Purification of recombinant human cPLA2 gamma and identification of C-terminal farnesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis.
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.
Jenkins
Christopher M
CM
Division of Bioorganic Chemistry, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Han
Xianlin
X
Yang
Jingyue
J
Mancuso
David J
DJ
Sims
Harold F
HF
Muslin
Anthony J
AJ
Gross
Richard W
RW
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054513:Group IV Phospholipases A2; D006801:Humans; D006868:Hydrolysis; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010171:Palmitoyl Coenzyme A; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D008245:Lysophospholipase; D010450:Endopeptidases