Human lysophospholipase

Details Human lysophospholipase Human lysophospholipase

1998-12-15

2001-11-20

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C424S094600

Reexamination Certificate

active

06319701

ABSTRACT:

FIELD OF THE INVENTION
This intention relates to nucleic acid and amino acid sequences of a human lysophospholipase and to the use of these sequences in the diagnosis prevention, and treatment of disorders associated with cell proliferation, inflammation, and immune response.
BACKGROUND OF THE INVENTION
Lysophospholipase (LPL) is a widely distributed enzyme which occurs in numerous isoforms. These isoforms vary in molecular mass, substrate metabolized, and optimum pH required for activity and they regulate the activity of intracellular lipids. Small isoforms, approximately 15-30 kD, function as hydrolases; large isoforms, those exceeding 60 kD function both as transacylases and hydrolases. LPLs hydrolyze lysophosphatidylcholine to produce saturated fatty acid and sn-glycero-3-phosphocholine, and they are regulated by lipid factors such as acylcarnitine, arachidonic acid and phosphatidic acid.
Sugimoto, H. et al. (1996; J. Biol. Chem. 271:7705-11) isolated a monomeric, 24 kD LPL from rat liver which hydrolyzed lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, and 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8.0. In an assay measuring LPL hydrolysis of 1-palmitoyl-glycero-3-phosphocholine, the substrate dependence curve was sigmoidal, the enzyme was active from pH 5.5-9.0, and activity was not affected by Ca
2
+, Mg
2
+, or EDTA. Km and Vmax were calculated to be 0.17 mM and 1.55 &mgr;M/min/mg.
The cDNA for this LPL was isolated, and the deduced amino acid sequence showed a conserved GXSXG motif and similarity to esterases from
Pseudomonas fluorescence
and
Spirulina platensis
. Transcripts encoding LPL were isolated from spleen, heart, brain, lung, stomach, testis, and liver. Experiments showed that DMSO treatment of an HL-60 (myelocytic leukemia) cell line induced granulocyte differentiation, produced a 3-fold increase in the 24 kD LPL, and correlated with the release of arachidonic acid.
The role of LPL in human tissues has been investigated in various research studies. When lysophosphatidylcholine is formed or imported into the cell membrane, it causes lysis. Selle, H. et al. (1993; Eur. J. Biochem. 212:411-16) characterized the role of LPL in the hydrolysis of lysophosphatidylcholine in erythrocyte membranes. Endresen, M. J. et al. (1993) Scand. J. Clin. Invest. 53:733-9) reported that the increased release of free fatty acids into the sera of pre-eclamptic women is attributable to the hydrolysis of lysophosphatidylcholine by LPL. In renal studies, LPL was shown to protect NA+,K+-ATPase from the cytotoxic and cytolytic effects of cyclosporin A (Anderson. R. et al. (1994) Toxicol. Appl. Pharmacol. 125:176-83).
The discovery of a human lysophospholipase 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 associated with cell proliferation, inflammation, and immune response.
SUMMARY OF THE INVENTION
The present invention features a human lysophospholipase hereinafter designated NHLP and characterized as having similarity to rat lysophospholipase.
Accordingly, the invention features a substantially purified NHLP having the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode NHLP. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features fragments of the polynucleotide sequences, expression vectors and host cells comprising polynucleotides that encode NHLP. The present invention also features antibodies which bind specifically to NHLP, and pharmaceutical compositions comprising substantially purified NHLP. The invention also features methods for reducing immune response using NHLP or its agonist, and for treating or preventing disorders associated with cancers and inflammation using an antagonist of NHLP.


REFERENCES:
patent: 5773276 (1998-06-01), Portilla et al.
Sugimoto, H. et al., “Purification, cDNA Cloning, and Regulation of Lysophospholipase from Rat Liver.”J.Biol.Chem. (1996) 271(13):7705-7711.
Selle, H. et al., “Glycerophosphocholine release in human erythrocytes. 1H spin-echo and 31P-NMR evidence for lysophospholipase.”Eur.J.Biochem. (1993) 212(2):411-416.
Endresen, M.J. et al., “Increased lipolytic activity of sera from pre-eclamptic women due to the presence of a lysophospholipase.”Scand.J.Clin.Lab Invest. (1993) 53(7):733-739.
Anderson, R. et al., “Alpha-tocopherol prevents cyclosporin A-mediated activation of phospholipase A2 and inhibition of Na+, K(+)-adenosine triphosphatase activity in cultured hamster renal tubular cells.”Toxicol.Appl.Pharmacol. (1994) 125(2):176-183.
Sugimoto, H. et al. (GI 1552244), GenBank Sequence Database (Accession D63885), National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, 20894.
Sugimoto, H. et al. (GI 1552243), GenBank Sequence Database (Accession D63885), National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, 20894.
Sugimoto, H. et al. “Purification, characterization, and inhibition by phosphatidic acid of lysophospholipase transacylase from rat liver.”J.Biol.Chem. (1994) 269(8):6252-6258.
GenBank Accession R59445 “yf55e02.r1 Homo sapienscDNA clons 25773 (5;)”, Submitted by R.K. Wilson (1995).
dbEST record ID “yf55e02.r1 Homo sapienscDNA clone 25773 (5′)” Record corresponds to GenBank Accession R12332 submitted by R.K. Wilson (1996).
Genbank Accession R59445 “yh17f05.r1 Homo sapienscDNA clone 37830 (5′)” Submitted by R.K. Wilson (1995).
dbEST record ID “yh17f05.r1 Homo sapienscDNA clone 37830 (5′)” Record corresponds to Genbank Accession R12332 submitted by R.K. Wilson (1996).
Boguski, M.S. et al., “Gene Discovery in dbEST”,Science, 265: 1993-1995 (1994).
Yan, Y.P. et al., “Molecular cloning and functional expression of human deoxyhypusine synthase cDNA based on expressed sequence tag information”,Biochem. J., 315: 429-434 (1996).
Cerretti, D.P. et al., “Isolation of Lerk-5: A Ligand of the EPH-Related Receptor Tyrosine Kinases”,Molecular Immunology, 32: 1197-1205 (1995).
Garsetti, D. et al., “Butyric acid-induced differentiation of HL-60 cells increases the expression of a single lysophospholipase”,Biochem J., 288: 831-837 (1992).
Weltzien, H.U., “Cytolytic and Membrane-Perturbing Properties of Lysophosphatidylcholine”,Biochimica et Biophysica Acta, 559: 259-287 (1979).
Wang, A. et al., “Regiospecificity and Catalytic Triad of Lysophospholipase I”,J. Biol. Chem., 272: 22030-22036 (1997).
Wang, A. et al., “Cloning, Expression, and Catalytic Mechanism of Murine Lysophospholipase I”,J. Biol. Chem., 272: 12723-12729 (1997).
She, H.S., et al., “The substrate specificities of four different lysophospholipase as determined by a novel flourescence assay”,Biochm J., 298: 23-29 (1994).
Taylor, J.K. et al., “Comparison of Intestinal Phospholipase A/Lysophospholipase and Sucrase-Isomaltase Genes Suggests a Common Structure for Enterocyte-Specific Promoters”,DNA and Cell Biology, 16: 1419-1428 (1997).
Database EMBL, Accession No. U89352, Mar. 7, 1997, Wang, A. et al.: “Mus Musculus lysophospholipase I mRNA, complete cds,”.
Database EMBL, Accession No. AA62396, Mar. 19, 1997, National Cancer Institute, Cancer Genome Anatomy Project (CGAP): “zs16b08.r1 NCI_CGAP Homo sapiens cDNA clone Image: 685335 5′ similar to WP: K04G2.5 CE06099 Esterase”.

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