Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-04-16
2001-03-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S320100, C435S325000, C435S252100, C435S006120, C536S023100, C536S023200, C536S023500, C536S024310, C536S024300
Reexamination Certificate
active
06204037
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to isolated and purified lipoxygenase proteins and nucleic acids. More particularly, the present invention relates to an isolated and purified second type of human 15S-lipoxygenase and an isolated and purified nucleic acid encoding the same, and to an isolated and purified nucleic acid encoding a mouse 8S-lipoxygenase.
Table of Abbreviations
15-Lox-1
Reticulocyte-type of 15S-lipoxygenase
15-Lox-2
Second type of human 15S-lipoxygenase
8-Lox
mouse 8S-lipoxygenase
PMA
Phorbol-12-myristate-13-acetate
H (P) ETE
Hydro (pero) xyeicosatetraenoic acid
HODE
Hydroxyoctadecadienoic acid
HPLC
High pressure liquid chromatography
PCR
Polymerase chain reaction
RACE
Rapid amplification of cDNA ends
BACKGROUND ART
The lipoxygenases are a structurally related family of non-heme iron dioxygenases that function in the production of fatty acid hydroperoxides. Three lipoxygenases have been identified and cloned in humans. Funk, C. D. (1993)
Prog. Nuc. Acid Res. Mol. Biol
. 45:67-98; Matsumoto et al. (1988)
Proc. Natl. Acad. Sci. USA
85: 26-30; Dixon et al. (1988)
Proc. Natl. Acad. Sci. USA
85: 416-420; Funk et al. (1990)
Proc. Natl. Acad. Sci. USA
87: 5638-5642; Izumi et al. (1990)
Proc. Natl. Acad. Sci. USA
87:7477-7481; Yoshimoto et al. (1990)
Biochem. Biophys. Res. Comm
. 172:1230-1235; Sigal et al. (1988)
Biochem. Biophys. Res. Comm
. 157:457-464). They oxygenate arachidonic acid in different positions along the carbon chain and form the corresponding 5S-, 12S- or 15S-hydroperoxides (hydroperoxy-eicosatetraenoic acids, HPETEs). The three enzymes are known mainly from the blood cell types in which they are strongly expressed—the 5S-lipoxygenase of leukocytes, the 12S-lipoxygenase of platelets, and the 15S-lipoxygenase of reticulocytes, eosinophils and macrophages. While these are the most widely recognized cellular sources, selective expression is well documented in other tissues. For example, both the 12S- and 15S-lipoxygenases are detected in skin. Nugteren et al. (1987)
Biochim. Biophys. Acta
921:135-141; Henneicke-von Zepelin et al. (1991)
J. Invest. Dermatol
. 97:291-297; Takahashi et al. (1993)
J. Biol. Chem
. 268:16443-16448; Hussain et al. (1994)
Amer. J. Physiol
. 266:C243-C253.
Potentially, the three cloned lipoxygenases could account for all enzymatic synthesis of arachidonate hydroperoxides in humans, but there are reasons to consider that other lipoxygenases may exist. For example, in the mouse there are five known lipoxygenases, three that correspond to the known human enzymes, Chen et al. (1994)
J. Biol. Chem
. 269:13979-13987; Chen et al. (1995)
J. Biol. Chem
. 270:17993-17999 and two others, Furstenberger et al. (1991)
J. Biol. Chem
. 266:15738-15745; Funk et al. (1996)
J. Biol. Chem
. 271:23338-23344.
Three of the five distinct mouse lipoxygenase enzymes are best known for their occurrence in different types of blood cells. In common with other mammals, a 5S-lipoxygenase is present in leukocytes and is responsible for synthesis of the pro-inflammatory mediators, the leukotrienes. Chen et al. (1995)
J. Biol. Chem
. 270:17993-17999; Chen et al. (1994)
Nature
372:179-182. A 12S-lipoxygenase is found in platelets and several other tissues including skin. Nugteren et al. (1987)
Biochim. Biophys. Acta
921:135-141; Chen et al. (1994)
J. Biol. Chem
. 269:13979-13987; Sun et al. (1996)
J. Biol. Chem
. 271:24055-24062.
A second type of 12S-lipoxygenase which is closely related in sequence to the human and rabbit “reticulocyte-type” of 15S-lipoxygenases occurs in certain macrophages. Sun et al. (1996) J. Biol. Chem. 271, 24055-24062. The fourth mouse lipoxygenase to be characterized is another enzyme to have 12S-lipoxygenase activity; it was cloned recently from mouse skin and has been classified as an epidermal 12S-lipoxygenase. van Dijk et al. (1995)
Biochim. Biophys. Acta
1259:4-8; Funk et al. (1996)
J. Biol. Chem
. 271:23338-23344. All four of these murine lipoxygenases enzymes have been characterized at the cDNA and genomic levels.
The fifth known mouse lipoxygenase was described originally in 1986 by Fürstenberger, Marks and colleagues as an enzyme in skin forming 8-HETE and inducible by phorbol ester treatment. Gschwendt et al. (1986)
Carcinogenesis
7:449-455. It was shown subsequently that this enzyme forms the 8S enantiomer (Hughes et al. (1991)
Biochim. Biophys. Acta
1081:347-354) and isolation of the corresponding hydroperoxide confirmed identification of the enzyme as a lipoxygenase. Fürstenberger et al. (1991)
J. Biol. Chem
. 266:15738-15745. Mouse skin is the only reported site of synthesis of 8S-HETE in animal tissues, and there is no indication from the literature pointing to a potential homologue of the mouse 8S-lipoxygenase in other mammals. Additionally, no nucleic acid, particularly a cDNA, which encodes this lipoxygenase has been characterized.
Despite the description in the art of the enzymes presented above, along with the catalytic activities covered by these enzymes, there remains an open question whether a lipoxygenase rather than a cytochrome P450 might account for the synthesis of 12R-hydroxy arachidonic acid (12R-HETE), Hammarström et al. (1975)
Proc. Natl. Acad. Sci. USA
72:5130-5134; Woollard, P. M. (1986)
Biochem. Biophys. Res. Commun
. 136(1):169-175; Baer et al. (1991)
J. Lipid Research
32:341-347; Holtzman et al. (1989)
J. Clin. Invest
. 84:1446-1453; Brash et al. (1996)
J. Biol. Chem
. 271:20549-20557, a prominent arachidonate metabolite in the skin disease of psoriasis and other proliferative dermatol (Hammarström et al. (1975)
Proc. Natl. Acad. Sci. USA
72:5130-5134; Baer et al. (1991)
J. Lipid Research
32:341-347; Baer et al. (1995)
J. Invest. Dermatol
. 104:251-255).
Therefore, what is needed, then, is further characterization of lipoxygenase enzymes in vertebrates, particularly in mammals, and more particularly in humans. A novel isolated and purified lipoxygenase and a nucleic acid encoding the same would have broad utility to due its role in arachidonic acid metabolism, a critical metabolic pathway.
DISCLOSURE OF THE INVENTION
A key aspect of this invention pertains to the discovery of a novel 15S-lipoxygenase (15-Lox-2) protein and nucleic acid encoding the 15-Lox-2 protein. Preferred nucleic acid and amino acid sequences for 15-Lox-2 are described in SEQ ID NO:1 and SEQ ID NO:2.
It is another aspect of this invention that the novel 15-Lox-2 protein acts in the metabolism of arachidonic acid to 15S-Hydro(pero)xyeicosatetraenoic acid.
Another key aspect of this invention is isolation and purification of a nucleic acid encoding mouse 8S-lipoxygenase (8-Lox). A preferred embodiment of this nucleic acid is described in SEQ ID NO:3.
Thus, in one aspect, the present invention provides an isolated and purified polynucleotide that encodes a lipoxygenase polypeptide wherein the lipoxygenase polypeptide includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. In a preferred embodiment, a polynucleotide of the present invention is a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes polypeptides designated 15-Lox-2 and 8-Lox. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequences of SEQ ID NO:1 or SEQ ID NO:3 or their homologues from other vertebrate species.
Yet another aspect of the present invention contemplates an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 10 contiguous bases of SEQ ID NO:1 wherein the polynucleotide hybridizes to a polynucleotide that encodes a lipoxygenase polypeptide wherein the lipoxygenase polypeptide includes an iron ligand comprising a serine. Preferably,
Boeglin William E.
Brash Alan R.
Jisaka Mitsuo
Achutamurthy Ponnathapu
Jenkins & Wilson, P.A.
Tung Peter P.
Vanderbilt University
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