Kinetic analysis of mouse retinal dehydrogenase type-2 (RALDH2) forretinal substrates

Isabelle Gagnon a, Gregg Duester b, Pangala V. Bhat a;�

a Laboratory of Nutrition and Cancer, Centre Hospitalier de l’Universite¤ de Montreal ^ Research Centre, Hotel-Dieu Campus,Department of Medicine, Universite¤ de Montreal, 3840 rue Saint-Urbain, Montreal, QC, Canada H2W 1T8

b Gene Regulation Program, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, USA

Received 2 October 2001; received in revised form 11 December 2001; accepted 13 December 2001

Abstract

Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that isessential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing theligands (all-trans and 9-cis RA) needed to bind and activate nuclear RA receptors, we determined the detailed kineticproperties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-trans retinal oxidation. The activity of the enzyme was lower at 37‡C compared to 25‡C. The efficiency of conversion of all-trans retinal to RA was 2- and 5-fold higher than 13-cis and 9-cis retinal, respectively. The Km for all-trans and 13-cisretinal were similar (0.66 and 0.62 WM, respectively). However, the Km of RALDH2 for 9-cis retinal substrate (2.25 WM)was 3-fold higher compared to all-trans and 13-cis retinal substrates. Among several reagents tested for their ability toeither inhibit or activate RALDH2, citral and para-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl2 activatedthe reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards variousreagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showeddistinct differences. Since RALDH2 has low Km and high catalytic efficiency for all-trans retinal, it may likely be involvedin the production of all-trans RA in vivo. ß 2002 Elsevier Science B.V. All rights reserved.

Keywords: Retinal dehydrogenase; Retinal oxidation; Retinoic acid

1. Introduction

Retinoic acid (RA), a naturally occurring metabo-lite of vitamin A, plays an important role in theregulation of embryonic development and tissue dif-ferentiation by binding to and activating RA recep-

tors (RARs) and retinoid X receptors (RXRs) [1^3].All-trans RA is the physiological ligand of RARswhereas 9-cis RA serves as a high-a⁄nity ligandfor RXRs [4]. The roles of these receptors in vitaminA function have been established in mice carryingtargeted mutations of RARs and RXRs [5^8].

RA is formed from retinal, generated from the re-versible dehydrogenation of retinol by NAD-depen-dent retinal dehydrogenase, belonging to the alde-hyde dehydrogenase (ALDH) super family [9].ALDHs that catalyze the oxidation of all-trans reti-nal to all-trans RA include rat RALDH1, mouse

0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 0 2 ) 0 0 2 1 3 - 3

Abbreviations: RALDH, retinaldehyde dehydrogenase;ALDH, aldehyde dehydrogenase; RA, retinoic acid; p-HMB,para-hydroxymercuribenzoic acid

* Corresponding author. Fax: +1-514-412-7152.E-mail address: bhatp@medclin.umontreal.ca (P.V. Bhat).

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AHD2, human ALDH1 (hALDH1), rat and mouseRALDH2, and human and mouse ALDH6 (alsocalled RALDH3) [9,10]. Among these ALDHs, ratRALDH1 and human ALDH1 have been well-char-acterized and have been shown to catalyze the oxi-dation of all-trans and 9-cis retinal with di¡erente⁄ciencies [11,12]. The other RALDHs, such asRALDH2 and RALDH3, remain largely uncharac-terized as to their particular role in the production of9-cis RA.

cDNAs of mouse and rat RALDH2 have beencloned [13,14]. The existence of a highly similarcDNA (s 95% deduced amino acid sequence iden-tity) in humans has been reported [15]. Several tissuesand cell types express RALDH2 during development[16,17], and overexpression of mouse RALDH2 inXenopus embryos results in high levels of RA pro-duction [18]. Targeted disruption of RALDH2 re-sults in embryonic death in the mouse [19]. It appearsfrom several studies that the tissue-speci¢c expressionand regulation of RALDH2 during developmentcontributes to RA homeostasis [16,17]. The catalytice⁄ciency of RALDH2 for all-trans retinal has beenfound to be 15-fold higher than that of RALDH1[14,20]. However, extensive kinetic characterizationof RALDH2 for various retinal substrates has notyet been carried out.

In an attempt to understand the role of RALDH2in the biosynthesis of all-trans and 9-cis RA and tocompare its properties to that of the well-character-ized RALDH1 and hALDH1, we undertook a de-tailed kinetic study of the enzyme. We found thatRALDH2 catalyzed all-trans, 9-cis and 13-cis retinalto corresponding RAs and, unlike RALDH1 andhALDH1, it showed low Km and high catalytic e⁄-ciency for all-trans retinal.

2. Materials and methods

2.1. Expression and puri¢cation of RALDH2 protein

Full-length cDNA of mouse RALDH2 was ob-tained as described earlier [18]. RALDH2 proteinwas expressed in the Escherichia coli strain BL-21DE3 as N-terminal fusions to Schistosoma gluta-thione-S-transferase (GST), using pGEX expressionvectors (Pharmacia Biotech, Uppsala, Sweden). Full-

length cDNA 1.6 kb of mouse RALDH2 was clonedinto the EcoRI site of pGEX-4T3-3. The recombi-nant plasmid was transformed into E. coli BL-21DE3 cells and its expression was induced with0.5 mM isopropyl-L-D-thiogalactopyranoside in ex-ponentially-growing bacteria and continued for 3 h.Bacterial pellets were sonicated for four times 10 s in20 mM Tris^HCl (pH 7.4) containing 1 mM EDTA,100 mM NaCl and a mixture of protease inhibitors.Protein extracts were collected after elimination ofdebris by centrifugation. Expressed GST^RALDH2protein complex was puri¢ed from the crude bacte-rial extract by GST-a⁄nity column (Pharmacia Bio-tech). RALDH2 protein was cleaved from GST-fu-sion protein by incubation with thrombin asdescribed by the manufacturer. The purity of therecombinant RALDH2 was veri¢ed by sodium do-decyl sulfate^polyacrylamide gel (10% acrylamide)electrophoresis (SDS^PAGE) [21] and visualized by0.05% Coomassie Brilliant blue.

2.2. Enzyme assays

RALDH2 activity toward retinal substrates wasassayed by measuring the formation of RA in thereaction mixture by high-pressure liquid chromatog-raphy (HPLC), as described previously [22]. Stan-dard reactions were performed at 25‡C in 100 mMphosphate bu¡er (pH 7.5) containing 0.02% Tween80, 161 mM dithiothreitol (DTT) and 603 WM NAD(¢nal volume 250 Wl). Protein concentrations in thereaction mixtures were 0.8^1 Wg. Reactions were ini-tiated by addition of the substrates (1 to 20 WM) all-trans, 13-cis and 9-cis retinal in 2.5 Wl dimethyl sulf-oxide. After the reactions, the retinoids were ex-tracted from the reaction mixture with 400 Wl of bu-tanol/acetonitrile, and an aliquot was subjected toHPLC. Kinetics were measured utilizing hyperbolicplots and double-reciprocal linear plots.

3. Results and discussion

SDS^PAGE of the recombinant a⁄nity-puri¢edRALDH2 showed a major 55 kDa band (Fig. 1).In addition, we also found a protein band at 80kDa (15% of the total puri¢ed fraction), probablyRALDH2 bound to GST-fusion protein. Repeated

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puri¢cation on the GST-a⁄nity column did not elim-inate the protein band completely. This enzyme prep-aration was used in all kinetic studies. RALDH2catalyzed all-trans retinal oxidation at a pH optimumof 9.0 (Fig. 2).

Formation of RAs from all-trans, 9-cis and 13-cisretinal at 37‡C and 25‡C was investigated to testwhether activation of RALDH2 at higher tempera-ture is needed for e⁄cient catalysis of these sub-strates. At 25‡C, RALDH2 converted all the retinalisomer substrates to respective RAs more e⁄cientlycompared to 37‡C (Fig. 3). Lower activity at 37‡Ccould be due to instability of the substrates at thistemperature as it is known that retinoids are unstable

at high temperatures [11]. The saturation kinetics ofRALDH2 with retinal isomers are shown in Fig. 4.All-trans retinal exhibited the highest activity fol-lowed by 13-cis and 9-cis retinal. The Km for all-transand 13-cis retinal were similar (Table 1). However,the Km for 9-cis retinal was 3.5-fold higher than thatof all-trans and 13-cis retinal (Table 1). RALDH2catalyzed all-trans retinal oxidation with 2- and 5-fold higher e⁄ciency compared to 13-cis and 9-cisretinal, respectively (Table 1).

Several reagents were tested for their ability toeither inhibit or stimulate the rate of oxidation ofall-trans retinal by RALDH2. The results are pre-sented in Table 2. p-HMB, a sulfhydryl-blocking re-

Fig. 1. Puri¢cation of recombinant RALDH2. Protein sampleswere analyzed by SDS^PAGE as described in Section 2. Lanes:1, protein standards; 2, bacterial cell lysate transformed withpGEX-4T-3 containing RALDH2 cDNA; 3, RALDH2 puri¢edfrom a GST-a⁄nity column. Ten-Wg protein samples were eachloaded on the gel.

Fig. 2. pH e¡ects on RALDH2 activity. Assays were performedat 25‡C using 10 WM all-trans retinal substrate. (F) Phosphatebu¡er (0.1 M), pH 7^8; (b) Tris^HCl bu¡er (0.1 M), pH 7.5^9.0; (R) glycine^NaOH bu¡er (0.05 M), pH 8.5^10.

Table 1Kinetic constants of RALDH1, hALDH1 and RALDH2 for retinal isomers

RALDH1a hALDH1a RALDH2

Retinalisomers

Km

(WM)Vmax

(nmol/min/mg)Vmax/Km Km

(WM)Vmax

(nmol/min/mg)Vmax/Km Km

(WM)Vmax

(nmol/min/mg)Vmax/Km

All-trans 9.8 16.04 1.64 2.24 9.16 4.09 0.66 4.31 6.5313-cis ^ ^ ^ 4.60 20.82 4.53 0.62 2.02 3.269-cis 5.7 22.92 4.02 5.52 30.81 5.58 2.25 2.6 1.16

Data from the saturation curves were treated with the Enz¢tter computer program. Values are the average of two independent deter-minations, where each point in the curve of each experiment is an average of three replicates.aValues are from the previous published data [11,12].

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agent, and citral, an ALDH inhibitor, strongly sup-pressed the enzyme reaction. Inhibition caused byp-HMB was completely reversed by addition of thesulfhydryl-blocking agent DTT (Table 2), indicatingthat RALDH2 requires sulfhydryl groups for activ-ity. We showed previously that all-trans retinol andchloral hydrate are potent inhibitors of RALDH1activity [11,21]. Therefore, we examined whetherthese two reagents inhibit RALDH2 activity. Neitherall-trans retinol nor chloral hydrate inhibited the en-zyme reaction (Table 2).

Earlier studies have established that magnesiuminhibits cytosolic class I ALDH activity and enhan-ces mitochondrial class 2 ALDH activity [23^25].Therefore, we investigated the e¡ect of MgCl2 onRALDH2 activity. MgCl2 enhanced RALDH2 activ-ity (Fig. 5). Forty percent stimulation of RALDH2activity was found with a MgCl2 concentration of2 mM.

Fig. 4. Saturation curves of RALDH2 with retinal isomers. Thereactions were performed at 25‡C with 0.8 and 1 Wg protein.All-trans (F), 13-cis (R) and 9-cis (b) retinal. Each point in thecurves represents the average of three replicates (less than 5%variation between each replicate).

Fig. 3. E¡ect of incubation temperature on RALDH2 all-transretinal oxidation activity. The enzyme was incubated with 10WM all-trans retinal at 37‡C and 25‡C for 10 and 60 min, re-spectively. Each point in the curves represents the average ofthree replicates.6

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Mouse RALDH2 shares 72% amino acid sequenceidentity with rat RALDH1 [13,26]. We noted thatthe kinetic properties of RALDH1 puri¢ed fromthe rat kidney and recombinant RALDH1 were sim-ilar [27]. The kinetic properties of rat RALDH1,hALDH1 (puri¢ed from human liver with s 99%purity) and mouse RALDH2 for retinal substratesare very di¡erent. RALDH1 catalyzes retinal oxida-tion at a pH optimum of 7.5 [22] whereas hALDH1and RALDH2 are more active at pH 8 and 9.0,respectively [11,12]. RALDH1 has greater catalytice⁄ciency for 9-cis retinal than for all-trans retinaland does not catalyze 13-cis retinal oxidation (Table1) [11], and hALDH1 catalyzes all three retinal iso-mers with equal e⁄ciency (Table 1) [12]. On the oth-er hand, RALDH2 shows higher catalytic e⁄ciencyfor all-trans retinal and also e⁄ciently catalyzes 13-cis retinal (Table 1). The Km and catalytic e⁄ciencyof RALDH2 for all-trans retinal is 15-fold and4-fold, respectively, higher than that of RALDH1,suggesting that RALDH2 may be involved in theproduction of all-trans RA in vivo.

It is noteworthy that RALDH2 also has the abilityto e⁄ciently catalyze 9-cis retinal oxidation. Thecrystal structure of RALDH2 shows the presenceof a disordered loop in its active site region, indicat-ing that it can accommodate all three retinal isomers

in this site [28], which is supported by the fact thatRALDH2 has a high a⁄nity for these retinal isomersand catalyzes them quite e⁄ciently. The catalytic ef-¢ciencies of RALDH2 and RALDH1 for retinal sub-strates correlate well with their abilities to synthesizeRAs in Xenopus embryos. Injection of RALDH2mRNA to Xenopus embryos stimulates much higherlevels of RA compared to the stimulation of RAsynthesis by RALDH1 mRNA in the same in vivosystem [18]. The relative roles of RALDH1 andRALDH2 in the production of all-trans and 9-cisRA in target tissues are not clear at present. Theirtissue expression pattern may shed some light on theroles of these enzymes in RA synthesis. RALDH2 isexpressed in adult reproductive organs and embryos,suggesting that it may function as a predominantregulator of RA synthesis during embryogenesis.The ubiquitous expression of RALDH1 in adult tis-sues [29,30,18] indicates that it may be the predom-inant regulator of RA synthesis in adulthood. Thespeci¢c expression of RALDH2 in midgestationalembryos and in reproductive organs implies thatthese tissues require high levels of RA [18]. The oc-currence of RALDH1 in several discrete locations inthe embryo suggests that these regions in the embryoneed low levels of RA.

Table 2E¡ect of various reagents on RALDH2 activity

Reagent Concentration(mM)

Retinoic acid produced(% of control)

None (control) 100All-trans retinol 0.001 122

0.002 1180.003 116

Chloral hydrate 0.01 900.05 1000.10 99

Citral 1 02 03 0

p-HMB (3DTT) 1 02 03 0

p-HMB (+DTT) 1 542 92

All incubations were carried out using standard assay condi-tions described in Section 2. The control rate of RA productionfrom all-trans retinal was 200 pmol/Wg protein per 60 min.

Fig. 5. Stimulatory e¡ect of MgCl2 on RALDH2 activity. Theassay was carried out with 0.8 Wg of recombinant protein, and10 WM all-trans retinal was used as substrate in the standard in-cubation. Each point represents the average of two replicates.

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Acknowledgements

This work was supported by CIHR grant MOP-36485 to P.V.B. and NIH grant AA07261 to G.D.We thank Ovid M. Da Silva for editing our manu-script.

References

[1] M. Maden, Role of retinoids in embryonic development, in:R. Blomho¡ (Ed.), Vitamin A in Health and Disease, MarcelDekker, New York, 1994, pp. 289^322.

[2] C. Hofmann, G. Eichele, Retinoids in development, in: M.B.Sporn, A.B. Roberts, D.S. Goodman (Eds.), The Retinoids:Biology, Chemistry, and Medicine, 2nd Edition, Raven, NewYork, 1994, pp. 387^441.

[3] P. Chambon, A decade of molecular biology of retinoic acidreceptors, FASEB J. 10 (1995) 940^954.

[4] D.M. Mangelsdorf, K. Umesono, R.M. Evans, The retinoidreceptors, in: M.B. Sporn, A.B. Roberts, D.S. Goodman(Eds.), The Retinoids: Biology, Chemistry and Medicine,2nd Edition, Raven, New York, 1994, pp. 319^349.

[5] D. Lohnes, M. Mark, C. Mendelsohn, P. Dolle, A. Dierich,P. Gory, A. Gansmuller, P. Chambon, Functions of the ret-inoic receptors (RARs) during development. I. Craniofacialand skeletal abnormalities in RAR double mutants, Devel-opment 120 (1994) 2723^2748.

[6] C. Mendelsohn, D. Lohnes, D. Daecimo, T. Lufkin, M.LeMeur, C. Chambon, M. Mark, Function of the retinoicacid receptors (RARs) during development. II. Multiple ab-normalities at various stages of organogenesis in RAR dou-ble mutants, Development 120 (1994) 2749^2771.

[7] J.M. Luo, H.M. Sucov, J.A. Bader, R.M. Evans, V. Giguere,Compound mutants for retinoic acid receptors (RAR) b andRAR 1 reveal developmental functions for multiple RARbisoforms, Mech. Dev. 55 (1996) 33^44.

[8] B. Mascrez, M. Mark, A. Dierich, N.B. Ghyselinck, P. Kast-ner, P. Chambon, The RXR ligand-dependent activationfunction 2 (AF-2) is important for mouse development, De-velopment 125 (1998) 4691^4707.

[9] G. Duester, Families of retinoid dehydrogenases regulatingvitamin A function. Production of visual pigment and reti-noic acid, Eur. J. Biochem. 267 (2000) 4315^4324.

[10] F.A. Mic, A. Molotkov, X. Fan, A.E. Cuenca, G. Duester,A retinaldehyde dehydrogenase that generates retinoic acid isexpressed in the ventral retina, otic vesicles and olfactory pitduring mouse embryogenesis, Mech. Dev. 97 (2000) 227^230.

[11] J. Labrecque, F. Dumas, A. Lacroix, P.V. Bhat, A novelisoenzyme of aldehyde dehydrogenase speci¢cally involvedin the biosynthesis of 9-cis and all-trans retinoic acid, Bio-chem. J. 305 (1995) 681^684.

[12] P.V. Bhat, H. Samaha, Kinetic properties of human livercytosolic aldehyde dehydrogenase for retinal isomers, Bio-chem. Pharmacol. 57 (1999) 195^197.

[13] D. Zhao, P. McCa¡ery, K.J. Ivins, R.L. Nerve, P. Hogans,W.W. Chin, U.C. Drager, Molecular identi¢cation of a ma-jor retinoic acid synthesizing a retinaldehyde-speci¢c dehy-drogenase, Eur. J. Biochem. 240 (1996) 15^22.

[14] X. Wang, P. Penzes, J.L. Napoli, Cloning of a cDNA encod-ing an aldehyde dehydrogenase and its expression in Esche-richia coli. Recognition of retinal as substrate, J. Biol. Chem.271 (1996) 16288^16293.

[15] Y. Ono, N. Fukuhara, O. Yoshie, TALi and LIM-only pro-teins synergistically induce retinaldehyde dehydrogenase 2expression in T-cell acute lymphoblastic leukemia by actingas cofactors for GATA3, Mol. Cell. Biol. 18 (1998) 6939^6950.

[16] K. Niederreither, P. McCa¡ery, U.C. Drager, P. Chambon,P. Dolle, Restricted expression and retinoic acid induceddown regulation of the retinaldehyde dehydrogenase type 2(RALDH-2) gene during mouse development, Mech. Dev.62 (1997) 67^78.

[17] Y. Zhai, S. Sperkova, J.L. Napoli, Cellular expression ofretinal dehydrogenase types 1 and 2: e¡ects of vitamin Astatus on testis mRNA, J. Cell Physiol. 186 (2001) 220^232.

[18] R.J. Haselbeck, I. Ho¡mann, G. Duester, Distinct functionsfor Aldh1 and Raldh2 in the control of ligand productionfor embryonic retinoid signaling pathways, Dev. Genet. 25(1999) 353^364.

[19] K. Niederreither, V. Subbarayan, P. Dolle, P. Chambon,Embryonic retinoic acid synthesis is essential for early mousepost-implantation development, Nat. Genet. 21 (1999) 444^448.

[20] P. Penzes, X. Wang, J.L. Napoli, Enzymatic characteristicsof retinal dehydrogenase type 1 expressed in Escherichia coli,Biochim. Biophys. Acta 1342 (1997) 175^181.

[21] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1989.

[22] P.V. Bhat, L. Poissant, A. Lacroix, Properties of retinal-ox-idizing enzyme activity in rat kidney, Biochim. Biophys.Acta 967 (1988) 211^217.

[23] F.M. Dickinson, G.J. Hart, E¡ects of Mg2þ, Ca2þ andMn2þ on sheep liver cytoplasmic aldehyde dehydrogenase,Biochem. J. 205 (1982) 443^448.

[24] A.F. Bennett, P.D. Buckley, L.F. Blackwell, Inhibition ofthe dehydrogenase activity of sheep liver cytoplasmic alde-hyde dehydrogenase by magnesium ions, Biochemistry 22(1983) 776^784.

[25] K. Takahashi, H. Weiner, Magnesium stimulation of horseliver aldehyde dehydrogenase. Changes in molecular weightand catalytic sites, J. Biol. Chem. 255 (1980) 8206^8209.

[26] P.V. Bhat, J. Labrecque, J.-M. Boutin, A. Lacroix, A. Yo-shida, Cloning of a cDNA encoding rat aldehyde dehydro-genase with high activity for retinal oxidation, Gene 166(1995) 303^306.

[27] P.V. Bhat, N. Chow Lan, J. Guimond, S. Mader, Retinaloxidizing enzyme activity of recombinantly expressed ratkidney and phenobarbital-induced aldehyde dehydrogenase,FASEB J. 12 (1998) A4870.

BBAPRO 36592 23-4-02

I. Gagnon et al. / Biochimica et Biophysica Acta 1596 (2002) 156^162 161

[28] A.L. Lamb, M.E. Newcomer, The structure of retinal dehy-drogenase type II at 2.7 A resolution: implications for reti-nal speci¢city, Biochemistry 38 (1999) 6003^6011.

[29] P.V. Bhat, M. Marcinkiewicz, Y. Li, S. Mader, Changingpatterns of renal retinal dehydrogenase expression parallel

nephron development in the rat, J. Histochem. Cytochem.46 (1998) 1025^1032.

[30] A. Frota-Ruchon, M. Marcinkiewicz, P.V. Bhat, Localiza-tion of retinal dehydrogenase type 1 in the stomach andintestine, Cell Tissue Res. 302 (2000) 397^400.

BBAPRO 36592 23-4-02

I. Gagnon et al. / Biochimica et Biophysica Acta 1596 (2002) 156^162162