Purification of recombinant human cPLA2 gamma and identification of C-terminal farnesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis.

Abstract

Cytosolic phospholipase A(2)gamma (cPLA(2)gamma) is a calcium-independent, membrane-associated phospholipase A(2) that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA(2)gamma containing an N-terminal His tag ((His)(6)cPLA(2)gamma) in Sf9 cells and quantitatively solubilized and purified the enzyme by sequential immobilized metal affinity and Mono Q column chromatographies. The final preparation appeared as a single 61 kDa band after SDS-PAGE/silver-staining, possessed high lysophospholipase activity (50 micromol min(-1) mg(-1)), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA(2)gamma with [(3)H]-mevalonolactone in the absence of statins and subsequent cleavage of prenyl groups with Raney nickel revealed that the enzyme is only farnesylated and is not geranylgeranylated. Analysis of CNBr-digested cPLA(2)gamma by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) mass spectrometry demonstrated the presence of a farnesyl moiety at Cys-538, cleavage of the Cys(538)-Cys(539) bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA(2)gamma to homogeneity and identify cPLA(2)gamma as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.