Identification, cloning, expression, and a purification of three novel human calcium-independent phospholipase a2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities

Abstract

Isolated novel and purified and characterized phospholipase A 2 , referred to herein as calcium-independent phospholipase A 2 zeta (iPLA 2 zeta) having SEQ. ID. NO: 2 (See FIG. 1 ), and nucleic acid sequence (SEQ. ID. NO: 4), and calcium-independent phospholipase A 2 eta (iPLA 2 eta) having SEQ. ID. NO: 3 (See FIG. 1 ), and nucleic acid sequences (SEQ. ID. NO: 5). For the first time herein, these novel enzymes have been isolated and characterized and are involved in the catalysis, synthesis and hydrolysis of lipids in a living mammalian cell. Moreover, these enzymes iPLA 2 zeta and iPLA 2 eta through the process of transesterification can catalyze the net anabolic synthesis of triglycerides through a variety of metabolic precursors (e.g. monoacylglycerol, diacylglycerol and acyl CoA).

1 . An isolated, purified, and characterized human calcium-independent phospholipase A 2  (iPLA 2 ) protein comprising a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 1. 
     
     
         2 . The protein of  claim 1  wherein the polypeptide has a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:11. 
     
     
         3 . The protein of  claim 2  wherein the polypeptide has a histidine tag. 
     
     
         4 . The protein of  claim 3  wherein the histidine tag comprises from 4 to 12 histidine residues. 
     
     
         5 . The protein of  claim 1  wherein the polypeptide is an iPLA 2 epsilon polypeptide having a sequence shown in SEQ ID NO: 1. 
     
     
         6 . The protein of  claim 1  wherein the polypeptide is an iPLA 2 zeta polypeptide having a sequence shown in SEQ ID NO: 2. 
     
     
         7 . The protein of  claim 1  wherein the polypeptide is an iPLA 2 eta polypeptide having a sequence shown in SEQ ID NO: 3. 
     
     
         8 . An isolated and characterized human gene comprising a calcium-independent phospholipase A 2  (iPLA 2 ) polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 10. 
     
     
         9 . The gene of  claim 8  wherein the polynucleotide is an iPLA 2 zeta polynucleotide having a sequence shown in SEQ ID NO: 4. 
     
     
         10 . The gene of  claim 8  wherein the polynucleotide is an iPLA 2 eta polynucleotide having a sequence shown in SEQ ID NO: 5. 
     
     
         11 . The gene of  claim 8  wherein the polynucleotide is an iPLA 2 epsilon polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10. 
     
     
         12 . A genetically engineered expression vector comprising the gene of  claim 8 . 
     
     
         13 . A genetically engineered cell capable of identifying substances which modulate iPLA 2 zeta expression, the genetically engineered cell comprising the gene of  claim 9 . 
     
     
         14 . The genetically engineered cell of  claim 13  further comprising a promoter operably linked to the gene of  claim 9  and a reporter gene. 
     
     
         15 . The genetically engineered cell of  claim 13  wherein the cell is an Sf9 cell. 
     
     
         16 . A genetically engineered cell capable of identifying substances which modulate iPLA 2 eta expression in a living cell, the genetically engineered cell comprising the gene of  claim 10 . 
     
     
         17 . The genetically engineered cell of  claim 16  further comprising a promoter operably linked to the gene of  claim 10  and a reporter gene. 
     
     
         18 . The genetically engineered cell of  claim 16  wherein the cell is an Sf9 cell. 
     
     
         19 . A genetically engineered cell capable of identifying substances which modulate iPLA 2 epsilon expression in a living cell, the genetically engineered cell comprising the gene of  claim 11 . 
     
     
         20 . The genetically engineered cell of  claim 19  wherein the cell is an Sf9 cell.