Biochemical characterization of human epidermal retinol dehydrogenase 2

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Abstract

The mRNA encoding a putative human enzyme named Epidermal Retinol Dehydrogenase 2 (RDH-E2) was found to be significantly elevated in psoriatic skin [Y. Matsuzaka, K. Okamoto, H. Tsuji, T. Mabuchi, A. Ozawa, G. Tamiya, H. Inoko, Identification of the hRDH-E2 gene, a novel member of the SDR family, and its increased expression in psoriatic lesion, Biochem. Biophys. Res. Commun. 297 (2002) 1171–1180]. This finding led the authors to propose that RDH-E2 may be involved in the pathogenesis of psoriasis through its potential role in retinoic acid biosynthesis and stimulation of keratinocyte proliferation. However, enzymatic activity for RDH-E2 has never been demonstrated. RDH-E2 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins, and is most closely related to the group of SDRs comprised of both NAD+- and NADP+-dependent enzymes with activities toward retinoid and steroid substrates. In this study, we began the characterization of RDH-E2 protein in order to determine whether it might play a role in retinoic acid biosynthesis. The results of this study show that, similarly to other SDR-type retinol dehydrogenases, RDH-E2 appears to be associated with the membranes of endoplasmic reticulum. Furthermore, RDH-E2 expressed in Sf9 insect cells as a fusion to the C-terminal His6-tag and purified using Ni2+-affinity chromatography recognizes all-trans-retinol and all-trans-retinaldehyde as substrates and exhibits a strong preference for NAD+/NADH as cofactors. Specific activity of RDH-E2 toward all-trans-retinoids is much lower than that of other retinoid-active SDRs, such as human RoDH4 or RDH10. The preference for NAD+ suggests that RDH-E2 is likely to function in the oxidative direction in vivo, further supporting its potential role in the oxidation of retinol to retinaldehyde for retinoic acid biosynthesis in human keratinocytes.

Introduction

The cDNA for RDH-E2 was originally cloned by Matsuzaka et al. as a result of their search for novel psoriasis-associated genes [1]. The authors carried out microarray analysis of the gene expression in the affected and unaffected skin of patients with psoriasis, and the normal skin of healthy individuals, and identified several genes that displayed increased expression in psoriatic skin. One of these genes was found to encode a previously unrecognized member of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins [1]. Based on its sequence similarity to retinal SDR2 (retSDR2, also known as pan1b, 17beta-HSD11) [2], [3], [4], the novel gene was named human epidermal retinol dehydrogenase 2 (RDH-E2) [1]. Real-time quantitative RT-PCR confirmed that the expression level of the RDH-E2 gene in psoriatic tissues was at least threefold higher than that in healthy skin [1].

Psoriasis vulgaris is a chronic autoimmune inflammatory skin disease characterized by hyperproliferation of keratinocytes [5]. Hyperproliferation of keratinocytes may be induced by retinoic acid [6], the activating ligand of nuclear transcription factors, retinoic acid receptors [7]. Therefore, the authors proposed that RDH-E2 may be involved in the pathogenesis of psoriasis through its potential role in retinoic acid biosynthesis. However, RDH-E2 protein has never been shown to possess an enzymatic activity and recognize retinoids as substrates. To determine whether RDH-E2 could play a role in retinoic acid biosynthesis, we initiated the characterization of its substrate and cofactor specificity. Here, we provide the first evidence that RDH-E2 might function as a retinol dehydrogenase.

Section snippets

Construction of expression vectors

A cDNA clone encoding RDH-E2 was obtained from the American Type Culture Collection (Manassas, VA, USA) (ATCC clone #7998610, IMAGE collection #5752917). For expression in eukaryotic cells, the full-length RDH-E2 cDNA in ATCC clone was PCR-amplified using forward primer 5′-AGC GAA TTC ATG TCT TTC AAC CTG CAA TCA T-3′ (EcoRI restriction site underlined) and reverse primer 5′-TCT CTC GAG CTT CTT CTT TTG GTC AAC-3′ (XhoI restriction site underlined). The PCR product was gel-purified, cleaved with

Expression and characterization of recombinant hRDH-E2 in human cells

To determine whether RDH-E2 is catalytically active, the recombinant protein was initially expressed in HEK293 cells as a fusion to the C-terminal FLAG tag. Analysis of the transfected cells by immunofluorescence revealed the presence of RDH-E2/FLAG in ∼30% of the cells (Fig. 1). RDH-E2 protein exhibited a punctuate localization pattern, surrounding the nucleus (Fig. 1B). This pattern was similar to that observed for cells stained with fluorescently labeled concanavalin A (Fig. 1C), suggesting

Discussion

The properties of RDH-E2 are of special interest because this protein may have a role in the progression of psoriatic skin disease [1], [12]. As shown in this study, RDH-E2 appears to be predominantly associated with the membranes of endoplasmic reticulum. According to various algorithms for prediction of transmembrane segments, at least three transmembrane helices can be detected in the amino acid sequence of RDH-E2, suggesting that it is an integral membrane protein. RDH-E2 is not

Conflict of interest statement

None.

Acknowledgements

This work was supported by the National Institute on Alcohol Abuse and Alcoholism Grant AA12153.

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