25934, a novel fatty acid desaturase and uses therefor

Details 25934, a novel fatty acid desaturase and uses therefor 25934, a novel fatty acid desaturase and uses therefor

2000-11-28

2004-02-03

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S252300, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06686185

ABSTRACT:

BACKGROUND OF THE INVENTION
Fatty acid desaturases are critical regulatory enzymes of unsaturated fatty acid biosynthesis and catalyze the conversion of a single bond between two carbon atoms (C—C) to a double bond (C═C) in a fatty acyl chain. The resultant double bond is often referred to as an unsaturated bond. Eukaryotic fatty acid desaturases, typically, are iron-containing enzymes that catalyze the NAD-(P)H and O
2
-dependent introduction of double bonds into methylene-interrupted fatty acid chains. Examination of the deduced amino acid sequence from mammals, fungi, insects, higher plants and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 or 4)H, HX(2 or 3), and HX(2 or 3)HH. This motif is also present in the bacterial membrane enzymes alkaline hydroxylase (omega-hydroxylase) and xylene monooxygenase.
There are three types of eukaryotic fatty acid desaturases, acyl-CoA, acyl-ACP, and acyl-lipid desaturases (Ntambi et al., Biochem. and Biophys. Res. Com. 266:1-4, 1999). In plants and cyanobacteria, acyl-lipid desaturases catalyzing most desaturation reactions and introduce unsaturated bonds into fatty acids that are in a lipid-bound form. Acyl-ACP desaturases are present in the plastids of plant cells and insert a double bond into fatty acids that are bound to acyl carrier protein (ACP). In animals, yeast and fungal cells, Acyl-CoA introduce unsaturated bonds into fatty acids that are bound to coenzyme A (CoA). A gene cloned from this family is stearoyl-CoA desaturase and this gene has been identified in many organisms including mice, rats, humans, yeast, ovines, and hamsters.
Fatty acid desaturases can introduce an unsaturated bond at a specific position in a fatty acyl chain, for example, at the &Dgr;6, &Dgr;9, or &Dgr;12 position. Desaturases are typically integral membrane proteins induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded (Ozols, J. (1997) MBC Vol. 8 (11): 2281-2290). Unsaturated fatty acids can be formed from a variety of fatty acids including palmitate and stearate resulting in the formation of unsaturated fatty acids palmitoleate (16:1), and oleate (18:1).
In mammals, the rate limiting step in the biosynthesis of monounsaturated fatty acids is the insertion of an unsaturated bond by stearoyl-CoA desaturase (SCD) in the &Dgr;9 position of the fatty acid. SCD preferentially catalyzes the synthesis of oleic acid. Oleate enriched low density lipoprotein (LDL) exhibits increased affinity for the vessel wall, and is therefore pro-atherogenic (Rudel, L. L. et al. (1997) J. Clin. Invest. 1:100(1):74-83). SCD involvement in generating atherogenic LDL variants and in regulating triglyceride synthesis is further supported by the finding that polyunsaturated fatty acids (PUFA), which protect against atherosclerosis, negatively regulate the expression of the SCD gene (Rudel, L L et al. (1995) Atheroscler. Thromb. Vasc. Biol. 15(12):2101-10; Ntambi, J M (1999) J. Lipid Res. 40(9):1549-58). Moreover, a mouse deficient for SCD exhibits significant reduction in triglycerides (Miyazaki, M. et al. (2000) J. Biol. Chem, in press).
Unsaturated fatty acids play an important role in normal and diseased organisms. For example, the degree of fatty acid unsaturation in cell membrane lipids determines membrane fluidity. Moreover, the production of monounsaturated fatty acids, which once complexed with lipoproteins such as LDL, show increased affinity for the vessels wall has profound implications for cardiovascular disorders caused by aberrant fatty acid metabolism. Examples of such disorders include atherosclerosis, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, among others.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of a novel fatty acid desaturase, referred to herein as “25934”. The nucleotide sequence of a cDNA encoding 25934 is shown in SEQ ID NO:1, and the amino acid sequence of a 25934 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 25934 protein or polypeptide, e.g., a biologically active portion of the 25934 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 25934 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167, wherein the nucleic acid encodes a full length 25934 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 25934 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 25934 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 25934 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 25934-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 25934 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 25934 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 25934 mediated or related disorders, e.g., a cardiovascular disorder. In another embodiment, the invention provides 25934 polypeptides having a 25934 activity. Preferred polypeptides are 25934 proteins including at least one desaturase domain, and, preferably, having a 25934 activity, e.g., a 25934 activity as described herein.
In other embodiments, the invention provides 25934 polypeptides, e.g., a 25934 polypeptide having the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number 2167; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167, wherein the nucleic acid encodes a full length 25934 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 25934 nucleic acid molecule described herein.
In a related aspect, the invention provides 25934 polypeptides or fragments operatively linked to non-25934 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 25934 polypeptides.
In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 25934 polypeptides or nucleic acids.
In still another aspect, the invention provid

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