Biochemical characterization of human epidermal retinol dehydrogenase 2
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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