Human glutathione peroxidase-6

Details Human glutathione peroxidase-6 Human glutathione peroxidase-6

1998-06-01

2001-05-15

Bansal, Geetha P. (Department: 1642)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Oxidoreductases

C424S094100, C435S192000, C514S012200, C530S350000

Reexamination Certificate

active

06231853

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a human glutathione peroxidase-6 and to the use of these sequences in the diagnosis, treatment, and prevention of reproductive, immune, and cell proliferative disorders.
BACKGROUND OF THE INVENTION
Eukaryotic cells extract energy and synthesize macromolecules by a complex series of oxidation-reduction reactions collectively referred to as aerobic metabolism. One consequence of aerobic metabolism is the production of free radicals in the form of superoxides (O
2
.
−
) and hydroxyl ions (OH.). Superoxides are produced within cells by mitochondria and the endoplasmic reticulum as a consequence of “leakage” of electrons onto O
2
from their correct paths in electron transfer chains. Hydroxyl ions are produced by ionizing radiation and by the reaction of O
2
.
−
with hydrogen peroxide (H
2
O
2
) at iron- or copper-containing sites. Free radicals, especially hydroxyl ions, are extremely reactive and can interact with almost all molecules, including proteins, carbohydrates, DNA, and lipids. These interactions can lead to the formation of nonradical hydroperoxides, such as phospholipid hydroperoxides. Interaction of hydroxyl ions with DNA may be a significant contributor to the age-dependent development of cancer. Cells also use free radicals and their derivatives in beneficial ways, such as cytochrome P45O-mediated oxidations, regulation of smooth muscle tone, and killing of microorganisms by macrophages and granulocytes. (Bast, A. et al. (1991) Am. J. Med. 91(3C): 2S-13S.)
Enzymatic antioxidants act as a defense against free radical damage. Most of the superoxides generated undergo dismutation by superoxide dismutase (SOD) generating hydrogen peroxide. H
2
O
2
can also be produced by the action of other oxidase enzymes. Hydrogen peroxide resembles water in its molecular structure and is very diffusible within and between cells. Hydrogen peroxide can be removed by catalase, but the most important enzyme in human cells for removing hydrogen peroxide and hydroperoxides is glutathione peroxidase (GSHPx). GSHPx removes peroxides by using them to oxidize reduced glutathione (GSH) to oxidized glutathione (GSSG).
The family of glutathione peroxidases encompass three tetrameric glutathione peroxidases (GPx1-3) and the monomeric phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4). Although the overall homology between tetrameric enzymes and GPx4 is less than 30%, a pronounced similarity has been detected in clusters involved in the active site and a common catalytic triad has been defined by structural and kinetic data. (Epp, O. et al. (1983) Eur. J. Biochem. 133: 51-69.) The family members show different tissue distributions. GPx1 is ubiquitously expressed in cells, whereas GPx2 is present in the liver and colon and GPx3 in blood plasma. GPx4 is found at low level in all tissues but is expressed at high level in the testis. These tissue localization patterns may be important for regulating the level and targets of glutathione peroxidase activity. (Ursini, F. et al (1995) Meth. Enzymol. 252: 38-53.)
GPx4 is unique in both its structure and activity. GPx4 is the only monomeric glutathione peroxidase found in mammals. It is also the only mammalian glutathione peroxidase to show high affinity for and reactivity with phospholipid hydroperoxides, and to be membrane associated. The inhibition of lipid peroxidation by GPx4 requires glutathione and physiological levels of vitamin E, suggesting a tandem mechanism for the antioxidant activities of GPx4 and vitamin E. GPx4 also has alternative transcription and translation start sites which determine its subcellular localization. (Esworthy, R. S. et al. (1994) Gene 144: 317-318; and Maiorino, M. et al. (1990) Meth. Enzymol. 186: 448-450.)
Antioxidant defenses exist as a balanced, coordinated system. Disruption in this balance leads to clinical manifestations. For example, an excess of SOD in relation to GSHPx may be relevant to the clinical condition known as Down's Syndrome. Low antioxidant levels or high O
2
.
−
and H
2
O
2
levels produce oxidative stress. Oxidative stress induced by phagocytes at sites of chronic inflammation lead to rheumatoid arthritis in the joints and inflammatory bowel diseases in the intestine. Asthma is also a manifestation of an inflammatory reaction in the lung and is related to oxygen free radical formation (Sies, H. (1991) Am. J. Med. 91 (3C): 31S-38S).
The discovery of a new glutathione peroxidase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of reproductive, immune, and cell proliferative disorders.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a new human glutathione peroxidase (GPx6), the polynucleotides encoding GPx6, and the use of these compositions for the diagnosis, treatment, or prevention of reproductive, immune, and cell proliferative disorders.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector comprising at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO: 1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a re

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