Aldehyde Dehydrogenase 6, a Cytosolic Retinaldehyde Dehydrogenase Prominently Expressed in Sensory Neuroepithelia during Development

Felix Grün,Yukihiro Hirose,Shimako Kawauchi,Toshihiko Ogura,Kazuhiko Umesono

doi:10.1074/jbc.m007376200

Felix Grün, Yukihiro Hirose + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m007376200

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Abstract

We have isolated the chick and mouse homologs of human aldehyde dehydrogenase 6 (ALDH6) that encode a third cytosolic retinaldehyde-specific aldehyde dehydrogenase. In both chick and mouse embryos, strong expression is observed in the sensory neuroepithelia of the head. In situ hybridization analysis in chick shows compartmentalized expression primarily in the ventral retina, olfactory epithelium, and otic vesicle; additional sites of expression include the isthmus, Rathke's pouch, posterior spinal cord interneurons, and developing limbs. Recombinant chick ALDH6 has a K(0.5) = 0.26 microm, V(max) = 48.4 nmol/min/mg and exhibits strong positive cooperativity (H = 1.9) toward all-trans-retinaldehyde; mouse ALDH6 has similar kinetic parameters. Expression constructs can confer 1000-fold increased sensitivity to retinoic acid receptor-dependent signaling from retinol in transient transfections experiments. The localization of ALDH6 to the developing sensory neuroepithelia of the eye, nose, and ear and discreet sites within the CNS suggests a role for RA signaling during primary neurogenesis at these sites.

Highlights

Genes with promoters that contain retinoic acid response elements (RAREs)1 are subject to regulation by the ligand-dependent nuclear transcription factors, the retinoic acid receptors RARs and RXRs
Screening of Aldehyde Dehydrogenase PCR Amplicons from Mouse and Chick Tissues—In the developing chick and mouse retina, ALDH1 is restricted to the dorsal neural retina, while RALDH2 is expressed in the pigmented retinal epithelium [31]
An additional retinaldehyde dehydrogenase activity, named V1 in mouse and C-V in chick, that is biochemically distinct from ALDH1 and RALDH2 has been detected in the ventral neural retina [22, 23]

Summary

EXPERIMENTAL PROCEDURES

Cloning of Mouse and Chick ALDH6 cDNAs—PCR amplicons of aldehyde dehydrogenases (1025 base pairs for mouse; 713 base pairs for chick) were amplified from 100 ng of oligo(dT)-primed mouse organ or chick embryonic day 4 (E4) ventral retina cDNA using 2.5 units of ExTaq polymerase (TaKaRa Shuzo, Kyoto, Japan) with 50 pmol each of forward and reverse degenerate primers: mouse ALDHdegF, 5Ј-GCW GGI TGG GCI GAY AAR ATY CAY GG-3Ј; mouse ALDHdegR, 5Ј-CCR TKI CCW GAC ATY TTR AAS CC-3Ј; chick ALDHdegF, 5Ј-CAR ATH ATH CCI TGG AAY TT-3Ј; chick ALDH6degR, 5Ј-AAI ATY TCY TCY TTI GCI AT-3Ј. Chick ALDH6 3Ј cDNA sequences (approximately 3 kilobase pairs) were cloned by sequential PCR with 20 pmol of oligo(dT)-M13-M4 primer and the nested gene-specific primers YHF4 (5Ј-GCC ATT GAA GAC AGA GGC CTG-3Ј), YHF6 (5Ј-TAC CGA GTA TGG ACT CAC-3Ј), and YHF7 (5Ј-AGC TTC TGC TTT GCA GTC G-3Ј). Aldehyde Dehydrogenase Assays—Recombinant chick ALDH6 enzyme was prepared from the expression vector pBAD-cALDH6 in DH5␣ bacterial cultures induced with 0.2% arabinose. Retinaldehyde dehydrogenase activity was assayed with 0.1– 0.4 ␮g of protein and 2 ␮M all-trans-retinaldehyde in 1 ml of enzyme buffer (50 mM HEPES, pH 8.0, 200 mM KCl, 2 mM NAD, 1 mM MgCl2, and 1 mM dithiothreitol) at 37 °C for 10 min. Kinetic data were fitted using the nonlinear regression analysis program Prism (GraphPad Software, Inc., San Diego, CA)

RESULTS

DISCUSSION

Retinal Octanalc Benzaldehydec Acetaldehydec Citralc