Cytosolic phospholipase A2-Gamma enzymes and polynucleotides encoding same

Abstract

The invention provides a novel calcium-independent cytosolic phospholipase A 2 -Gamma enzyme, polynucleotides encoding such enzyme and methods for screening unknown compounds for anti-inflammatory activity mediated by the arachidonic acid cascade.

1 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: 
 (a) the nucleotide sequence of SEQ ID NO:1 from nucleotide  312  to nucleotide  1934 ;    (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2;    (c) a nucleotide sequence :encoding a fragment of the amino acid sequence of SEQ ID NO:2 having activity in a mixed micelle assay with 1 palmitoy-2[] 14 C]-arachidonyl-phosphatidylcholine    (d) a nucleotide; sequence capable of hybridizing with the sequence of (a), (b) or (c) which encodes a peptide having activity in a mixed mice ile assay with 1-palmtoyl-2-[ 14 ]-arachidonyl-phosphatidylcholine;    (e) allelic variants of the sequence of (a); and    (f) species homologues of the sequence of (a) or (b)).    
     
     
         2 . An expression vector comprising the polynucleotide of  claim 1  and an expression control sequence.  
     
     
         3 . A host cell transformed with the vector of  claim 2 .  
     
     
         4 . A process for producing phospholipase enzyme, said process comprising: 
 (a) establishing a culture of the host cell of  claim 3  in a suitable culture medium; and    (b) isolating said enzyme from said culture.    
     
     
         5 . A composition comprising a peptide made according to the process of  claim 4 .  
     
     
         6 . A composition comprising a peptide encoded by the polynucleotide of  claim 1 .  
     
     
         7 . A composition comprising a peptide comprising an amino acid sequence selected from the group consisting of: 
 (a) the amino acid sequence of SEQ ID NO:2;    (b) a fragment of the amino acid sequence of SEQ ID NO:2 having activity in a mixed micelle assay with 1-palmitoyl-2-[ 14 C]-arachidonyl-phosphatidylcholine; and    (c) species homologues of (a);    such peptide being substantially free of other mammalian proteins.    
     
     
         8 . A method for identifying an inhibitor of phospholipase activity, said method comprising: 
 (a) combining a phospholipid, a candidate inhibitor compound, and a composition comprising a phospholipase enzyme peptide; and    (b) observing whether said phospholipase enzyme peptide cleaves said phospholipid and releases fatty acid thereby,    wherein said composition is the composition of  claim 7 .    
     
     
         9 . An inhibitor of phospholipase activity identified according to the method of  claim 8 .  
     
     
         10 . A pharmaceutical composition comprising a therapeutically effective amount of the inhibitor of  claim 9  and a pharmaceutically acceptable carrier.  
     
     
         11 . A method of reducing inflammation comprising administering a pharmaceutical composition of  claim 10  to a mammalian subject.  
     
     
         12 . A composition comprising an antibody which binds to the peptide of the composition of  claim 7 .  
     
     
         13 . The composition of  claim 12  wherein said antibody is polyclonal.  
     
     
         14 . The composition of  claim 12  wherein said antibody is monoclonal.  
     
     
         15 . The polynucleotide of  claim 1  comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 312 to nucleotide 1934.  
     
     
         16 . The polynucleotide of  claim 1  comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.  
     
     
         17 . The composition of  claim 7  wherein said protein comprises the amino acid sequence of SEQ ID NO:2.  
     
     
         18 . The composition of  claim 1  wherein said polynucleotide comprises a polynucleotide which encodes the amion acid sequence of SEQ ID NO:2 from amino acid 6 to amino acid 242.  
     
     
         19 . The composition of  claim 1  wherein said polynucleotide comprises a polynucleotide which encodes the amion acid sequence of SEQ ID NO:2 from amino acid 366 to amino acid 535.  
     
     
         20 . The composition of  claim 7  wherein said peptide comprises the amino acid sequence of SEQ ID NO:2 from amino acid 6 to amino acid 242.  
     
     
         21 . The composition of  claim 7  wherein said peptide comprises the amino acid sequence of SEQ ID NO:2 from amino acid 366 to amino acid 535.