Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-17
2001-06-05
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023200, C536S023100, C536S023500, C435S320100, C435S252300, C435S325000, C435S196000, C435S069100
Reexamination Certificate
active
06242179
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of human phosphatases and to the use of these sequences in the diagnosis, prevention, and treatment of immune disorders and diseases associated with cancer.
BACKGROUND OF THE INVENTION
The protein phosphorylation/dephosphorylation cycle is one of the major regulatory mechanisms employed by eukaryotic cells to control cellular activities. It is estimated that more than 10% of the active proteins in a typical mammalian cell are phosphorylated. During protein phosphorylation/dephosphorylation, phosphate groups are transferred from adenosine triphosphate molecules to a protein by protein kinases and are removed from a protein by protein phosphatases.
Protein phosphatases function in cellular signaling events that regulate cell proliferation and differentiation, cell-to-cell contacts, the cell cycle, and oncogenesis. Three evolutionary distinct protein phosphatase families have been identified. These include the serine/threonine phosphatases, the protein tyrosine phosphatases, and the acid/alkaline phosphatases (Carbonneau H. and Tonks N. K. (1992) Annu. Rev. Cell Biol. 8:463-93).
Phosphatidic acid phosphate (PAP) is metabolized to diacylglycerol in the classical pathway of glycerolipid biosynthesis by dephosphorylating phosphatidic acid. Phosphatidic acid and its metabolic derivative, lysophosphatidic acid, are known to be potent mitogens and activators when exogenously added to different cells. Two isoforms of PAP exist in rat liver. The first, designated PAP1, is associated with the cytosol and microsomes, and appears to be responsible for glycerolipid biosynthesis. The second isoform PAP2 is bound to the plasma membrane and is involved in cellular signal transduction. The activities of the two PAP isoforms appear to undergo different activity alterations in several liver diseases (Day, C. P (1993) Clin. Sci. (Lond.) 85:281-287).
Phospholipase D (PLD) is activated in a variety of cells by hormones and growth factors. Activated PLD results in the generation of phosphatidic acid (PA) by hydrolysis of PAP, which can be further metabolized by PA phosphohydrolase to diacylglycerol (DG). The generation of PA by PLD in neutrofils has been linked to the activation of the respiratory burst enzyme, NADPH oxidase. During phosphorylation-dependent oxidase activation, PA, but not DG, induces phosphorylation of a wide range of proteins, the most prominent of which is the NADPH oxidase component p47-phox (phagocytic oxidase component). The absence of p47-phox is implicated in a genetic defect called p47-phox-deficient chronic granulomatous disease (P47-GCD). P-47-GCD is caused by a GT deletion at the beginning of exon 2. The GT deletion has been found in 11 becteriophage and 15 YAC clones.
The discovery of two new human protein phosphatases and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of immune disorders and diseases associated with cancer.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human phosphatases, referred to collectively as “HPA” and individually as “HPA-1” and “HPA-2.” In one aspect, the invention provides a substantially purified polypeptide, HPA, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:4.
The invention further provides a substantially purified variant of HPA having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:4, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide sequence encoding the polypeptide comprising any of the amino acid sequences described above. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide sequence encoding the polypeptide comprising these amino acid sequences.
Additionally, the invention provides a composition comprising a polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:4, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:4. The invention further provides an isolated and purified polynucleotide sequence which hybridizes under stringent conditions to one of these polynucleotide sequences, as well as an isolated and purified polynucleotide sequence which is complementary to one of these polynucleotide sequences.
The invention also provides an isolated and purified polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:5. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to one of these polynucleotide sequences, as well as an isolated and purified polynucleotide sequence which is complementary to one of these polynucleotide sequences.
The invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In another aspect, the expression vector containing the polynucleotide sequence 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, SEQ ID NO:4, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:4, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide sequence encoding HPA 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 HPA having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:4, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:4 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:4, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:4, as well as a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of HPA.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of HPA.
The invention also provides a method for detecting a polynucleotide encoding HPA in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence which encodes the polypeptide comprising SEQ ID NO:1, SEQ ID NO:4, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:4 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding HPA in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
REFERENCES:
patent: WO 98/46730 (1998-10-01), None
A.R. Rodaway et al., “Characterization of the 47-Kilodalton Autosomal Chronic Granulomatous Disease Protein: Tissue-Specific Expression and Transcriptional Control By Retinoic Acid” Mol. Cell. Biol. 10(10):5388-5396, Oct. 1990.
GenBank entry H97570, Dec. 1995.
GenBank entry N25122, Dec. 1995.
GenBank entry AA043085, Sep. 1996.
Kai KM, et al.: “Cloning and characterization of two human isozymes of Mg2+independent phosphatidic acid phosphatases” Journal of Biological Chemistry, vol. 272,
Corley Neil C.
Hillman Jennifer L.
Lal Preeti
Shah Purvi
Incyte Pharmaceuticals Inc.
Incyte Pharmaceuticals, Inc.
Prouty Rebecca E.
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