c.583ΔC allele and both mutations appeared in the period after the European origin of the Ashkenazi Jewish population (i.e. after Jews migrated into Europe from Palestine about 2000 years ago). (2) DCXR expression in cancer Data regarding the expression of DCXR in cancerous tissues are rare. Only in a few different organs including prostate, skin (melanoma), and liver, has a possible association of DCXR with cancer been investigated. Despite this limited information, the strikingly different expression pattern of DCXR in prostate cancer versus melanoma and liver cancer implies a highly tissue-specific regulation. While an increased DCXR level has been detected in prostate cancer (Cho-Vega et al., 2007a), in melanoma (Cho-Vega et al., 2007b) and in hepatocellular carcinoma (HCC) DCXR was down-regulated (Liu et al., 2006). In 58% of analyzed HCC cases from Chinese patients a reduced level of DCXR mRNA (in this study called human carbonyl reductase 2 or HCR2) was observed. A similar expression pattern was detected for DCXR protein content, albeit only a small number of specimens were examined: three out of four tumour samples showed a substantially decreased DCXR protein content compared to tumour-free tissue. Moreover, DCXR expression was associated with tumour stage: Edmondson’s grade III/IV carcinoma displayed a further reduction in the DCXR expression compared to earlier (grade I/II) tumour samples. Because oxidative stress promotes the development of HCCs, the authors hypothesized that a disturbance of the DCXR-mediated detoxification system might favour the onset and progression of HCCs. Further clinical and biochemical data are needed to substantiate this interpretation. Contrasting with the reduction of DCXR expression in HCC, in early-stage and hormonally treated prostate cancer an up-regulation of DCXR on both protein and mRNA levels has been reported (Cho-Vega et al., 2007a). Remarkably, the authors detected an unexplained ‘shift’ of the subcellular localization of DCXR in cancerous cells compared to healthy tissue: while normal prostate cells express DCXR in the cytoplasmic membrane, in cancerous tissue, DCXR was predominantly localized in the cytoplasm and nucleus. A similar altered subcellular localization was also detected in melanoma (Cho-Vega et al., 2007b). Importantly, in this study, the investigators made an unexpected observation that suggested a possible novel function of DCXR, not attributable to the enzyme’s catalytic activity but possibly based on protein–protein interactions: a possible role in cell adhesion (see Section III.2a).


Human DCXR – another ‘moonlighting protein’? The mechanisms leading to the over- or underabundance of DCXR in malignant lesions remain unresolved and deserve further investigation, as does the elucidation of the unexplored transcriptional regulation of DCXR.

9

(A) Tight junctions

(a) DCXR expression in melanoma: a function in cell adhesion and metastasis? The establishment of the normal epithelium requires strong adhesive cell to cell and cell to extracellular matrix (ECM) junctions, which differ in their localization and molecular composition. Adherens junctions (AJs) mediate cell–cell contacts via transmembrane cadherins (e.g. E-cadherin), which are bound to intracellular anchors (e.g. 𝛽-catenin) (Fig. 2). Tight junctions (TJs) that mediate cell–cell contacts via transmembrane claudins and occludins seal the gaps between epithelial cells, thereby functioning as a diffusion barrier and conferring the epithelium with selective permeability. By contrast, hemidesmosomes are cell–ECM adhesions that anchor cells to the basement membrane (Fig. 2). They are distinct from AJs and TJs with respect to their localization and molecular structure: while AJs and TJs are attached to the actin cytoskeleton, hemidesmosomes are anchorage sites for keratin intermediate filaments, which are linked to transmembrane adhesion proteins (integrins and type XVII collagens) via an intracellular anchor protein, plectin (Simpson, Patel & Green, 2011). The integrity of intercellular junctions between epithelial cells lining the surface of organs is of central importance for a functional blood–organ barrier. Mechanisms that regulate the loss of cell–cell contacts occurring during the malignant transformation of benign neoplasms have been intensively studied due to their important role in the development of metastases. The epithelial-to-mesenchymal transition (EMT) is a cellular remodelling process by which differentiated epithelial cells undergo morphological changes, lose their cell–cell contacts, and become de-differentiated. Despite being indispensible for normal physiological processes like wound healing and organogenesis, the EMT has received most attention due to its involvement in tumour metastasis (Thiery et al., 2009). Owing to its high metastatic behaviour, malignant melanoma is recognized as the most deadly type of skin cancer. It develops in pigment cells of the skin, the melanocytes, and may arise from a benign nevus. Understanding the mechanisms governing the regulation of cell adhesion in this type of cancer may help to develop novel treatment strategies. A possible cell-adhesion function of DCXR was concluded from the observation that DCXR co-localized with 𝛽-catenin and E-cadherin in prostate carcinoma cells (Velculescu et al., 1999). Later analysis of the subcellular localization of DCXR in different cell types of normal skin and in benign or malignant

Adherens junction

Hemidesmosome

Basal lamina

Extracellular matrix

Adherens junction

(B)

Cytoplasm

Actin Actin

E-cadherin α

β DC XR

DCXR

β

cell membrane

Intercellular space

α

β-catenin

Fig. 2. Schematic representation of cell–cell and cell– extracellular matrix contacts in polarized epithelial cells. (A) In a typical vertebrate polarized epithelial cell, tight junctions occupy the most apical part of the cell, while adherens junctions are found in the middle region. Hemidesmosomes reside at the basement membrane (basal lamina) and anchor the cell to the extracellular matrix. (B) In adherens junctions, the extracellular part of the transmembrane protein E-cadherin establishes contact with the neighbouring cell and the cytoplasmic part is bound to catenins (𝛼-catenin, 𝛽-catenin, etc.) of which 𝛼-catenin is linked to the actin cytoskeleton. According to Cho-Vega et al. (2007b), immunofluorescence labelling showed that DCXR co-localizes with E-cadherin and 𝛽-catenin as well as with other cell-adhesion molecules such as PECAM-1 in endothelial cells.

melanocytic lesions showed a similar co-localization with cell-adhesion molecules, providing more evidence for this novel function of DCXR (Cho-Vega et al., 2007b). Using double immunofluorescence and confocal microscopy, it was shown that in normal skin, DCXR resides in the intercellular membranes of keratinocytes, in association with E-cadherin and 𝛽-catenin, components of AJs (Fig. 2). In nevi, benign melanocytic lesions, DCXR expression was greater than in malignant melanomas, indicating down-regulation during the malignant transformation of melanocytes. While the majority of analyzed nevi displayed DCXR-staining of the cytoplasmic membrane, in some melanomas (20–30%) this membranous expression had changed to aberrant expression around the nucleus, described as ‘perinuclear (Golgi-like)


10 expression’ (Cho-Vega et al., 2007b) (Fig. 3). A possible role of DCXR in cell adhesion was concluded from the observation that in 33% of primary and metastatic melanomas with a dishesive growth pattern DCXR was predominantly expressed in the perinuclear region, suggesting that the shift of DCXR localization from the cytoplasmic membrane to the region near the nucleus might be associated with the observed loss of cell adhesion. Accordingly, in melanomas with obviously intact cell–cell contacts i.e. showing a cohesive growth pattern, DCXR protein expression was detected in the cytoplasmic membrane (Fig. 3). The mechanism responsible for the localization of DCXR to the plasma membrane has not yet been determined. DCXR-staining has also been observed in other intercellular junctions: in dermal endothelial cells, DCXR was co-localized with the endothelial adhesion molecule CD31 (the platelet endothelial cell adhesion molecule; PECAM-1). By contrast, DCXR was not detectable at the interface between basal cells and the basement membrane, where hemidesmosomes establish cell to basal lamina junctions. As AJs and hemidesmosomes differ in their protein composition, this observation implies a preference of DCXR for association with either E-cadherin, 𝛽-catenin or other adhesion molecules such as PECAM-1, but not for proteins forming the hemidesmosomes (e.g. integrins or plectins). Further studies are needed to decipher the molecular mechanisms that lead to the altered subcellular localization of DCXR in transformed cells that may depend on altered post-translational modification such as phosphorylation by kinases activated in the course of an EMT. Whether the observed co-localization of DCXR with adhesion molecules involves a physical association with, for example, E-cadherin or 𝛽-catenin, also remains to be investigated. (3) Idiopathic male infertility: DCXR and its role in fertilization Mammalian spermatozoa produced in the testis are non-motile and not able to fertilize. During their transit along the excurrent duct system, composed of the vasa efferentia, epididymis and vas deferens, spermatozoa undergo a process referred to as post-testicular sperm maturation (Cooper, 1986), characterized by modifications in the composition of macromolecules on the sperm’s surface. Sperm maturation is finally completed by further complex biochemical and physiological changes occurring during ejaculation and in the female reproductive tract, collectively called capacitation (Austin & Bishop, 1958; Zaneveld et al., 1991), yielding spermatozoa that are able to penetrate and fertilize the egg. The epididymis is commonly divided into three major segments (the proximal caput, the middle corpus and the distal cauda; Fig. 4). The caput and corpus have been identified as sites where sperm maturation takes

Bettina Ebert and others place, while the cauda serves as a storage area for mature sperm. The epididymis expresses numerous proteins, which are released into its luminal compartment; as the maturing spermatozoa transit through the epididymis from the proximal to the distal region, they acquire new surface proteins essential for fertilization. One of these proteins, DCXR, initially called sperm surface protein P34H (named as the protein isolated from epididymal sperm migrating at 34 kDa in Western blot analyses) (Boué et al., 1994). The fertilization process includes the species-specific binding of capacitated spermatozoa to the outer membranous shell (zona pellucida; ZP) of the oocyte. This induces the acrosome reaction in bound sperm cells, a secretory process which leads to the exocytosis of the acrosomal content including enzymes into the egg, allowing the sperm to penetrate the ZP and finally resulting in fusion with the oocyte plasma membrane. The acrosome is a single secretory vesicle located apically on the sperm cell. Due to the essential role of epididymal sperm maturation in human fertility, epididymal proteins like DCXR have been recognized as potential targets for the development of male contraceptives (Sipila et al., 2009). (a) DCXR (P34H) is involved in sperm maturation and gamete interaction In humans and most mammals the species-specific interaction between male and female gametes depends on the presence of a variety of macromolecules attached to the sperm’s surface. In a search for a human protein homologous to the hamster sperm protein P26h, Boué et al. (1994) identified DCXR on human epididymal spermatozoa using immunochemistry and Western blotting. Interestingly, the polyclonal antibody they used, directed against hamster P26h, which is not identical with hamster Dcxr (‘P31h’) (St-Cyr et al., 2004), recognized human DCXR, showing that both proteins share common epitopes. Similarly to P26h, DCXR resides on the acrosomal cap of mature epididymal spermatozoa and plays an important role in gamete interaction, a prerequisite for successful fertilization. When human spermatozoa were incubated with anti-P26h immune serum, the ability of those spermatozoa to bind to the ZP of human oocytes was significantly decreased. By contrast, neither binding to the plasma membrane nor penetration of zona-free hamster oocytes were affected in those spermatozoa, indicating that DCXR solely determines the ability of spermatozoa to bind to the ZP but does not influence later events in the fertilization process including membrane fusion or penetration. (b) DCXR is differentially expressed along the male reproductive tract Once the importance of DCXR in epididymal sperm maturation was recognized, studies were undertaken


Human DCXR – another ‘moonlighting protein’? (A)

Intact cell adhesion - cohesive growth: nevus

DCXR

11

(B)

Loss of cell adhesion

- dishesive growth: metastatic melanoma

DCXR

AJ

Fig. 3. Altered subcellular localization of DCXR in metastatic melanoma. In several epithelial cell types like prostate carcinoma, normal skin, benign and malignant melanocytic lesions, DCXR is co-localized with the transmembrane protein E-cadherin and its cytoplasmic anchor 𝛽-catenin (see Fig. 2), both of which constitute adherens junctions (AJ). (A) In benign nevi and melanomas with intact cell–cell contacts, DCXR is found predominantly in the cytoplasmic membrane, while in some primary and metastatic melanomas with dishesive growth pattern (B), DCXR is localized in the perinuclear region near the Golgi apparatus. These observations suggest a function of DCXR in cell adhesion, although the mechanisms causing this altered subcellular localization are unknown. This illustration summarizes findings described in Cho-Vega et al. (2007b).

to establish the surface localization of this protein on human spermatozoa from donors with physiologically normal epididymides (Boué et al., 1996). Cryosections from different regions of the male reproductive tract were prepared and immunohistochemical analyses of those tissues including spermatozoa showed that DCXR expression increased from the testis to the distal epididymis. While no DCXR protein expression was found on spermatozoa from the seminiferous tubules and the vasa efferentia, DCXR staining was detected on spermatozoa in the caput segment of the epididymis, increasing in intensity from the proximal corpus to the cauda region. In ejaculated sperm, DCXR staining was much lower compared to spermatozoa from the cauda but after initiation of the capacitation process, DCXR labelling was re-established. This observation suggested masking of DCXR by seminal fluid components, so-called ‘de-capacitation factors’, coating the surface of the spermatozoon at the time of ejaculation. After the capacitation process, when the spermatozoon is ready to interact with the ZP, DCXR becomes accessible again (Fig. 5). Finally, after the acrosome reaction, where the outer acrosomal membrane is shed, DCXR is undetectable, confirming its localization on the acrosomal cap of spermatozoa.

(c) DCXR on the sperm surface as a predictor of male fertility Approximately one-third of cases with proven male-factor infertility are of unknown cause and considered idiopathic (Mosher & Pratt, 1991). Because DCXR is involved in sperm–ZP binding, a process indispensible for successful fertilization, levels of spermatozoa-bound DCXR from patients with idiopathic infertility were compared to those from fertile donors (Boue & Sullivan, 1996). From 16 infertile donors 9 (56%) displayed low DCXR expression: less than 30% of values determined in samples from fertile men. A sperm–ZP binding assay revealed that the ability of spermatozoa to bind to the ZP depends on the presence of DCXR on their surface. When DCXR levels on spermatozoa were low or undetectable, their ZP-binding ability was dramatically decreased. In order to determine the frequency of spermatozoaassociated DCXR deficiency, semen samples of an unselected study population consisting of both fertile and infertile individuals (‘infertility evaluation group’) were analyzed for DCXR content and compared to a control group consisting of fertile donors only (Moskovtsev et al., 2007). Western blot analyses of semen samples from the infertility evaluation group showed that



12

Epididymal principal cells Vasa efferentia

Caput Lumen

or

Testis

Fig. 4. Sites of post-testicular sperm maturation: the epididymis. Immature spermatozoa are produced in the testis and enter the epididymis via the vasa efferentia. The proximal (caput or head) and middle (corpus or body) section of the epididymis are sites where epididymal sperm maturation takes place, while the distal (cauda or ‘tail’) section serves as sperm reservoir. The cross section of the epididymal ducts (right) shows the epididymal principal cells with stereocilia. These cells produce and secrete proteins including DCXR, which are essential for epididymal sperm maturation.

most of them were DCXR positive (86.7%) while only 13.3% (14 out of 105 patients) were DCXR negative. As expected, in the fertile control group only one sample was DCXR negative (0.9%), confirming the role of DCXR as a fertility marker. Levels of sperm-bound DCXR can serve as a powerful biomarker to predict whether standard in vitro fertilization (IVF) will be successful: in 97% of cases (97 from 100 donors) in which DCXR-negative semen samples were used in the IVF process, no embryos could be obtained (Sullivan et al., 2006). Cryopreservation of human semen samples is routinely performed in fertility clinics, however this process can cause cryoinjuries to sperm cells resulting in reduced semen quality. Because the freezing process affects membrane integrity and membrane-bound proteins, the effect of cryopreservation on DCXR levels on human sperm cells has been investigated (Desrosiers et al., 2006). The results indicate that loss of semen quality after cryopreservation can be partly attributed to the loss of membrane-bound DCXR, which was reduced to 50% of that in fresh samples. In conclusion, DCXR may serve as a powerful biomarker to predict male fertility. However, it should be noted that because many other factors determine fertilization ability, the presence of DCXR on the sperm

surface alone is not sufficient to prove fertility; in some cases normal levels of this protein were determined in donors of proven infertility (Boue & Sullivan, 1996). Nevertheless, future investigations are needed to unravel the molecular mechanisms that determine the DCXR-dependent interaction between sperm and ZP that might pave the way for the development of new male contraceptives. (d) Vasectomy affects DCXR expression in the human epididymis Vasectomy is a widely used male contraceptive method, however recovery of fertility after surgical reversal (vasovasostomy) is not always complete. This unwanted infertility in men who had undergone vasovasostomy was demonstrated to result in some cases from low levels of DCXR on spermatozoa in those individuals (Guillemette et al., 1999). It was shown that vasectomy affects both histology and gene expression patterns (Sullivan et al., 2011) of the epididymis (Legare et al., 2001). They reported a greatly enlarged epididymal lumen and a decreased height of the epithelium lining the ducts from the distal caput to the distal cauda region. Remarkably, DCXR mRNA expression was proportional to epithelium height in both normal and vasectomized men, explaining the low levels of DCXR on sperm from


Human DCXR – another ‘moonlighting protein’? Caput

Corpus

Cauda

Male reproductive tract

Epididymal sperm maturation

DCXR expression increases

DCXR

1. Zona pellucida interaction

3. DCXR undetectable after acrosome reaction (outer membrane is shed)

Oocyte

Acrosome reaction

2. Acrosome reaction

zona pel luc ida

Capacitated spermatozoa

Female reproductive tract

Capacitation

Ejaculated spermatozoa Masking of DCXR with unknown factors from seminal fluid

Fig. 5. Localization of DCXR on spermatozoa during epididymal sperm maturation, capacitation and acrosome reaction. After leaving the testis, immature spermatozoa transit through the epididymis, where they undergo epididymal sperm maturation characterized by the accumulation of surface proteins (including DCXR) on their acrosomal cap. Spermatozoa from the testis and vasa efferentia were DCXR-negative; DCXR staining was first detected in spermatozoa from the caput section of the epididymis, increasing in intensity from the corpus through the cauda. After ejaculation, DCXR-labelling decreased, most likely due to interaction with seminal fluid components (de-capacitation factors). In capacitated spermatozoa, DCXR became detectable again, playing an important role in the sperm–zona pellucida interaction. Finally, after the acrosome reaction, spermatozoa were DCXR-negative, indicating that DCXR was bound to the membrane covering the acrosome. This illustration summarizes the findings described in Boué et al. (1996).

vasectomized men who had a thinner epithelium, as protein synthesis also correlates with epithelial height. Finally, Legare et al. (2001) proposed that surgical ligation of the vas deferens results in an intraluminal pressure in the epididymis that leads to histological remodelling of the epithelium. Interestingly, this process includes de-differentiation of the epididymis that might be attributed to a lack of androgens and other testicular factors which normally promote the terminal differentiation of the epididymal epithelium.

13 From these observations it can be concluded that DCXR expression and secretion in the human epididymis is associated with cellular differentiation. (e) Sperm maturation and cell–cell contacts: epididymal principal cells from infertile donors display characteristics of an EMT It is now well established that post-testicular sperm maturation depends on an intact blood–epididymis barrier (BEB), which itself requires functional intercellular junctions. Consequently, altered expression of genes coding for cell junctional proteins in the principal cells of the epididymis will affect epididymal sperm maturation and hence the sperm’s ability to fertilize. To identify alterations in the expression of cell-adhesion molecules associated with human infertility, Dubé et al. (2010a,b) established several immortalized caput epididymal cell lines from either a fertile patient (FHCE, fertile human caput epididymal cell line) or an infertile patient with obstructive azoospermia (IHCE, infertile human caput epididymal cell line). In one of the ‘infertile’ cell lines, clone IHCE1, several epididymal markers including DCXR were absent at the mRNA level, as well as differentiation markers (Dubé et al., 2010b). Although both cell lines (FHCE and IHCE) expressed the epithelial marker cytokeratin, the flattened cell shape of IHCE1 indicated their de-differentiated state, characteristic of cells undergoing an EMT. The cells from the infertile patient were unable to form tight junctions as evidenced by a low transepithelial electrical resistance (TEER) compared to FHCE. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of genes encoding tight junctional (e.g. claudins, CLDN1, 4, 7, 8) and adherens junctional proteins (e.g. E-cadherin, CDH1) showed that these genes were not expressed at the mRNA level in IHCE1, explaining the lack of functional tight junctions. A comprehensive analysis of the transcriptome of both cell lines further revealed that several genes involved in the regulation of cellular junctions were differentially expressed. In accordance with the observed de-differentiated appearance of IHCE1, genes known to promote an EMT [e.g. the E-cadherin repressors Slug, Twist basic helix-loop-helix transcription factor (TWIST), and 𝛽-catenin] were up-regulated in this cell line, while the level of the mesenchymal marker vimentin was elevated (Fig. 6). From these observations it seems obvious that the production of infertile spermatozoa resulted from the lack of proper differentiation of the epididymal principal cells. Apparently, without differentiation, the principal cells are unable to express and secrete DCXR, which is critical for the establishment of an intraluminal epididymal milieu needed for sperm maturation. These findings confirm the observations made in epididymides from vasectomized men and substantiate the suggestion that DCXR expression depends on cellular differentiation.



14 (A)

(B)

Differentiated principal cells (FHCE)

De-differentiated principal cells (IHCE)

DCXR

DCXR: normal

DCXR: down-regulated

epithelial marker cyokeratin

mesenchymal marker vimentin

cell adhesion intact: TEER

loss of cell adhesion: TEER claudins, E-cadherin

differentiation markers: RAR-β, TR-β

differentiation markers: RAR-β, TR-β hormone receptors: AR, ER-α, ER-β EMT mediators: β-catenin, Slug, TWIST

Fig. 6. Differential gene expression in epididymal principal cells from fertile (FHCE) and infertile (IHCE) patients. While the epididymal principal cell line established from the caput section of a fertile patient (FHCE; A) expressed normal levels of DCXR, had intact cell–cell contacts and was well differentiated, the respective cell line from an infertile patient (IHCE; B) appeared de-differentiated, was unable to form tight junctions as evidenced by low transepithelial electrical resistance (TEER), and showed reduced DCXR expression. Loss of cell adhesion in IHCE cells was also reflected by reduced expression of the tight junctional and adherens junctional proteins claudins and E-cadherin. The down-regulation of several nuclear receptors (RAR-𝛽, TR-𝛽, AR, ER-𝛼, and ER-𝛽) suggests that androgen-, estrogen-, thyroid hormone and retinoid-dependent cellular differentiation was absent in these cells. Moreover, the flattened cell shape of IHCE cells together with the expression of the mesenchymal marker vimentin and the E-cadherin repressors 𝛽-catenin, Slug and TWIST, indicates that these cells are undergoing an epithelial to mesenchymal transition (EMT). Clearly, the proper cellular differentiation of epididymal principal cells is necessary for the expression and excretion of DCXR protein into the epididymal lumen and consequently determines the fertility of epididymal spermatozoa. AR, androgen receptor; ER-𝛼, estrogen receptor-alpha; ER-𝛽, estrogen receptor-beta; FHCE, fertile human caput epididymal cell line; IHCE, infertile human caput epididymal cell line; RAR-𝛽, retinoid acid receptor beta; TR-𝛽, thyroid hormone receptor-beta. This illustration summarizes the findings reported in Dubé et al. (2010a,b).

IV. DHS-21, A DCXR ORTHOLOG FROM C. ELEGANS MODULATES LONGEVITY AND REPRODUCTION – ANOTHER MULTIFUNCTIONAL PROTEIN? Although DCXR is widely distributed across the animal kingdom (see Section VI), there is only one invertebrate protein that has been characterized at a functional level to date: in the nematode C. elegans a DCXR ortholog (Q21929; DCXR_CAEEL) was identified (Kisiela et al., 2011). The protein was later comprehensively characterized in vitro and in vivo (Son le

et al., 2011) and according to WormBase nomenclature (http://www.wormbase.org) designated DHS-21, the protein product of the dhs-21 gene (WBGene00000984). DHS-21 is the only putative DCXR in C. elegans and despite the great evolutionary distance between humans and nematodes, both DCXR and DHS-21 share a remarkably high degree of sequence identity (68%) at the amino acid level. In addition, critical amino acids constituting the cofactor (NADPH) and substrate-binding motifs are well conserved between human DCXR and DHS-21 (Son le et al., 2011) suggesting similar catalytic properties. Recombinant DHS-21 showed activity with several sugars including the prototypical DCXR substrate l-xylulose (K m = 0.66 mM; k cat = 4.9 s−1 ). As for human DCXR, DHS-21 showed high activity towards 𝛼-dicarbonyl compounds: 1,4-dibromo-2,3-butanedione was metabolized with the highest turnover number (k cat = 85.8 s−1 ) of all compounds tested, although with a rather low substrate affinity (K m = 8.54 mM). In contrast to the human enzyme that can utilize both NADPH and NADH although NADPH is favoured, DHS-21 only uses NADPH as cofactor. No activity on l-xylulose was measured with NADH. Injection of a plasmid encoding a dhs-21-GFP fusion protein driven by the dhs-21 promoter revealed that DHS-21 is predominantly expressed in the intestine and the reproductive organs (uterine seam, spermathecal–uterus valve and gonadal sheath cells) of the worm. (1) DHS-21 is expressed on spermatids and plays a role in reproduction The high abundance of DHS-21 in the reproductive organs of C. elegans implied a function for reproduction, prompting investigations of this possible role by measuring brood size and egg-laying behaviour (Son le et al., 2011). In adult dhs-21 null mutants, egg laying was impaired: these animals retained an excess of eggs in the uterus compared to wild-type worms. The absence of functional dhs-21 decreased the brood size to 70% of that in wild-type animals. However, mechanisms linking the absence of functional dhs-21 with the observed reproductive changes remain to be deciphered. Because DHS-21 converts xylulose to xylitol, which acts as an osmolyte, the authors speculated that DHS-21 might affect the egg-laying apparatus by controlling its osmotic pressure (Son le et al., 2011). In a C. elegans strain that produced an excess of males [him-8(e1489)], immunofluorescence staining showed the expression of DHS-21 protein on the plasma membrane of immature sperm cells (spermatids) (Son le et al., 2011). This is reminiscent of the detection of human DCXR (P34H) on male gametes. However, in humans DCXR has been detected on the surface of spermatozoa that have undergone post-testicular maturation during epididymal transit, but it is not present on immature testicular spermatozoa.


Human DCXR – another ‘moonlighting protein’? Although reproductive mechanisms differ fundamentally between humans and C. elegans, sperm surface proteins essential for oocyte recognition and fertilization have been identified in C. elegans (Singson, Hang & Parry, 2008); no such function has been investigated for DHS-21 in C. elegans to date. DCXR bound to the human sperm surface is most likely transferred extracellularly from epididymosomes in contrast to the apparent intracellular expression of DHS-21 in C. elegans spermatids. Whether the function of DHS-21 requires its localization on the male C. elegans gamete or whether its presence is simply due to the protein’s affinity to cell-membrane components is a challenging question that remains to be solved. (2) DHS-21 expression increases the lifespan of C. elegans Because decreased defence against oxidative damage accompanies ageing processes and DCXR detoxifies 𝛼-dicarbonyl compounds, which are AGE precursors, Son le et al. (2011) investigated the effect of DHS-21 on longevity of C. elegans. The decreased lifespan of dhs-21 null mutants (15.3 days) compared to that of wild-type animals (18.1 days) suggests that DHS-21 expression is essential for normal lifespan in C. elegans. Similar to human DCXR, DHS-21 might form a tetramer to become a functional protein; the amino acids that mediate the interactions between monomers are highly conserved in C. elegans.

V. Cbr2, A CARBONYL REDUCTASE CLOSELY RELATED TO DCXR Similar to DCXR, carbonyl reductase 2 is a member of the SDR superfamily that forms an active homotetramer, a characteristic reflected by alternative names such as mouse tetrameric carbonyl reductase. It should be noted that despite being the official designation recommended by the UniProt database, ‘Cbr2’ is very rarely used in publications, probably for historical reasons. Instead, the most commonly used names for mouse Cbr2 are ‘mouse lung carbonyl reductase’ (MLCR) and ‘adipocyte protein 27’ (AP27); Cbr2 from golden hamster is usually termed ‘hamster sperm protein P26h’; pig Cbr2 is named ‘pig lung carbonyl reductase’ (PLCR); and P25b is the established name for the bovine homolog. Except for humans and other primates, in which the gene is lacking, Cbr2 is widely distributed among mammals. To date, its biochemical properties have been documented only for the proteins from golden hamster (Ishikura et al., 2001b), mouse (Nakanishi et al., 1995), pig (Hara et al., 1992; Oritani et al., 1992) and guinea pig (Matsuura et al., 1988). Table 1 provides an overview of

15 alternative names and UniProt identifiers for Cbr2 and DCXR from the most investigated species. Confusion regarding the assignment of proteins to either the Cbr2 or DCXR subfamily is reflected by some misleading entries in public databases such as UniProt, where Dcxr from golden hamster (DCXR_MESAU) is incorrectly named ‘sperm antigen P26h’ and the homolog from guinea pig (DCXR_CAVPO) is referred to as ‘Protein P26h’, leading to confusion with hamster Cbr2 that was originally named P26h. In other mammalian species such as dog and pig, some other tetrameric carbonyl reductases have been described as ‘pig peroxisomal tetrameric carbonyl reductase’ (PTCR) (Usami et al., 2003), ‘pig heart peroxisomal carbonyl reductase’ (PerCR) (Tanaka et al., 2008), and ‘dog liver carbonyl reductase’ (Endo et al., 2007) – potentially sources of confusion with Cbr2 or DCXR. In fact, all of these are homologs of human dehydrogenase/reductase SDR family member 4 (DHRS4). One characteristic that distinguishes DHRS4 enzymes from Cbr2 is their capability to metabolize retinoids, which are not substrates for Cbr2. Also, DHRS4 is localized to the peroxisomes, while Cbr2 is mainly found in mitochondria (MLCR, PLCR, hamster P26h) and associated with the peri-acrosomal membrane (hamster P26h, bovine P25b) on mature spermatozoa (see Sections V.2 and V.3). (1) Cbr2 from mouse (Mus musculus): ‘mouse lung carbonyl reductase (MLCR, AP27)’ In analogy to Cbr2 from pig (Oritani et al., 1992) and guinea pig (Nakayama et al., 1986) large amounts of Cbr2 have been detected in mouse lung, which is why the protein was designated ‘mouse lung carbonyl reductase’ (MLCR). Immunocytochemical localization revealed that MLCR is predominantly found in non-ciliated Clara cells and to a lesser extent in ciliated cells of bronchioles as well as in type II alveolar pneumocytes (Matsuura et al., 1994). Similar to Cbr2 from hamster (P26h), MLCR has been identified as a mitochondrial enzyme (Matsuura et al., 1994) that contains a non-cleavable mitochondrial targeting signal in its N-terminal sequence (Nakanishi et al., 1995). Initial characterization of the purified protein from mouse lung revealed its tetrameric structure, a low substrate specificity for aliphatic and aromatic carbonyl compounds as well as the ability to use both NADPH and NADH as cofactor, of which NADPH is preferred (Nakayama et al., 1986). Cloning of the corresponding cDNA and partial protein sequencing provided evidence for the identity of MLCR with ‘adipocyte protein P27’ (AP27) (Nakanishi et al., 1995), until then only known as putative protein expressed in murine adipocytes at the transcript level (Navre & Ringold, 1988). Investigation of tissue distribution showed, as expected, high expression of MLCR mRNA in mouse lung, but also in liver and to a lesser extent in fat


16 and testis (Nakanishi et al., 1995). A more comprehensive catalytic characterization of the recombinant MLCR demonstrated reductase activity with ketones (e.g. 4-nitroacetophenone, acetone, menadione), aldehydes (e.g. 1-propanal, pyridine-3-aldehyde) and 3-keto steroids (e.g. 5𝛽-pregnane-3,20-dione, 5𝛽-androstan-17𝛽-ol-3-one = 5𝛽-DHT) as well as dehydrogenase activity with cyclohex-2-en-1-ol (CHX) (Nakanishi et al., 1995). Re-investigation of the catalytic properties of MLCR later identified further substrates for the carbonyl reductase (e.g. 2-butanone) and dehydrogenase [e.g. (S)-2-butanol] activity of the enzyme (Ishikura et al., 2001b). Most interestingly, both the native enzyme from mouse lung and the recombinant MLCR were activated in the presence of arachidonic acid (20:4), a characteristic feature also reported for Cbr2 from pig lung (PLCR) that displayed a 5.7-fold activation by this unsaturated fatty acid (Hara et al., 1992). Site-directed mutagenesis of MLCR revealed that the positively charged side-chain of Lys17 within the cofactor binding site is probably involved in the interaction with the negatively charged carboxylate group of fatty acids that act as activators (Nakanishi et al., 1996). Because arachidonic acid is an important second messenger that is released enzymatically from cell membranes and can be metabolized to prostaglandins that trigger an inflammatory response as well as adipocyte differentiation (prostaglandin J2; PGJ2 ), both MLCR and PLCR seem to sense chemical cellular microenvironments that require prompt regulation (i.e. activation) by the enzyme’s catalytic activities. However, the physiological significance of this regulation remains to be investigated. Long before the tetrameric structure of human DCXR was resolved, crystallization of MLCR provided experimental evidence that this protein is a homotetramer (Tanaka et al., 1995). The crystal structure of MLCR in complex with cofactor NADPH and 2-propanol elucidated that Lys17 and Arg39, which are well conserved among NADPH-preferring SDRs but not among NADH-preferring ones, interact with the 2′ -phosphate group of NADPH, thus explaining the preference for NADPH over NADH (Tanaka et al., 1996). Apart from its supposed function in the metabolism of endogenous or inhaled carbonyl compounds in the lung, expression of mouse Cbr2 (MLCR) in the mouse olfactory epithelium, especially in sustentacular support cells, suggests that Cbr2 might aid in the clearance of inhaled xenobiotic carbonyls in this tissue as well (Yu et al., 2005). Moreover, mouse Cbr2 (AP27) appears to have a regulatory function, because suppression of Cbr2 in mouse adipogenic cell lines (TA1 and 3T3-L1) by antisense RNA prevented morphological differentiation of those cells, indicating a role for this gene in adipocyte differentiation (Wenz et al., 1992). Whether this

Bettina Ebert and others function is based on the metabolism of unknown signalling molecules (lipid mediators, androgens?) or results from interaction with other regulatory proteins (e.g. kinases, transcription factors) remains to be investigated. Interestingly, in contrast to its hamster (P26h) or bovine (P25b) counterpart, a possible role in the fertilization process has not been substantiated for mouse Cbr2 to date, although a sperm protein with immunoreactive properties similar to that of P26h has been shown to reside on the acrosomal region of mouse spermatozoa (Begin et al., 1995). This protein was detected by anti-P26h antiserum and appears as a double band in Western blot analysis with a major signal of 26 kDa and a minor one of slightly smaller size. As the UniProt database contains two entries for mouse Cbr2 (CBR2_MOUSE and Q542P5_MOUSE; see Table 1), this double band might be interpreted as two Cbr2 isoforms present on mouse spermatozoa, but this hypothesis needs to be confirmed experimentally. (2) Cbr2 from golden hamster (Mesocricetus auratus): ‘hamster sperm protein P26h’ Several studies have addressed the involvement of P26h in the fertilization process as well as its tissue-dependent expression, distribution along the male reproductive tract and localization on male gametes. Indeed, the majority of publications investigating hamster P26h are related to its involvement in reproduction rather than its catalytic function. More than 10 years before the role of DCXR (P34H) in the human fertilization process was discovered, a hamster sperm glycoprotein, P26h, was described to act in the binding of spermatozoa to the egg’s extracellular coat (Sullivan & Bleau, 1985). The enzyme was later purified from spermatozoa collected form the distal cauda (Coutu, Des Rosiers & Sullivan, 1996) and cloning of the full-length hamster P26h cDNA from a hamster testicular cDNA library allowed its assignment to the SDR superfamily (Gaudreault et al., 1999). The new sperm protein displayed a high degree of sequence homology with Cbr2 from mouse (MLCR) and Cbr2 from pig (PLCR) of 86 and 85%, respectively (Gaudreault et al., 1999). Similar to human DCXR (P34H), P26h accumulates on the heads of maturing spermatozoa during their transit through the epididymis (Robitaille, Sullivan & Bleau, 1991) and can be extracted by treatment with detergent (Nonidet P-40) (Berube & Sullivan, 1994). The protein occurs in the epididymal fluid both in soluble form and bound to prostasome-like particles (‘epididymosomes’), by phosphatidylinositol (Robitaille et al., 1991; Legare et al., 1999a). Epididymosomes are small membranous vesicles that are involved in the transfer of hamster P26h to the sperm surface (Legare et al., 1999a), a mechanism that has been also reported for the bovine counterpart, P25b (see Section V.3).


Human DCXR – another ‘moonlighting protein’? Interestingly, the epididymal expression of P26h is controlled by androgens (Berube et al., 1996), which have been identified as substrates for oxidoreductase activity of the recombinant enzyme (see Section V.2a). Active immunization of male hamsters against P26h resulted in infertility (Berube & Sullivan, 1994), proving an important role of P26h in the fertilization process. Immunocytochemistry experiments repeatedly showed that P26h-specific staining was restricted to the acrosomal cap of mature hamster spermatozoa (Berube & Sullivan, 1994; Begin et al., 1995; Legare et al., 1999a). By contrast, Nagdas, Winfrey & Olson (2006) showed that hamster P26h was exclusively localized to the midpiece of spermatozoa from the cauda epididymis, while no staining was observed on the head region. Moreover, P26h was detected in total cauda sperm lysate but was absent in both the plasma membrane fraction of spermatozoa and cauda epididymal fluid, also in contrast to previous findings. Extraction of spermatozoa with detergent, DTT, or both, and subsequent fractionation revealed that hamster P26h is a mitochondrial protein that was not extractable from spermatozoa by detergent but was released from mitochondria by the disulfide-reducing agent DTT. The authors concluded that P26h is a disulfide-stabilized protein that might be associated with the mitochondrial capsule in sperm tails and proposed a mitochondria-specific function for P26h (Nagdas et al., 2006). It seems that both groups detected isoforms of hamster P26h, thus one possible explanation for their conflicting results might be the application of different fixation/permeabilization methods or different antibodies that were produced in-house. The respective P26h proteins used for antibody production were either extracted from spermatozoa with detergent (Berube & Sullivan, 1994) or purified from isolated sperm tails (Nagdas et al., 2006). Interestingly, the antibodies from each group exclusively detected P26h in that particular region of the sperm from which the respective immunizing protein was extracted (sperm acrosome versus sperm flagellum). In this context it is interesting that in contrast to acrosome-bound P26h, mitochondria-associated P26h isolated from testis is not a phosphatidylinositol-anchored protein (Ishikura et al., 2001b). Both groups also determined different isoelectric points (pI ) for either sperm-tail-derived P26h (pI = 9.0) or P26h extracted from sperm heads (pI = 8.3). Although the UniProt database reports only one P26h protein in golden hamster (Q9Z245_MESAU), the existence of a second isoform cannot be ruled out. Alternatively, post-translational modifications might be a possible explanation for these conflicting data. (a) Catalytic properties of hamster P26h Catalytic characterization of recombinant hamster P26h and comparison with MLCR showed that both enzymes

17 share similar characteristics (e.g. broad substrate specificity, activation by arachidonic acid, sensitivity to inhibitors, and tetrameric structure) but differ in their coenzyme preference: NADH for P26h but NADPH for MLCR (Ishikura et al., 2001b). From all compounds tested, the aromatic ketone 4-nitroacetophenone, which was only moderately metabolized by MLCR, was by far the best substrate for hamster P26h. By contrast, the reduction of 2-butanone, an established substrate for MLCR, was catalyzed by both enzymes with comparable efficiencies (Ishikura et al., 2001b). Both proteins can catalyze reductions as well as oxidation reactions, but P26h displayed higher catalytic activities for oxidations of, for example, secondary alcohols such as (S)-2-butanol and the unsaturated cyclic alcohol cyclohex-2-en-1-ol (CHX). Moreover, hamster P26h can oxidize steroid diols such as 5𝛼-androstane-3𝛼,17𝛽-diol, while no such reaction is catalyzed by the mouse enzyme (Ishikura et al., 2001b). The clearest difference between these enzymes was the capability of P26h to catalyze efficiently the reduction of potent androgens including 5𝛼-dihydrotestosterone (5𝛼-DHT) and 5𝛽-DHT while MLCR showed only low activity with these steroids. Investigation of the tissue distribution and subcellular localization of P26h using NAD + -linked CHX dehydrogenase assay and Western blot analyses, indicated that the enzyme was primarily present in the epididymis and predominantly expressed in the mitochondrial fraction of the testis (Ishikura et al., 2001b), supporting the mitochondrial localization of P26h described previously (Nagdas et al., 2006) in mature spermatozoa. Because whole-tissue lysates including spermatids/ spermatozoa were analyzed by Ishikura et al. (2001b) and their method did not allow for the discrimination between epithelial mitochondria and sperm-associated mitochondria, detection of hamster P26h in both testis and epididymis was interpreted to result from mitochondria of either testicular spermatids or epididymal spermatozoa, rather than from mitochondria of the epithelia (testis, epididymis) itself. Nonetheless, it was concluded that P26h might act to control the level of potent androgens (e.g. 5𝛼-DHT), which regulate processes associated with spermatogenesis in spermatogenic cells in the testis. In addition, it was also hypothesized that hamster P26h might represent a detoxification system that removes lipid-peroxidationderived carbonyl compounds within the mitochondria of testicular cells. However, because classical lipid-peroxidation-derived breakdown products such as 4-hydroxynonenal (4-HNE) and malondialdehyde have not been tested as potential substrates for P26h, this proposal remains to be experimentally substantiated. Because the mechanism responsible for the function of P26h in sperm–ZP interaction is still unknown, catalytic characterization of hamster P26h raised the question of whether the acrosomal-bound protein remains


18 enzymatically active. Partial purification of P26h from cauda epididymal spermatozoa revealed that the protein reduced menadione, indicating that P26h must be present in an active tetrameric form (Montfort, Frenette & Sullivan, 2002). Most interestingly, inhibition of P26h-dependent carbonyl reductase activity in spermatozoa with diclofenac or phenylbutazone decreased the sperm–ZP binding capacity in vitro to nearly 50%. However, the authors were cautious in drawing conclusions; they pointed out that their results do not demonstrate that carbonyl reductase activity of P26h is a prerequisite to mediate binding of sperm to ZP ligands, although a link between enzyme activity and gamete interaction was obvious. This requires further detailed investigation. It is worth noting that hamster P26h (Cbr2) and human P34H (DCXR) were originally assumed to be homologous until another related protein of 31 kDa (P31h) was discovered in the principal cells of the hamster epididymis (St-Cyr et al., 2004). Cloning and sequencing of hamster P31h cDNA revealed identity with hamster Dcxr that was first described as ‘hamster diacetyl reductase’ and initially characterized in 2001 (Ishikura et al., 2001a). The enzyme was later compared to Dcxr homologues from mouse, rat and human (Nakagawa et al., 2002). Interestingly, P31h was predominantly expressed in the cytoplasmic compartment of the epididymal epithelium. In contrast to the human form P34H, involvement of hamster P31h in sperm–ZP interaction seems unlikely, because it was undetectable on mature spermatozoa (St-Cyr et al., 2004). (3) Cbr2 from cow (Bos taurus): ‘P25b’ By using hamster P26h antiserum, a detergentextractable sperm surface protein of 25 kDa was identified on bull sperm (Parent et al., 1999). The protein, designated P25b, is a bovine homolog of P26h and its function in the sperm–ZP interaction has been well documented (Parent et al., 1999). Similar to hamster P26h, bovine P25b is a glycosyl phosphatidylinositol (GPI)-anchored protein that accumulates on the sperm’s acrosomal cap during epididymal maturation. Again, epididymosomes are involved in the transfer of P25b to the sperm surface and cholesterol/sphingolipid-enriched lipid microdomains present on the epididymosomes have been shown to be important for this transfer (Frenette & Sullivan, 2001; Sullivan, Frenette & Girouard, 2007). Apart from bovine P25b, the hamster P26h antiserum detected a second, 21 kDa sperm protein (‘P21b’) that was not detergent-extractable and was associated with insoluble intracellular structures of the flagellum, reminiscent of the flagellar and acrosomal localization of P26h on hamster sperm. In contrast to P25b, levels of P21b on spermatozoa were not associated with fertility of bulls (Parent et al., 1999).

Bettina Ebert and others Although bovine P25b has been well described at a functional level with regard to reproduction, the gene has not been cloned yet and its catalytic properties as well as mechanisms of action in fertilization are still unexplored. It should therefore be pointed out that from our present knowledge and until protein-sequencing data become available, it is not possible to state with certainty whether P25b is a DCXR (Q1JP75 or A5PJR3) or a Cbr2 (G3N1E7; see Table 1).

VI. PHYLOGENETIC RELATIONSHIP BETWEEN DCXR AND Cbr2 Because of the early divergence of the SDR superfamily, unrelated SDR members usually share very low sequence similarities, often as low as 15–30%. Therefore, the remarkably high resemblance between Cbr2 and DCXR (e.g. 65% amino acid identity between Dcxr and Cbr2 from mouse) suggests a close evolutionary ancestry for these genes. Their common evolutionary roots might be reflected by shared characteristic traits, although both proteins vary in some features, indicating their rate of divergence. Cbr2 and DCXR have a similar tetrameric structure and broad substrate specificity for carbonyl compounds, but they differ with respect to their catalytic activity for distinct classes of chemical compounds. For example, DCXR has a preference for 𝛼-diketones (e.g. 2,3-hexanedione) over other dicarbonyls lacking an 𝛼-carbonyl group (e.g. 2,5-hexanedione) and does not reduce ketones such as 2-butanone, 4-nitroacetophenone, acetone, and menadione, all of which are substrates for Cbr2 from hamster (P26h) and mouse (MLCR) (Ishikura et al., 2001b; Nakagawa et al., 2002). On the other hand, unlike Cbr2, rodent and human DCXR reduce monosaccharides like l-xylulose (Nakagawa et al., 2002). Another distinguishing feature is the activity of Cbr2 with 3-oxo-steroids (e.g. 5𝛽-androstane-3,17-dione and 5𝛼-dihydrotestosterone) (Nakanishi et al., 1995; Ishikura et al., 2001b), which has been not observed for DCXR (Nakagawa et al., 2002). Both proteins are thought to function as a clearance/ defence mechanism for a diverse set of carbonyl compounds that include endogenous substances arising from normal cellular metabolism (sugars, sugardegradation products, ketones) as well as signalling molecules such as steroid hormones, and also exogenous compounds including food flavourings (e.g. 2,3hexanedione) or diesel-exhaust-derived contaminants (e.g. 9,10-phenanthrenequinone). In addition, at least for human DCXR and Cbr2 from hamster (P26h) and cow (P25b), both enzymes have other functions such as species-specific gamete interaction. Whether this latter function requires catalytic activity needs to be further substantiated. To illustrate the distribution of DCXR and Cbr2 among animal species and to trace the origin of Cbr2,


Human DCXR – another ‘moonlighting protein’? protein sequences for both genes were retrieved from public databases and bioinformatically processed as described in (M. Kisiela, B. Ebert & E. Maser, unpublished data) to construct a phylogenetic tree (Fig. 7). Most sequences were taken from fully sequenced and annotated genomes. However, some species with partially sequenced genomes [e.g. pig (Sus scrofa), sheep (Ovis aries), the naked mole-rat (Heterocephalus glaber )] also were included. Thus, conclusions about gene-loss events cannot be drawn from this analysis, because this would require the use of complete genomes. Our phylogenetic analysis of DCXR and Cbr2 proteins shows the wide distribution of DCXR among species from bacteria to mammals, while Cbr2 is restricted to placental and marsupial mammals only (Fig. 7A). The majority of DCXR protein sequences are found in prokaryotes (Kisiela et al., 2011), but for the sake of clarity, only eukaryotic organisms are presented in the tree. Rhizobium etli DCXR was used as an outgroup to root the tree (Fig. 7A). DCXR proteins are present in single-celled eukaryotes like the marine choanoflagellate Monosiga brevicollis, the protozoan oyster parasite Perkinsus marinus and also in simple multicellular organisms like Cnidaria (e.g. Nematostella vectensis), sponges (Amphimedon queenslandica), Urochordata (the sea squirt Ciona intestinalis), hemichordates (the acorn worm Saccoglossus kowalevskii), echinoderms (the purple sea urchin Strongylocentrotus purpuratus) and nematodes (Caenorhabditis spp.) (Fig. 7A). A large range of insects also express DCXR protein including Drosophila spp., bees, wasps, mosquitoes, beetles, and the aphid Acyrthosiphon pisum; together representing 37 DCXR protein sequences. This might simply reflect the large number of insects for which sequencing data are available (especially Drosophila spp.). Interestingly, 25 DCXR proteins were found in eutherian mammals, while only two proteins have been identified in marsupial animals. (1) Cbr2 has arisen from a gene duplication event Gene duplication provides the genetic raw material necessary to evolve new gene functions (Ohta, 1989; Zhang, 2003). Tandem duplications that produce two initially identical copies (paralogs) mostly arise from unequal crossing-over during meiosis. Comparison of amino acid sequences of their coded proteins showed that in duplicate gene pairs one duplicate evolves faster than the other (Conant & Wagner, 2003). This asymmetric evolution or asymmetric sequence divergence seems to apply especially to recent duplicates because in older duplicates this effect may have been erased by a subsequent period of purifying selection (Zhang, Gu & Li, 2003). If the new gene confers a new advantageous function (neofunctionalization), it may become fixed in the genome (Ohno, 1970). However, recently duplicated genes that are not evolving under constraint will

19 accumulate mutations over time, leading to nonfunctional pseudogenes (Lynch & Conery, 2000). Thus, the long-term fate of most new non-essential genes is their removal from the population (Otto & Yong, 2002). Figure 7(B) shows that Cbr2 emerged as a result of a gene duplication event in the ancestral DCXR gene, when the Theria (therian mammals) diverged into Metatheria (marsupial mammals) and Eutheria (placental mammals) an estimated 165 million years ago (Luo et al., 2011). Cbr2 is restricted to mammalian species such as rodents (rat, mouse, hamster, guinea pig, naked mole-rat), carnivores (cat, dog, pacific walrus), and even-toed ungulates (Cetartiodactyla) such as the killer whale Orca orcinus, ruminants [sheep, cow and yak (Bos grunniens)] and pig, all of which are placental mammals. In marsupial mammals only data for the Tasmanian devil (Sarcophilus harrisii) and the gray short-tailed opossum (Monodelphis domestica) are available; both animals express Cbr2. Cbr2 protein is absent from a variety of placental mammals [e.g. Florida manatee (Trichechus manatus), hedgehog (Erinaceus europaeus), bottlenosed dolphin (Tursiops truncatus), giant panda (Ailuropoda melanoleuca), horse (Equus caballus)] and all primate species investigated (Fig. 7B). From the characteristics of DCXR and Cbr2 it is likely that one copy of the duplicated DCXR ancestor gene retained DCXR-specific properties (also found in older lineages like C. elegans) such as the ability to reduce sugars, the preference for 𝛼-diketones and its association with membranes. The other copy, now known as Cbr2, lost its activity towards sugars but gained the ability to reduce/oxidize androgens, aldehydes and ketones (neofunctionalization). The emerging Cbr2 also acquired an altered subcellular localization; Cbr2 is localized to mitochondria instead of being cytoplasmic or cell-membrane-associated like DCXR. Despite their differences, both DCXR and Cbr2 retained the common property of being transferred to maturing spermatozoa by epididymosomes within the epididymis. As far as can be judged from the available data, both DCXR and Cbr2 play an important role in fertilization. The absence of Cbr2 in primates might indicate taxon-specific functions that were lost in primate species. Alternatively, in humans, other proteins might have served these functions. For example, proteins from the aldo-keto reductase (AKR) superfamily have a wide substrate spectrum for carbonyl compounds (Jin & Penning, 2007) and could replace the steroid-metabolizing activity of Cbr2 (Penning et al., 2000; Penning & Byrns, 2009). Finally, the properties of Cbr2 that resulted in the retention of this gene in most marsupial and placental mammals in contrast to the evolutionarily ‘younger’ primate species remains another interesting aspect for future investigations. Such properties might relate to: (i) fat cell differentiation; (ii) body fat mobilization during fasting or hibernation; or (iii) olfactory sensing.



20

Rhizobium etli CFN 42 (defined outgroup)

(A)

Wasps & bees (11) Drosophila ssp. (12) & mosquitoes (4) Tribolium castaneum (5) & I xodes scapularis (1) C. elegans (3) & Perkinsus marinus (1) Acyrthosiphon pisum (5) Eutheria

Theria

D

DCXR subgroup (25)

Metatheria + Eutheria

Metatheria

CBR2 group (14)

Marsupials (2)

Sauropsids (birds, reptiles) (3) Frogs & toads (2) Fish (4) Ciona ssp. & Capsaspora sp. (3) Hemichordates & Echinoderms (3) Sponges (1)

D Duplication node (165 mya) Nematostella sp. & Monosiga sp. (2)

(B)

D

DCXR only Duplication node

DCXR

Primates

DCXR+Cbr2

Rodents

D

165 mya

Cbr2

Theria

Trichechus manatus latirostris Procavia capensis Otolemur garnettii Erinaceus europaeus Papio anubis Macaca mulatta Pan troglodytes Pan paniscus Gorilla gorilla gorilla Pongo abelii Pongo abelii Nomascus leucogenys Homo sapiens Callithrix jacchus Callithrix jacchus Heterocephalus glaber Pteropus alecto Tupaia chinensis Cavia porcellus Mus musculus Rattus norvegicus Mesocricetus auratus Cricetulus griseus Cavia porcellus Ictidomys tridecemlineatus Bos taurus Bos taurus Odobenus rosmarus divergens Canis lupus familiaris Felis catus Tursiops truncatus Orcinus orca Echinops telfairi Ailuropoda melanoleuca Equus caballus Ictidomys tridecemlineatus Bos taurus Bos grunniens Heterocephalus glaber Cricetulus griseus Cricetulus griseus Mesocricetus auratus Rattus norvegicus Rattus norvegicus Mus musculus Orcinus orca Sus scrofa Felis catus Canis lupus familiaris Odobenus rosmarus divergens Cavia porcellus Ovis aries Sarcophilus harrisii Monodelphis domestica

Rodents

Cbr2 only

Fig. 7. Legend on next page. Biological Reviews (2014) 000–000 © 2014 The Authors. Biological Reviews © 2014 Cambridge Philosophical Society

Human DCXR – another ‘moonlighting protein’? Studies on mouse adipogenic cells have shown that Cbr2 regulates adipocyte differentiation (see Section V.1). Possibly, Cbr2 might be involved in the metabolism of lipid mediators (e.g. prostaglandins) or hormones (e.g. androgens) that trigger fat cell differentiation. The synchronized regulation of adipocyte differentiation versus adipocyte proliferation is indispensable for proper storage of body fat and weight gain, processes which are essential for survival of hibernators and animals with a high metabolic rate (e.g. rodents). Moreover, it might be speculated that Cbr2 has an important metabolic function in thermogenic brown adipose tissue, which is rich in mitochondria (where Cbr2 is found) and that serves to maintain body temperature. Brown adipose tissue is predominantly found in hibernating animals but is only sparsely expressed in adult humans. Likewise, proper mobilization of body fat guarantees survival under conditions of food shortage and during hibernation. Long-term fasting leads to elevated plasma concentrations of ketone bodies, which are used as energy source. Acetone, a known Cbr2 substrate (see Section V.1) is a by-product of this process and is removed via the airways. Interestingly, the production of ketone bodies takes place in mitochondria, and Cbr2 is highly expressed in mouse lung. Lastly, the olfactory sense is known to be degenerated in humans compared with rodents and other animals. In mice, Cbr2 has been detected in the olfactory epithelium (see Section V.1), where it possibly metabolizes odourants that may carry carbonyl functions (e.g. diacetyl = butter flavour). The effective clearance of odorants is crucial to maintaining high olfactory sensitivity. Unlike humans, most animals depend on olfaction for successful foraging, this might explain the differential loss of Cbr2 in humans and other primates compared to most other mammals.

VII. CONCLUSIONS (1) From a review of the published literature it is evident that although DCXR has been characterized, many

21 open questions remain. In particular: (i) the transcriptional regulation of DCXR in normal and cancerous tissues, and (ii) the molecular mechanisms underlying its role in gamete interaction and possible role in cell adhesion and metastasis. These unresolved questions hold the promise of discovering new properties of DCXR making this ‘old’ protein an interesting new target for future studies. (2) The catalytic characterization of DCXR has been exclusively performed with recombinant protein, which is a common method used to investigate possible physiological roles of an uncharacterized protein. However, its low substrate affinity, especially for l-xylulose, stands in contrast to its demonstrated function in vivo, implying the presence of differences between the recombinant and native protein or is possibly associated with its reported rapid inactivation by oxidation (Zhao et al., 2009). The same applies to recombinant mouse Dcxr that exhibits very low substrate affinities for AGE precursors (e.g. 3-deoxyglucosone) in contrast to its role in intact animals where Dcxr-overexpression promotes clearance of this reactive carbonyl compound. (3) Unexplained tissue-specific differences in the molecular weight of human DCXR imply posttranslational modification that might alter its biochemical characteristics. Re-investigations of the catalytic properties of DCXR either in a cellular context or as purified protein would help to further clarify the functions of DCXR in vivo. (4) Pentosuria is a benign condition caused by the absence of DCXR with no severe disorders or increased mortality reported for affected individuals. Thus DCXR clearly is not an essential protein. Other backup systems obviously can largely compensate for the absence of this enzyme. However, l-xylulose reductase activity is unique to DCXR and cannot be replaced by other proteins as shown by elevated l-xylulose levels in blood and urine of pentosuric individuals. (5) We have traced the evolutionary roots of both DCXR and Cbr2 and show that these tetrameric proteins are likely to be the product of a gene duplication event that occurred before the eutherian/metatherian

Fig. 7. (A) Schematic representation of the distribution of DCXR and Cbr2 proteins in the animal kingdom. Bacterial DCXR from Rhizobium etli was used as an outgroup to root the tree. The number of identified protein sequences is given in parentheses after each group of species. Cbr2 derived from a gene duplication event (indicated by duplication node D) that occurred in the ancestral DCXR gene before the eutherian/metatherian divergence. Protein sequences were obtained from public databases [NCBI RefSeq, Ensembl (release 71) and UniProtKB (release 2013_04)] using a specific hidden Markov model (HMM) as described in (M. Kisiela, B. Ebert & E. Maser, unpublished data). Dating of the gene duplication event was achieved by reconciliation of gene and species trees using the program notung 2.6. (B) Detail of highlighted boxed regions in A showing the distribution of DCXR and Cbr2 protein sequences among therian mammals (Theria) after the gene duplication event (indicated by duplication node D) of the ancestral DCXR gene. (Species names in red indicate the presence of Cbr2 proteins only, green indicates species that have DCXR but no Cbr2 and black denotes species that have both DCXR and Cbr2 proteins. See also text for details. For colours please refer to the online version). All primate species are devoid of Cbr2 protein sequences. Note that this cladogram contains sequences from incomplete genomes, therefore conclusions about gene-loss events can not be drawn with confidence. Biological Reviews (2014) 000–000 © 2014 The Authors. Biological Reviews © 2014 Cambridge Philosophical Society


22 divergence an estimated 165 million years ago. Although neofunctionalization of the offspring gene (Cbr2) resulted in distinct catalytic properties and subcellular localization, both proteins seem to have retained their multifunctional nature. At least as far as hamster P26h and DCXR are concerned, both of which are important in fertilization, this multifunctionality is composed of both catalytic and non-catalytic functions. (6) Multifunctionality that involves both catalytic and non-catalytic functions has been reported for several other SDR proteins including dehydrogenase/reductase SDR family member 2 (DHRS2; SDR25C1) and WW domain-containing oxidoreductase (WWOX; SDR41C1), potentially implying that this feature might be characteristic of SDR proteins. Similar to DCXR, DHRS2 reduces 𝛼-diketones but also actively regulates gene transcription at the level of protein–protein interactions. By binding to and inhibiting murine double minute oncogene (Mdm2), the negative regulator of the p53 tumour suppressor, DHRS2 suppresses cell proliferation by promoting accumulation and stabilization of p53 (Deisenroth et al., 2010). WWOX functions as a tumor suppressor (Aqeilan & Croce, 2007) and regulates apoptosis (Lai et al., 2005). Moreover, by being a ‘substrate’ for glycogen synthase kinase-3 beta (GSK3𝛽), an enzyme involved in tau-phosphorylation, down-regulation of WWOX has been shown to induce the formation of hyperphosphorylated tau (Sze et al., 2004; Wang et al., 2012) that precedes the emergence of neurofibrillary tangles. The role of WWOX in the pathogenesis of Alzheimer’s disease is supported by the reduced expression of WWOX in hippocampi of those patients (Sze et al., 2004). The extent to which the non-catalytic functions of DCXR and Cbr2 involve functional control based on kinase-related phosphorylations or other post-translational modifications (myristoylations and other acetylations, etc.) and the associated altered protein–protein interactions remains another challenging question to be resolved.

VIII. LIST OF ABBREVIATIONS 2ME = 4-HNE = 5𝛼-DHT = 5𝛽-DHT = AGE = AJ = AKR = AP27 = AR = BEB = Cbr2 = CDH1 = CD31 = CHX =

2-mercaptoethanol 4-hydroxynonenal 5𝛼-dihydrotestosterone 5𝛽-androstan-17𝛽-ol-3-one advanced glycation end product adherens junction aldo-keto reductase adipocyte protein 27 (= Cbr2 mouse) androgen receptor blood–epididymis barrier carbonyl reductase 2 E-cadherin cluster of differentiation 31 (= PECAM-1) cyclohex-2-en-1-ol

CLDN1 = CML = DCR = DCXR = DHRS2 =

claudin 1 N𝜖 -(carboxymethyl)-lysine dicarbonyl reductase (= DCXR human) dicarbonyl/l-xylulose reductase dehydrogenase/reductase SDR family member 2 DHRS4 = dehydrogenase/reductase SDR family member 4 DHS-21 = DCXR from C. elegans DTT = dithiothreitol ECM = extracellular matrix EMT = epithelial-to-mesenchymal transition ER-𝛼/𝛽 = estrogen receptor alpha/beta FHCE = fertile human caput epididymal cell line GPI = glycosyl phosphatidylinositol GSH = glutathione GSK3𝛽 = glycogen synthase kinase-3 beta HCC = hepatocellular carcinoma HCR2 = human carbonyl reductase 2 (= DCXR human) IHCE = infertile human caput epididymal cell line IVF = in vitro fertilization KIDCR = kidney dicarbonyl reductase (= DCXR human) Mdm2 = murine double minute oncogene MLCR = mouse lung carbonyl reductase (= Cbr2 mouse) P21b = bovine sperm protein of 21 kDa (widely uncharacterized) P25b = Cbr2 from cow P26h = Cbr2 from golden hamster P31h = Dcxr from golden hamster P34H = sperm surface protein, synonymous with human DXCR PAH = polycyclic aromatic hydrocarbon PECAM-1 = platelet endothelial cell adhesion molecule PerCR = pig heart peroxisomal carbonyl reductase (= DHRS4 pig) PGJ2 = prostaglandin J2 PLCR = pig lung carbonyl reductase (= Cbr2 pig) PTCR = pig peroxisomal tetrameric carbonyl reductase (= DHRS4 pig) RAGE = receptor for advanced glycation end products RAR-𝛽 = retinoid acid receptor beta ROS = reactive oxygen species RT-PCR = reverse transcription polymerase chain reaction SDR = short-chain dehydrogenase/reductase SDS = sodium dodecyl sulphate SNP = single nucleotide polymorphism TEER = transepithelial electrical resitance Tg = transgenic TJ = tight junction TR-𝛽 = thyroid hormone receptor beta


Human DCXR – another ‘moonlighting protein’? TWIST = Twist basic helix-loop-helix transcription factor UUO = unilateral ureteral obstruction WWOX = WW domain-containing oxidoreductase XR = l-xylulose reductase ZP = zona pellucida

IX. ACKNOWLEDGEMENTS This project was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; MA 1704/12-1).

X. REFERENCES Aqeilan, R. I. & Croce, C. M. (2007). WWOX in biological control and tumorigenesis. Journal of Cellular Physiology 212, 307–310. Asami, J., Odani, H., Ishii, A., Oide, K., Sudo, T., Nakamura, A., Miyata, N., Otsuka, N., Maeda, K. & Nakagawa, J. (2006). Suppression of AGE precursor formation following unilateral ureteral obstruction in mouse kidneys by transgenic expression of alpha-dicarbonyl/L-xylulose reductase. Bioscience, Biotechnology, and Biochemistry 70, 2899–2905. Austin, C. R. & Bishop, M. W. (1958). Capacitation of mammalian spermatozoa. Nature 181, 851. Begin, S., Berube, B., Boue, F. & Sullivan, R. (1995). Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein. Molecular Reproduction and Development 41, 249–256. Berube, B., Lefievre, L., Coutu, L. & Sullivan, R. (1996). Regulation of the epididymal synthesis of P26h, a hamster sperm protein. Journal of Andrology 17, 104–110. Berube, B. & Sullivan, R. (1994). Inhibition of in vivo fertilization by active immunization of male hamsters against a 26-kDa sperm glycoprotein. Biology of Reproduction 51, 1255–1263. Boué, F., Berube, B., De Lamirande, E., Gagnon, C. & Sullivan, R. (1994). Human sperm-zona pellucida interaction is inhibited by an antiserum against a hamster sperm protein. Biology of Reproduction 51, 577–587. Boué, F., Blais, J. & Sullivan, R. (1996). Surface localization of P34H an epididymal protein, during maturation, capacitation, and acrosome reaction of human spermatozoa. Biology of Reproduction 54, 1009–1017. Boue, F. & Sullivan, R. (1996). Cases of human infertility are associated with the absence of P34H an epididymal sperm antigen. Biology of Reproduction 54, 1018–1024. Cho-Vega, J. H., Tsavachidis, S., Do, K. A., Nakagawa, J., Medeiros, L. J. & McDonnell, T. J. (2007a). Dicarbonyl/L-xylulose reductase: a potential biomarker identified by laser-capture microdissection-micro serial analysis of gene expression of human prostate adenocarcinoma. Cancer Epidemiology, Biomarkers & Prevention: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology 16, 2615–2622. Cho-Vega, J. H., Vega, F., Schwartz, M. R. & Prieto, V. G. (2007b). Expression of dicarbonyl/L-xylulose reductase (DCXR) in human skin and melanocytic lesions: morphological studies supporting cell adhesion function of DCXR. Journal of Cutaneous Pathology 34, 535–542. Conant, G. C. & Wagner, A. (2003). Asymmetric sequence divergence of duplicate genes. Genome Research 13, 2052–2058. Cooper, T. G. (1986). The epididymis: sperm maturation and fertilisation. In The male gamete: from basic science to clinical applications (ed. C. Gagnon), pp. 268–272. Cache River Press, Vienna. Coutu, L., Des Rosiers, P. & Sullivan, R. (1996). Purification of P26h: a hamster sperm protein. Biochemistry and Cell Biology = Biochimie et Biologie Cellulaire 74, 227–231. Deisenroth, C., Thorner, A. R., Enomoto, T., Perou, C. M. & Zhang, Y. (2010). Mitochondrial Hep27 is a c-Myb target gene that inhibits Mdm2 and stabilizes p53. Molecular and Cellular Biology 30, 3981–3993. Desrosiers, P., Legare, C., Leclerc, P. & Sullivan, R. (2006). Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertility and Sterility 85, 1744–1752. Dubé, E., Dufresne, J., Chan, P. T., Hermo, L. & Cyr, D. G. (2010a). Assessing the role of claudins in maintaining the integrity of epididymal tight junctions using novel human epididymal cell lines. Biology of Reproduction 82, 1119–1128.

23 Dubé, E., Hermo, L., Chan, P. T. & Cyr, D. G. (2010b). Alterations in the human blood-epididymis barrier in obstructive azoospermia and the development of novel epididymal cell lines from infertile men. Biology of Reproduction 83, 584–596. El-Kabbani, O., Carbone, V., Darmanin, C., Ishikura, S. & Hara, A. (2005). Structure of the tetrameric form of human L-Xylulose reductase: probing the inhibitor-binding site with molecular modeling and site-directed mutagenesis. Proteins 60, 424–432. El-Kabbani, O., Chung, R. P., Ishikura, S., Usami, N., Nakagawa, J. & Hara, A. (2002). Crystallization and preliminary crystallographic analysis of human L-xylulose reductase. Acta Crystallographica. Section D, Biological Crystallography 58, 1379–1380. El-Kabbani, O., Ishikura, S., Darmanin, C., Carbone, V., Chung, R. P., Usami, N. & Hara, A. (2004). Crystal structure of human L-xylulose reductase holoenzyme: probing the role of Asn107 with site-directed mutagenesis. Proteins 55, 724–732. Endo, S., Matsunaga, T., Nagano, M., Abe, H., Ishikura, S., Imamura, Y. & Hara, A. (2007). Characterization of an oligomeric carbonyl reductase of dog liver: its identity with peroxisomal tetrameric carbonyl reductase. Biological & Pharmaceutical Bulletin 30, 1787–1791. Frenette, G. & Sullivan, R. (2001). Prostasome-like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface. Molecular Reproduction and Development 59, 115–121. Gaudreault, C., Le gare, C., Berube, B. & Sullivan, R. (1999). Hamster sperm protein, p26h: a member of the short-chain dehydrogenase/reductase superfamily. Biology of Reproduction 61, 264–273. Guillemette, C., Thabet, M., Dompierre, L. & Sullivan, R. (1999). Some vasovasostomized men are characterized by low levels of P34H, an epididymal sperm protein. Journal of Andrology 20, 214–219. Hara, A., Oritani, H., Deyashiki, Y., Nakayama, T. & Sawada, H. (1992). Activation of carbonyl reductase from pig lung by fatty acids. Archives of Biochemistry and Biophysics 292, 548–554. Ishikura, S., Isaji, T., Usami, N., Kitahara, K., Nakagawa, J. & Hara, A. (2001a). Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase. Chemico-Biological Interactions 130-132, 879–889. Ishikura, S., Usami, N., Kitahara, K., Isaji, T., Oda, K., Nakagawa, J. & Hara, A. (2001b). Enzymatic characteristics and subcellular distribution of a short-chain dehydrogenase/reductase family protein, P26h, in hamster testis and epididymis. Biochemistry 40, 214–224. Ishikura, S., Isaji, T., Usami, N., Nakagawa, J., El-Kabbani, O. & Hara, A. (2003a). Identification of amino acid residues involved in substrate recognition of L-xylulose reductase by site-directed mutagenesis. Chemico-Biological Interactions 143-144, 543–550. Ishikura, S., Usami, N., El-Kabbani, O. & Hara, A. (2003b). Structural determinant for cold inactivation of rodent L-xylulose reductase. Biochemical and Biophysical Research Communications 308, 68–72. Jeffery, C. J. (2003). Moonlighting proteins: old proteins learning new tricks. Trends in Genetics: TIG 19, 415–417. Jin, Y. & Penning, T. M. (2007). Aldo-keto reductases and bioactivation/detoxication. Annual Review of Pharmacology and Toxicology 47, 263–292. Kawada, N., Moriyama, T., Ando, A., Fukunaga, M., Miyata, T., Kurokawa, K., Imai, E. & Hori, M. (1999). Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction. Kidney International 56, 1004–1013. Kisiela, M., El-Hawari, Y., Martin, H. J. & Maser, E. (2011). Bioinformatic and biochemical characterization of DCXR and DHRS2/4 from Caenorhabditis elegans. Chemico-Biological Interactions 191, 75–82. Lai, F. J., Cheng, C. L., Chen, S. T., Wu, C. H., Hsu, L. J., Lee, J. Y., Chao, S. C., Sheen, M. C., Shen, C. L., Chang, N. S. & Sheu, H. M. (2005). WOX1 is essential for UVB irradiation-induced apoptosis and down-regulated via translational blockade in UVB-induced cutaneous squamous cell carcinoma in vivo. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 11, 5769–5777. Lane, A. B. (1985). On the nature of L-xylulose reductase deficiency in essential pentosuria. Biochemical Genetics 23, 61–72. Lane, A. B. & Jenkins, T. (1985). Human L-xylulose reductase variation: family and population studies. Annals of Human Genetics 49, 227–235. Legare, C., Berube, B., Boue, F., Lefievre, L., Morales, C. R., El-Alfy, M. & Sullivan, R. (1999a). Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein. Molecular Reproduction and Development 52, 225–233. Legare, C., Gaudreault, C., St-Jacques, S. & Sullivan, R. (1999b). P34H sperm protein is preferentially expressed by the human corpus epididymidis. Endocrinology 140, 3318–3327. Legare, C., Thabet, M., Picard, S. & Sullivan, R. (2001). Effect of vasectomy on P34H messenger ribonucleic acid expression along the human excurrent duct: a reflection on the function of the human epididymis. Biology of Reproduction 64, 720–727.


24 Liu, S., Ma, L., Huang, W., Shai, Y., Ji, X., Ding, L., Liu, Y., Yu, L. & Zhao, S. (2006). Decreased expression of the human carbonyl reductase 2 gene HCR2 in hepatocellular carcinoma. Cellular & Molecular Biology Letters 11, 230–241. Luo, Z. X., Yuan, C. X., Meng, Q. J. & Ji, Q. (2011). A Jurassic eutherian mammal and divergence of marsupials and placentals. Nature 476, 442–445. Lynch, M. & Conery, J. S. (2000). The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155. Matsunaga, T., Kamiya, T., Sumi, D., Kumagai, Y., Kalyanaraman, B. & Hara, A. (2008). L-Xylulose reductase is involved in 9,10-phenanthrenequinone-induced apoptosis in human T lymphoma cells. Free Radical Biology & Medicine 44, 1191–1202. Matsuura, K., Bunai, Y., Ohya, I., Hara, A., Nakanishi, M. & Sawada, H. (1994). Ultrastructural localization of carbonyl reductase in mouse lung. The Histochemical Journal 26, 311–316. Matsuura, K., Nakayama, T., Nakagawa, M., Hara, A. & Sawada, H. (1988). Kinetic mechanism of pulmonary carbonyl reductase. The Biochemical journal 252, 17–22. Montfort, L., Frenette, G. & Sullivan, R. (2002). Sperm-zona pellucida interaction involves a carbonyl reductase activity in the hamster. Molecular Reproduction and Development 61, 113–119. Mosher, W. D. & Pratt, W. F. (1991). Fecundity and infertility in the United States: incidence and trends. Fertility and Sterility 56, 192–193. Moskovtsev, S. I., Jarvi, K., Legare, C., Sullivan, R. & Mullen, J. B. (2007). Epididymal P34H protein deficiency in men evaluated for infertility. Fertility and Sterility 88, 1455–1457. Nagdas, S. K., Winfrey, V. P. & Olson, G. E. (2006). Identification of a hamster sperm 26-kilodalton dehydrogenase/reductase that is exclusively localized to the mitochondria of the flagellum. Biology of Reproduction 75, 197–202. Nakagawa, J., Ishikura, S., Asami, J., Isaji, T., Usami, N., Hara, A., Sakurai, T., Tsuritani, K., Oda, K., Takahashi, M., Yoshimoto, M., Otsuka, N. & Kitamura, K. (2002). Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney. The Journal of Biological Chemistry 277, 17883–17891. Nakanishi, M., Deyashiki, Y., Nakayama, T., Sato, K. & Hara, A. (1993). Cloning and sequence analysis of a cDNA encoding tetrameric carbonyl reductase of pig lung. Biochemical and Biophysical Research Communications 194, 1311–1316. Nakanishi, M., Deyashiki, Y., Ohshima, K. & Hara, A. (1995). Cloning, expression and tissue distribution of mouse tetrameric carbonyl reductase. Identity with an adipocyte 27-kDa protein. European Journal of Biochemistry/FEBS 228, 381–387. Nakanishi, M., Kakumoto, M., Matsuura, K., Deyashiki, Y., Tanaka, N., Nonaka, T., Mitsui, Y. & Hara, A. (1996). Involvement of two basic residues (Lys-17 and Arg-39) of mouse lung carbonyl reductase in NADP(H)-binding and fatty acid activation: site-directed mutagenesis and kinetic analyses. Journal of Biochemistry 120, 257–263. Nakayama, T., Yashiro, K., Inoue, Y., Matsuura, K., Ichikawa, H., Hara, A. & Sawada, H. (1986). Characterization of pulmonary carbonyl reductase of mouse and guinea pig. Biochimica et Biophysica Acta 882, 220–227. Navre, M. & Ringold, G. M. (1988). A growth factor-repressible gene associated with protein kinase C-mediated inhibition of adipocyte differentiation. The Journal of Cell Biology 107, 279–286. Odani, H., Asami, J., Ishii, A., Oide, K., Sudo, T., Nakamura, A., Miyata, N., Otsuka, N., Maeda, K. & Nakagawa, J. (2008). Suppression of renal alpha-dicarbonyl compounds generated following ureteral obstruction by kidney-specific alpha-dicarbonyl/L-xylulose reductase. Annals of the New York Academy of Sciences 1126, 320–324. Ohno, S. (1970). Evolution by Gene Duplication. Springer-Verlag, Berlin. Ohta, T. (1989). Role of gene duplication in evolution. Genome/National Research Council Canada = Genome/Conseil National de Recherches Canada 31, 304–310. Oritani, H., Deyashiki, Y., Nakayama, T., Hara, A., Sawada, H., Matsuura, K., Bunai, Y. & Ohya, I. (1992). Purification and characterization of pig lung carbonyl reductase. Archives of Biochemistry and Biophysics 292, 539–547. Otto, S. P. & Yong, P. (2002). The evolution of gene duplicates. Advances in Genetics 46, 451–483. Parent, S., Lefièvre, L., Brindle, Y. & Sullivan, R. (1999). Bull subfertility is associated with low levels of a sperm membrane antigen. Molecular Reproduction and Development 52, 57–65. Penning, T. M., Burczynski, M. E., Jez, J. M., Hung, C. F., Lin, H. K., Ma, H., Moore, M., Palackal, N. & Ratnam, K. (2000). Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. The Biochemical Journal 351, 67–77. Penning, T. M. & Byrns, M. C. (2009). Steroid hormone transforming aldo-keto reductases and cancer. Annals of the New York Academy of Sciences 1155, 33–42. Persson, B., Kallberg, Y., Bray, J. E., Bruford, E., Dellaporta, S. L., Favia, A. D., Duarte, R. G., Jornvall, H., Kavanagh, K. L., Kedishvili,

Bettina Ebert and others N., Kisiela, M., Maser, E., Mindnich, R., Orchard, S., Penning, T. M., Thornton, J. M., Adamski, J. & Oppermann, U. (2009). The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative. Chemico-Biological Interactions 178, 94–98. Pierce, S. B., Spurrell, C. H., Mandell, J. B., Lee, M. K., Zeligson, S., Bereman, M. S., Stray, S. M., Fokstuen, S., MacCoss, M. J., Levy-Lahad, E., King, M. C. & Motulsky, A. G. (2011). Garrod’s fourth inborn error of metabolism solved by the identification of mutations causing pentosuria. Proceedings of the National Academy of Sciences of the United States of America 108, 18313–18317. Robitaille, G., Sullivan, R. & Bleau, G. (1991). Identification of epididymal proteins associated with hamster sperm. The Journal of Experimental Zoology 258, 69–74. Simpson, C. L., Patel, D. M. & Green, K. J. (2011). Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nature Reviews Molecular Cell Biology 12, 565–580. Singson, A., Hang, J. S. & Parry, J. M. (2008). Genes required for the common miracle of fertilization in Caenorhabditis elegans. The International Journal of Developmental Biology 52, 647–656. Sipila, P., Jalkanen, J., Huhtaniemi, I. T. & Poutanen, M. (2009). Novel epididymal proteins as targets for the development of post-testicular male contraception. Reproduction 137, 379–389. Son le, T., Ko, K. M., Cho, J. H., Singaravelu, G., Chatterjee, I., Choi, T. W., Song, H. O., Yu, J. R., Park, B. J., Lee, S. K. & Ahnn, J. (2011). DHS-21, a dicarbonyl/L-xylulose reductase (DCXR) ortholog, regulates longevity and reproduction in Caenorhabditis elegans. FEBS Letters 585, 1310–1316. St-Cyr, A., Legare, C., Frenette, G., Gaudreault, C. & Sullivan, R. (2004). P26h and dicarbonyl/L-xylulose reductase are two distinct proteins present in the hamster epididymis. Molecular Reproduction and Development 69, 137–145. Sudo, T., Ishii, A., Asami, J., Uematsu, Y., Saitoh, M., Nakamura, A., Tada, N., Ohnuki, T., Komurasaki, T. & Nakagawa, J. (2005). Transgenic mice over-expressing dicarbonyl/L-xylulose reductase gene crossed with KK-Ay diabetic model mice: an animal model for the metabolism of renal carbonyl compounds. Experimental Animals/Japanese Association for Laboratory Animal Science 54, 385–394. Sullivan, R. & Bleau, G. (1985). Interaction of isolated components from mammalian sperm and egg. Gamete Research 12, 101–116. Sullivan, R., Frenette, G. & Girouard, J. (2007). Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit. Asian Journal of Andrology 9, 483–491. Sullivan, R., Legare, C., Thabet, M. & Thimon, V. (2011). Gene expression in the epididymis of normal and vasectomized men: what can we learn about human sperm maturation? Journal of Andrology 32, 686–697. Sullivan, R., Legare, C., Villeneuve, M., Foliguet, B. & Bissonnette, F. (2006). Levels of P34H, a sperm protein of epididymal origin, as a predictor of conventional in vitro fertilization outcome. Fertility and sterility 85, 1557–1559. Sze, C. I., Su, M., Pugazhenthi, S., Jambal, P., Hsu, L. J., Heath, J., Schultz, L. & Chang, N. S. (2004). Down-regulation of WW domain-containing oxidoreductase induces Tau phosphorylation in vitro. A potential role in Alzheimer’s disease. The Journal of Biological Chemistry 279, 30498–30506. Tanaka, N., Aoki, K., Ishikura, S., Nagano, M., Imamura, Y., Hara, A. & Nakamura, K. T. (2008). Molecular basis for peroxisomal localization of tetrameric carbonyl reductase. Structure 16, 388–397. Tanaka, N., Nonaka, T., Nakanishi, M., Deyashiki, Y. & Hara, A. (1995). Crystallization of mouse lung carbonyl reductase complexed with NADPH and analysis of symmetry of its tetrameric molecule. Journal of Biochemistry 118, 871–873. Tanaka, N., Nonaka, T., Nakanishi, M., Deyashiki, Y., Hara, A. & Mitsui, Y. (1996). Crystal structure of the ternary complex of mouse lung carbonyl reductase at 1.8 A resolution: the structural origin of coenzyme specificity in the short-chain dehydrogenase/reductase family. Structure 4, 33–45. Thiery, J. P., Acloque, H., Huang, R. Y. & Nieto, M. A. (2009). Epithelial-mesenchymal transitions in development and disease. Cell 139, 871–890. Usami, N., Ishikura, S., Abe, H., Nagano, M., Uebuchi, M., Kuniyasu, A., Otagiri, M., Nakayama, H., Imamura, Y. & Hara, A. (2003). Cloning, expression and tissue distribution of a tetrameric form of pig carbonyl reductase. Chemico-Biological Interactions 143–144, 353–361. Velculescu, V. E., Madden, S. L., Zhang, L., Lash, A. E., Yu, J., Rago, C., Lal, A., Wang, C. J., Beaudry, G. A., Ciriello, K. M., Cook, B. P., Dufault, M. R., Ferguson, A. T., Gao, Y., He, T. C., Hermeking, H., Hiraldo, S. K., Hwang, P. M., Lopez, M. A., Luderer, H. F., Mathews, B., Petroziello, J. M., Polyak, K., Zawel, L., Kinzler, K. W., et al. (1999). Analysis of human transcriptomes. Nature Genetics 23, 387–388. Wang, H. Y., Juo, L. I., Lin, Y. T., Hsiao, M., Lin, J. T., Tsai, C. H., Tzeng, Y. H., Chuang, Y. C., Chang, N. S., Yang, C. N. & Lu, P. J. (2012). WW domain-containing oxidoreductase promotes neuronal differentiation


Human DCXR – another ‘moonlighting protein’? via negative regulation of glycogen synthase kinase 3beta. Cell Death and Differentiation 19, 1049–1059. Wang, Y. M. & Van Eys, J. (1970). The enzymatic defect in essential pentosuria. The New England Journal of Medicine 282, 892–896. Wenz, H. M., Hinck, L., Cannon, P., Navre, M. & Ringold, G. M. (1992). Reduced expression of AP27 protein, the product of a growth factor-repressible gene, is associated with diminished adipocyte differentiation. Proceedings of the National Academy of Sciences of the United States of America 89, 1065–1069. Yu, T. T., McIntyre, J. C., Bose, S. C., Hardin, D., Owen, M. C. & McClintock, T. S. (2005). Differentially expressed transcripts from phenotypically identified olfactory sensory neurons. The Journal of Comparative Neurology 483, 251–262.

25 Zaneveld, L. J., De Jonge, C. J., Anderson, R. A. & Mack, S. R. (1991). Human sperm capacitation and the acrosome reaction. Human Reproduction 6, 1265–1274. Zhang, J. (2003). Evolution by gene duplication: an update. Trends in Ecology & Evolution 18, 292–298. Zhang, P., Gu, Z. & Li, W. H. (2003). Different evolutionary patterns between young duplicate genes in the human genome. Genome Biology 4, R56. Zhao, H. T., Endo, S., Ishikura, S., Chung, R., Hogg, P. J., Hara, A. & El-Kabbani, O. (2009). Structure/function analysis of a critical disulfide bond in the active site of L-xylulose reductase. Cellular and Molecular Life Sciences: CMLS 66, 1570–1579.

(Received 10 July 2013; revised 21 February 2014; accepted 19 March 2014 )


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Carbohydrate metabolism in male infertility and female fertility-control patients.

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Another DRAM involved in autophagy and cell death.

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Dose-dependent effects of caffeine in human Sertoli cells metabolism and oxidative profile: relevance for male fertility.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

Genetic alterations affecting cholesterol metabolism and human fertility.

Phospholipid hydroperoxide glutathione peroxidase is involved in the maintenance of male fertility under cryptorchidism in mice.

Lectin cell adhesion molecules (LEC-CAMs): a new family of cell adhesion proteins involved with inflammation.

Fertility regulation in the male.

Carbonyl reduction pathways in drug metabolism.

Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

Fungal metabolism and detoxification of fluoranthene.