Purification of recombinant human cPLA2γ and identification of C-terminal famesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis

Christopher M. Jenkins, Xianlin Han, Jingyue Yang, David J. Mancuso, Harold F. Sims, Anthony J. Muslin, Richard W. Gross

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Cytosolic phospholipase A2γ (cPLA2γ) is a calcium-independent, membrane-associated phospholipase A2 that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA2γ containing an N-terminal His tag ((His)6cPLA2γ) 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 μmol min-1 mg-1), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA2γ with [3H]-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 cPLA2γ 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 Cys538-Cys539 bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA2γ to homogeneity and identify cPLA 2γ as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.

Original languageEnglish (US)
Pages (from-to)11798-11807
Number of pages10
JournalBiochemistry
Volume42
Issue number40
DOIs
StatePublished - Oct 14 2003
Externally publishedYes

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Cytosolic Phospholipases A2
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Purification
Processing
Lysophospholipase
Palmitoyl Coenzyme A
Prenylation
Membranes
Sf9 Cells
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Immobilized Enzymes
Column chromatography
Silver Staining
Phospholipases A2
Enzymes
Post Translational Protein Processing
Nickel
Silver
Ionization
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification of recombinant human cPLA2γ and identification of C-terminal famesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis. / Jenkins, Christopher M.; Han, Xianlin; Yang, Jingyue; Mancuso, David J.; Sims, Harold F.; Muslin, Anthony J.; Gross, Richard W.

In: Biochemistry, Vol. 42, No. 40, 14.10.2003, p. 11798-11807.

Research output: Contribution to journalArticle

Jenkins, Christopher M. ; Han, Xianlin ; Yang, Jingyue ; Mancuso, David J. ; Sims, Harold F. ; Muslin, Anthony J. ; Gross, Richard W. / Purification of recombinant human cPLA2γ and identification of C-terminal famesylation, proteolytic processing, and carboxymethylation by MALDI-TOF-TOF analysis. In: Biochemistry. 2003 ; Vol. 42, No. 40. pp. 11798-11807.
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abstract = "Cytosolic phospholipase A2γ (cPLA2γ) is a calcium-independent, membrane-associated phospholipase A2 that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA2γ containing an N-terminal His tag ((His)6cPLA2γ) 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 μmol min-1 mg-1), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA2γ with [3H]-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 cPLA2γ 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 Cys538-Cys539 bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA2γ to homogeneity and identify cPLA 2γ as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.",
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AU - Jenkins, Christopher M.

AU - Han, Xianlin

AU - Yang, Jingyue

AU - Mancuso, David J.

AU - Sims, Harold F.

AU - Muslin, Anthony J.

AU - Gross, Richard W.

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N2 - Cytosolic phospholipase A2γ (cPLA2γ) is a calcium-independent, membrane-associated phospholipase A2 that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA2γ containing an N-terminal His tag ((His)6cPLA2γ) 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 μmol min-1 mg-1), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA2γ with [3H]-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 cPLA2γ 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 Cys538-Cys539 bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA2γ to homogeneity and identify cPLA 2γ as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.

AB - Cytosolic phospholipase A2γ (cPLA2γ) is a calcium-independent, membrane-associated phospholipase A2 that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA2γ containing an N-terminal His tag ((His)6cPLA2γ) 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 μmol min-1 mg-1), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA2γ with [3H]-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 cPLA2γ 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 Cys538-Cys539 bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA2γ to homogeneity and identify cPLA 2γ as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.

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