Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1997-09-29
2001-01-09
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C536S023200
Reexamination Certificate
active
06171837
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
BACKGROUND OF THE INVENTION
All-trans- and 9-cis-retinoic acid are active retinoids for regulating expression of retinoid responsive genes, serving as ligands for two classes of ligand-dependent transcription factors, the retinoic acid receptors and retinoid X receptors. Little is known, however, regarding 9-cis-retinoic acid formation. We have obtained a 1.4-kilobase cDNA clone from a normalized human breast tissue library, which when expressed in CHO cells encodes a protein that avidly catalyzes oxidation of 9-cis-retinol to 9-cis-retinaldehyde. This protein also catalyzes oxidation of 13-cis-retinol at a rate approximately 10% of that of the 9-cis isomer but does not catalyze all-trans-retinol oxidation. NAD+ was the preferred electron acceptor for oxidation of 9-cis-retinol, although NADP+ supported low rates of 9-cis-retinol oxidation. The rate of 9-cis-retinol oxidation was optimal at pHs between 7.5 and 8. Sequence analysis indicates that the cDNA encodes a protein of 319 amino acids that resembles members of the short chain alcohol dehydrogenase protein family. mRNA for the protein is most abundant in human mammary tissue followed by kidney and testis, with lower levels of expression in liver, adrenals, lung, pancreas, and skeletal muscle. We propose that this cDNA encodes a previously unknown stereospecific enzyme, 9-cis-retinol dehydrogenase, which probably plays a role in 9-cis-retinoic acid formation.
Retinoids (vitamin A and its analogs) are essential dietary substances that are needed by mammals for reproduction, normal embryogenesis, growth, vision, and maintaining normal cellular differentiation and the integrity of the immune system (1-5). Within cells, retinoids regulate gene transcription acting through ligand-dependent transcription factors, the retinoic acid receptors (RARs)
1
, and the retinoid X receptors (RXRs) (6,7). All-trans-retinoic acid binds only to RARs with high affinity, whereas its 9-cis isomer binds with high affinity to both RARs and RXRs. The actions of all-trans- and 9-cis-retinoic acid in regulating cellular responses are distinct and not interchangeable.
In contrast to the great explosion of information regarding the actions of retinoid receptors in regulating gene transcription, information regrading how the abundant precursor retinol is physiologically activated to form the ligands needed to activate retinoid receptors is only slowly emerging (see Refs. 8 and 9 for recent reviews). It is clear that the pathway for conversion of retinol to retinoic acid involves first the oxidation of retinol to retinaldehyde and then the oxidation of retinaldehyde to retinoic acid. Numerous enzymes that are able to catalyze either retinol or retinaldehyde oxidation have been identified, purified, and/or characterized (8-10). These enzymes are members of four distinct families: the alcohol dehydrogenases, the short chain alcohol dehydrogenases, the aldehyde dehydrogenases, and cytochrome P-450s (8-10). At present, the most attention has focused on enzymes responsible for the oxidation of all-trans-retinol to all-trans-retinaldehyde (11-15). Several recent reports have indicated that both alcohol dehydrogenases and short chain alcohol dehydrogenases may be responsible for catalyzing all-trans-retinol oxidation (11-15), but the exact in vivo roles of each of these dehydrogenases in all-trans-retinoic acid formation remains controversial (8).
9-cis-Retinoic acid has been reported to be present in mammalian tissues and cells (16-18), but it has not been convincingly established how 9-cis-retinoic acid is formed within tissues and cells. Urbach and Rando have reported that liver microsomes can nonenzymatically catalyze the isomerization of all-trans-retinoic acid to the 9-cis isomer (19). Others have demonstrated that 9-cis-&bgr;-carotene can be converted to 9-cis-retinoic acid within rat tissues (20). However, this latter pathway cannot be an essential one for 9-cis-retinoic acid formation because rats maintained on a &bgr;-carotene-free purified diet containing only retinol as a precursor for retinoic acid formation are normal. In this communication, we report the characterization of a cDNA clone for a novel human enzyme that we have designated 9-cis-retinol dehydrogenase (9cRDH) and that catalyzes in a stereospecific manner the oxidation of 9-cis-retinol to 9-cis-retinaldehyde, a first enzymatic step needed for 9-cis-retinoic acid formation. Because it has been established that 9-cis-retinaldehyde can be further oxidized to 9-cis-retinoic acid by abundant tissue retinaldehyde dehydrogenases (21-23), it is possible that 9cRDH catalyzes a key oxidation step in the formation of 9-cis-retinoic acid.
SUMMARY OF THE INVENTION
This invention provides an isolated nucleic acid molecule encoding a mammalian 9-cis-retinol dehydrogenase. In a preferred embodiment the isolated nucleic acid is a cDNA molecule which encodes a human 9-cis-retinol dehydrogenase. In an embodiment the cDNA molecule encodes a mouse 9-cis-retinol dehydrogenase. In another preferred embodiment the isolated nucleic acid molecule has the nucleotide sequence shown in
FIG. 1A
(SEQ. ID NO: 1). In another embodiment the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ. ID NO: 6. In yet another embodiment the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ. ID NO: 7. In still another embodiment the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ. ID NO: 8.
This invention provides a purified 9-cis-retinol dehydrogenase. In an embodiment a 9-cis-retinol dehydrogenase is encoded by the isolated nucleic acid molecule encoding a mammalian 9-cis-retinol dehydrogenase. In a preferred embodiment the 9-cis-retinol dehydrogenase has the amino acid sequence set forth in
FIG. 1B
(SEQ. ID NO: 2).
This invention provides a mouse 9-cis-retinol dehydrogenase, wherein the nucleic acid molecule encoding the 9-cis-retinol dehydrogenase has the nucleotide sequence shown in any of SEQ. ID NOS: 6, 7, and 8.
This invention also provides a vector comprising the isolated mammalian nucleic acid molecule encoding a mammalian 9-cis-retinol dehydrogenase. In an embodiment the vector is a plasmid.
In an embodiment, a full-length cDNA nucleic acid molecule encoding a human 9-cis-retinol dehydrogenase is inserted by EcoR1/Not1 into a pCDNA3 plasmid (Invitrogen®) and the resulting plasmid is designated as pCDNA3-1. In an embodiment, a full-length cDNA nucleic acid molecule encoding a mouse liver 9-cis-retinol dehydrogenase is inserted by EcoR1/Not1 into a pCDNA3 plasmid (Invitrogen®) and the resulting plasmid is designated as pCDNA3-2. In an embodiment, a full-length cDNA nucleic acid molecule encoding a mouse kidney 9-cis-retinol dehydrogenase is inserted by EcoR1/Not1 into a pCDNA3 plasmid (Invitrogen®) and the resulting plasmid is designated as pCDNA3-3. In an embodiment, a full-length cDNA nucleic acid molecule encoding a mouse testis 9-cis-retinol dehydrogenase is inserted by EcoR1/Not1 into a pCDNA3 plasmid (Invitrogen®) and the resulting plasmid is designated as pCDNA3-4.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of an isolated nucleic acid molecule encoding a mammalian 9-cis-retinol dehydrogenase.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a sequence of the isolated nucleic acid molecule encoding a mammalian 9-cis-retinol dehydrogenase.
This invention provides a method of detecting expression of a mammalian 9-cis-retin
Blaner William S.
Gamble Mary V.
Mertz James R.
Zott Roseann Piantedosi
Cooper & Dunham LLP
Low Christopher S. F.
Srivastava Devesh
The Trustees of Columbia University in the City of New York
White John P.
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