Isolated nucleic acid molecule which codes for a 32 KDA...

Details Isolated nucleic acid molecule which codes for a 32 KDA... Isolated nucleic acid molecule which codes for a 32 KDA...

1996-10-11

2001-08-28

Pak, Michael (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S325000, C536S023100, C536S023500

Reexamination Certificate

active

06280997

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a protein having 11-cis retinol dehydrogenase activity, and which forms a complex with a specific portion of a membrane receptor for plasma retinol-binding protein (RBP) expressed, e.g., in retinal pigment epithelium (RPE), and more specifically a 32 kDa protein having 11-cis retinol dehydrogenase activity, which forms a complex with a 63 kDa RBP-binding membrane protein. The invention also involves isolation of the 32 kDa protein (p32), as well as nucleic acid molecules coding for p32 or complementary to coding sequences therefor, in addition to various applications of these materials.
BACKGROUND AND PRIOR ART
Retinoids (vitamin A-derivatives) have important physiological functions in a variety of biological processes. During embryonic growth and development, as well as during growth and differentiation of adult organisms, retinoids act as hormones and participate in the regulation of gene expression in a number of cell types. See Lied et al. Trends Genet., 17:427-433 (1992). It is believed that the effects of these molecules are mediated through two classes of nuclear ligand-controlled transcription factors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), Benbrook et al., Nature, 333:669-672 (1988); Brand et al., Nature, 332:850-853 (1988); Giguere et al., Nature, 330:624-629 (1987); Mangelsdorf et al., Nature, 345:224-229 (1990); Mangelsdorf, et al. Genes Dev. 6:329-344 (1992); Petkovich et al. Nature 330:440-450 (1987); and Zelent et al., Nature 339:714-717 (1989).
Apart from their role as hormones in cellular growth and differentiation, retinoids are also involved in the visual process as the stereo isomer 11-cis retinaldehyde of retinaldehyde is the chromophore of the visual pigments. See, e.g. Bridges,
The Retinoids
, Vol. 2, pp 125-176, Academic Press, Orlando, Fla., (1984).
Under normal physiological conditions most cells, both ocular and non-ocular, obtain all-trans retinol as their major source of retinoids. Despite the many different metabolic events taking place in different tissues, it is known that a common extracellular transport machinery for retinol has evolved. Specifically, in plasma, retinol is transported by plasma retinol binding protein (RBP). See Goodman et al.,
The Retinoids
, Academic Press, Orlando Fla., Volume 2, pp. 41-88 (1984). The active derivatives of retinol, i.e., retinoic acid in non-ocular tissues and mostly 11-cis retinaldehyde for ocular tissues, are generated by cellular conversion of trans-retinol, using specific mechanisms. To date, none of these mechanisms have been fully defined at the molecular level and several of the enzymes involved have only been identified by enzymatic activities. See Lion et al., Biochem. Biophys. Acta. 384:283-292 (1975); Zimmermann et al., Exp. Eye Res. 21:325-332 (1975); Zimmermann, Exp. Eye Res. 23:159-164 (1976) and Posch et al., Biochemistry 30:6224-6230 (1991).
Polarized retinal pigment epithelial cells (polarized RPE) are unique with regard to retinoid uptake since all-trans retinol enters these cells via two different mechanisms. Retinol accumulated from RBP is taken up through the basolateral plasma membrane, while all-trans retinol, presumably taken up from the interstitial retinol-binding protein (IRBP) following bleaching of the visual pigments, may enter through the apical plasma membrane. See Bok et al., Exp. Eye Res. 22:395-402 (1976); Alder et al., Biochem. Biophys. Res. Commun. 108:1601-1608 (1982); Lai et al., Nature 298:848-849 (1982); and Inu et al., Vision Res. 22:1457-1468 (1982).
The transfer of retinol from RBP to cells is not fully understood. In a number of cell types, including RPE, specific membrane receptors for RBP have been identified. This is consistent with a receptor-mediated uptake mechanism for retinol. For example, isolated retinol binding protein receptors, nucleic acid molecules coding for these receptors and antibodies binding to the receptor have been taught, in references relating to the first of the two mechanisms. See Bavik et al., J. Biol. Chem. 266:14978-14985 (1991); Bavik, et al. J. Biol. Chem. 267:23035-23042 1992; Bavik et al., J. Biol. Chem. 267:20540-20546 (1993); and copending U.S. application Ser. No. 083,539 and International Publication WO 93/23538, all of which are incorporated by reference herein. See also Heller, J. Biol. Chem. 250:3613-3619 (1975); and Bok et al., Exp. Eye Res. 22:395-402 (1976).
Retinol uptake on the apical side of the RPE for the regeneration of 11-cis retinaldehyde, is less well characterized. Regardless of the origin of all-trans retinol, however, the synthesis and apical secretion of 11-cis retinaldehyde seems to be the major pathway for accumulated retinol in the RPE. At present, it is not known whether similar mechanisms are used with regard to cellular retinol uptake through the basolateral and the apical plasma membranes. Available data do show that functional receptors for RBP are exclusively expressed on the basolateral plasma membrane of RPE-cells. Bok et al., Exp. Eye Res. 22:395-402 (1976).
It is also known that RPEs express a 63 kDa protein (p63). This molecular weight, and all others, is by reference to SDS-PAGE, unless stated otherwise. It has also been shown by chemical cross-linking that this protein may be part of an oligomeric protein complex which functions as a membrane receptor for plasma retinol-binding protein (RBP) in RPEs, or a component of the retinoid uptake machinery in RPE cells. See Bavik et al, J. Biol. Chem. 266:14978-14875 (1991); Bavik et al., J. Biol, Chem. 267:23035-23042 (1992), U.S. patent application Ser. No. 083,539 and PCT application WO93/23538. The p63 protein has been isolated and the corresponding cDNA cloned. See Bavik et al., J. Biol. Chem. 267:20540-20546 (1993). Nothing in these references suggests the existence of the protein which is a feature of this invention.
SUMMARY OF THE INVENTION
In accordance with this invention, RPE membrane associated proteins which have a molecular weight of about 32 kd, as determined by SDS-PAGE, are disclosed. These proteins, referred to as “p32,” form oligomeric protein complexes with the previously characterized p63 protein, a component of the membrane receptor for RBP. Also disclosed are nucleic acid molecules which code for the p32 protein. Sequence analysis shows that the p32 protein belongs to the family of short chain alcohol dehydrogenases, and exhibits 11-cis retinol dehydrogenase activity, the enzyme which catalyzes the stereospecific conversion of 11-cis-retinol into 11-cis retinaldehyde in the presence of cofactor NAD+.
As will be shown, p32 has many important uses. For example, owing to its membrane bound 11-cis -retinol dehydrogenase activity, which catalyzes the conversion of 11-cis-retinol to 11-cis-retinaldehyde, a major metabolic step in retinoid metabolism in RPE-cells, retinoid accumulation and metabolism which may lead to retinitis pigmentosa, may be directly or indirectly tied to the presence of p32, and/or its activation or inhibition. As p32 has also been found to be a member of the short chain alcohol dehydrogenase super family, many known alcohol dehydrogenase inhibitors (and activators) are available to develop activity assays, and thus diagnostic materials for retinol uptake, and ocular retinoid metabolism.
Particular features of the invention include nucleic acid molecules which encode mammalian forms of these proteins, such as human, bovine, and murine forms. Also a part of the invention are probes, based upon extended sequences as described herein.
These and other aspects of this invention are more fully discussed in the following specification with accompanying drawings.


REFERENCES:
patent: 5508164 (1996-04-01), Kausch et al.
Simon et al. (1995) J. Biol. Chem. 270:1107-1112.*
Chai et al., “Cloning of a cDNA for a Second Retinol Dehydrogenase Type II”, J. Biol. Chem. 270 (47): 28408-28412 (Nov. 1995).
Suzuki et al., “Identification and immunohistochemistry of retinol dehydrogenase from bovine retinal pigment epithelium”, Biochim. et Bioph

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