Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1999-02-26
2001-08-21
Achutamurthy, Ponnathapu (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S196000, C435S252300, C435S320100, C536S023200, C530S350000, C530S300000
Reexamination Certificate
active
06277373
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a new phosphatidyl-inositol 4,5-bisphosphate 5-phosphatase and to the use of these sequences in the diagnosis, prevention, and treatment of disorders of cell proliferation and cell signaling.
BACKGROUND OF THE INVENTION
Protein phosphorylation is the ubiquitous strategy used to control the activities of eukaryotic cells. It is estimated that 10% of the proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate which confers activation and is transferred from adenosine triphosphate molecules to a protein by protein kinases is subsequently removed from the protein by protein phosphatases. In this way, the phosphatases control most cellular signaling events that regulate cell growth and differentiation, cell-to-cell contacts, the cell cycle, and oncogenesis.
The inositol polyphosphate phosphatases include several monomer enzymes of different molecular weights (115-160 kD, 69-75 kD, and 32-43 kD) which hydrolyze inositol polyphosphates. Such phosphatases are known to remove the phosphate from the 3, 4, or 5 position of the inositol ring of several different substrates. For example, membrane-bound polyphosphoinositide phosphatase from rat brain utilizes phosphatidylinositol (4)phosphate, phosphatidylinositol (3)phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2) as substrates. Phosphatidyl-inositol 4,5-bisphosphate 5 phosphatase preferentially removes the position 5 phosphate from PIP2, but its affinity for other positions or substrates is not fully known (Hope H. M. and L. J. Pike (1994) J. Biol. Chem. 269:23648-54; Jefferson and Majerus (1995; J. Biol. Chem. 270:9370-9377).
Hydrolysis of PIP2 produces diacylglycerol (DAG) and inositol triphosphate (IP3), both of which act as second messengers in the cell signaling pathways. DAG and IP3 release Ca2+ from intracellular stores (endoplasmic/sarcoplasmic reticulum) and promote Ca2+ influx from the extracellular fluid. The phosphatases that catalyze the removal of phosphate are positively and negatively regulated by the local concentration of various ions including CA
−
, Mg
−
, and Li
−
.
Jefferson and Majerus (supra) expressed and studied a type II polyphosphate 5 phosphatase cloned from a human erythroleukemia cell cDNA library. Their protein was 942 amino acids in length; hydrolyzed inositol 1,4,5-triphosphate, inositol 1,4-bisphosphate, and PIP2; shared two potential binding/catalytic motifs (amino acids 472-483 and 545-570) with other phosphatases; and had a 3′CPNL motif that suggested isoprenylation and membrane association. They showed that antibodies raised against a recombinant 75 kD inositol (1,4,5) triphosphate 5 phosphatase also depleted phosphatidyl-inositol 4,5-bisphosphate 5 phosphatase activity from the cytosolic and membrane fractions of platelets; and they suggested that tissue specific expression and/or proteolytic processing of the larger phosphatases may be the source of the cytosolic phosphatases.
The discovery of a new human phosphatidylinositol 4,5-bisphosphate 5-phosphatase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of disorders of cell proliferation and cell signaling.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, phosphatidylinositol 4,5-bisphosphate 5-phosphatase (PBPP), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet 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 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding PBPP 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 PBPP having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immune disorder comprising administering to a subject in need of such treatment an effective amount of an antagonist of PBPP.
The invention also provides a method for treating or preventing a cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist of PBPP.
The invention also provides a method for treating or preventing a neuronal disorder comprising administering to a subject in need of such treatment an effective amount of an antagonist of PBPP.
The invention also provides a method for detecting a polynucleotide which encodes PBPP in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding PBPP in the biological sample. In one aspect the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.
REFERENCES:
patent: 5716806 (1998-02-01), Meissner et al.
patent: WO 95/14772 (1995-01-01), None
Rath Hope, H.M. et al., Purification and characterization of a polyphosphoinositide phosphatase from rat brain.J.Biol.Chem. (1994) 269:23648-23654.
Jefferson, A.B. et al., Properties of Type II Inositol Polyphosphate 5-Phosphatase.J.Biol.Chem. (1995) 270:9370-9377. (GI 1019103).
Nussbaum, R.L., (GI 1399104), GenBank Sequence Database (Accession U45975), National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, 20894.
Nussbaum, R.L. et al., (GI 1420919), GenBank Sequence Database (Accession U57627), National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, 20894. (GI 1420920).
Matzaris, M. et al., Identification and characterization of the phosphatidylinositol-(4,5)-bisphosphate 5-phosphatase in human plat
Corley Neil C.
Hillman Jennifer L.
Lal Preeti
Shah Purvi
Achutamurthy Ponnathapu
Incyte Genomics Inc.
Incyte Genomics, Inc.
Saidha Tekchand
LandOfFree
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