Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-30
2001-07-31
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S198000, C435S252300, C435S320100, C435S021000, C536S023200, C536S023500, C530S350000
Reexamination Certificate
active
06268135
ABSTRACT:
BACKGROUND OF THE INVENTION
Phospholipases are involved in the signaling pathway in which a cellular response such as proliferation or secretion is produced consequent to an extracellular stimulus. Activation of mammalian phosphoinositide-specific Phospholipase C (PLC) by a receptor-linked G-protein results in the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to release the second messengers 1,2-diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3). DAG activates protein kinase C (PKC), and IP3 releases calcium from stores in the endoplasmic reticulum. Sustained response to the stimulus arises from processing of phosphatidylcholine (PC) by either PLC, which generates DAG directly, or by PLC, which gives phosphatidic acid (PA); PA is then hydrolyzed to DAG.
Phospholipase A2 (PLA2) represents a class of heat-stable, calcium-dependent enzymes catalyzing the hydrolysis of the 2-acyl bond of 3-n-phosphoglycerides. This enzyme is named Phospholipase A2 to denote its 2-acyl specificity (Uthe 1971). Each protein is composed of dimeric subunits, &agr;, &bgr;, respectively (Wells 1971). PLA2s hydrolyze the sn-2 ester bond on L-glycerophospholipids. At present there are three commonly recognized categories of PLA2s: a large (85 kDa) Ca++ dependent cytosolic PLA2 (Leslie et al:
Biochem Biophys. Acta
963:476-492 (1988); Clark et al.:
Proc. Natl. Acad. Sci. USA
87:7708-7712 (1990); Kramer et al. 1991), a smaller (40 kDa) Ca++ independent PLA2 found in myocardium (Hazen et al.:
J. Biol. Chem
. 265:10622-10630 (1990)), and a less well defined group of secreted enzymes found in platelets, synovial fluid, and in some insect and lizard venoms (Verheij et al.:
Rev. Physiol. Biochem. Phramacol
. 91:91-103 (1981); Dennis:
The Enzymes
16:307-353 (1983); Kramer et al.
J. Biol. Chem
. 264:5768-5775 (1989)). BV-PLA2 is one of many PLA2s in it's class of small, secreted, Ca++ dependent PLA2 enzymes (Dawson: Chemical Studies of Structural Features in Staphylococcus Nuclease T′, in
Form and Function of Phospholipids
, (Ansell, Hawthorne and Dawson eds.), Elsevier, Amsterdam, 97 (1973)).
The first cystolic forms of PLA2 have been implicated in the release of arachidonic acid from cell membranes. This release in arachidonic acid has been reported to be indirectly involved in the inflammatory response. Several types of PLA2s such as Secretory Phospholipase A
2
, Phospholipase A
2
, and Human Extracellular Group II Phospholipase A
2
are involved in inflammatory diseases such as Rheumatic disease, acute pancreatitis, and skin inflammation, respectively. Arachidonic acid in mast cells, macrophages, monocytes, eosinophils and basophils is released from membrane phospholipids by the activation of phospholipase A2. After its release, arachidonic acid undergoes metabolism via two major pathways: the cyclooxygenase pathway (which produces various prostaglandins and thromboxanes) and the 5-lipoxygenase pathway (which produces leukotrienes). Leukotrienes are “slow reacting substances of anaphylaxis” and have been named A, B, C, D, and E leukotrienes and these leukotrienes subtypes play a crucial role in asthma.
Phospholipases are targets of a variety of body system modulating agents one of which is endothelin, an endothelium derived vasoactive polypeptide which is the most potent vasoconstrictor identified to date. This protein modulates vascular smooth muscle tone as well as participating in the long term control of the cell cycle involved in the chronic remodeling of the vascular tree. Additionally, endothelin interacts with phospholipases via guanine nucleotide regulatory proteins using a common guanine nucleotide modulating machinery. Thus, phospholipases are an important component of the complex array of effectors which regulate the response of organs, such as the heart, to disease states and injury recovery.
Given the importance of such phospholipases in the regulation of lipid metabolism, signal transduction, and cell cycle control, there exists a need to identify novel phospholipases which function as modulators in these processes such as the suppression of inflammation and oncogenesis and whose aberrant function can result in disorders arising from improper signal transduction such as cancer, inappropriate levels of phospholipase metabolites which mediate the anaphalactic response in respiratory disorders such as asthma, as well as improper responses to disease states such as chronic heart failure which direct tissues into a remodeling paradigm. Further, phospholipases may be potential drug target candidates in a variety of disease areas, including anticancer drugs, cardiovascular drugs, and anti-inflammatory drugs.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules, refered herein as “Cardiovascular System Associated Phospholipase” (“CSAPL”) proteins. The CSAPL nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cardiac cellular processes; lipid metabolism, e.g., release of arachidonic acid from cell membranes; inflammatory response. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding CSAPL proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of CSAPL-encoding nucleic acids.
In one embodiment, a CSAPL nucleic acid molecule of the invention is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more homologous to a nucleotide sequence (e.g., to the entire length of the nucleotide sequence) including SEQ ID NO:1, SEQ ID NO:3 or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or SEQ ID NO:3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-690 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In another preferred embodiment, the nucleic acid molecule comprises a fragment of at least 594 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or a complement thereof In another embodiment, an CSAPL nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, an CSAPL nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 30%, 35%, 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human CSAPL. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2. In yet another preferred embodiment, the nucleic acid molecule is at least 594 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 594 nucleotides in length and encodes a protein having an CSAPL activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably CSAPL nucleic acid molecules, which specifically detect CSAPL nucleic acid molecules relative to nucleic acid molecules encoding non-CSAPL proteins. For example, in one embodiment, such a nucleic acid molecule is at least 350, 400, 450, 500, 550, 593, 600, 650, 700, 750, or 800 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof. In a particularly preferred embodiment, the nucleic acid molecule comprises a fragment o
DeConti, Jr. Giulio A.
Lahive & Cockfield LLP
Mandragouras Amy E.
Millennium Pharmaceuticals Inc.
Saidha Tekchand
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