Mass spectrometry analysis of modified bee venom phospholipase A2 in correlation with increased activity

S. Pasha, A.K. Mahalka, A. Reis, A.R. Pitt, P.K.J. Kinnunen, C.M. Spickett

Research output: Contribution to journalMeeting abstract

Abstract

Oxidative stress and inflammatory conditions result in the formation of carbonyl-containing lipid derivatives. These reactive carbonyl groups can interact with amine groups on proteins via the formation of Schiff base adducts, modifications that can lead to changes to the proteins structure and influence overall activity. Phospholipase A2 (PLA2) modified with 1-palmitoyl-2-(9'oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) forms Schiff bases that trigger an increase in protein activity, possibly through changes in enzyme conformation, protein-protein interaction, or peptide-protein heteroligomerisation (Code et al., 2010). However, the site of carbonyl modification that is responsible for the change in PLA2 activity remains unknown, so further studies were required to address this question. In this study, PLA2 from Apis mellifera modified with physiological concentrations of PoxnoPC (15μM) was analysed by mass spectrometry. Fragmentation data was used to identify the nature and sites of modification in the modified PLA2. No modifications containing PoxnoPC were found in the protein; however, modifications containing 9-oxononanoic acid (ON) were found on a number of lysine residues including Lys14 and Lys66. These residues are located in the vicinity of the catalytic site, the calcium binding site, and the carboxyl terminal end loop of the PLA2 structure, suggesting that PLA2 catalysed the breakdown of PoxnoPC to ON, although it is not known whether this occurred before or after adduct formation. The modifications of lysine residues found could account for the generation of highly reactive protofibril intermediates that increase the enzyme's overall activity (Code et al., 2010) which supports the need for detailed quantitative mass spectrometry and 3D mapping studies to complement these findings. References: Code, C. et al., 2010. Biochim Biophys Acta 1798 (8); 1593-1600. Acknowledgments: AR was supported by a Marie Curie Intra-European Fellowship within the 7th European Community Framework Program (IEF 255076). This work is supported by the EPSRC (EP/I017887) and Aston University.
LanguageEnglish
Article number0578
PagesS213-S214
Number of pages2
JournalFree Radical Biology and Medicine
Volume53
Issue numberSupplement 1
DOIs
Publication statusE-pub ahead of print - 2 Sep 2012
EventSociety for Free Radical Research International 16th Biennial Meeting - Imperial College London, London, United Kingdom
Duration: 6 Sep 20129 Sep 2012

Fingerprint

Bee Venoms
Phospholipases A2
Mass spectrometry
Mass Spectrometry
Proteins
Schiff Bases
Lysine
Protein Conformation
Bees
European Union
Enzymes
Oxidative stress
Enzyme activity
Amines
Catalytic Domain
Oxidative Stress
Conformations
Binding Sites
Calcium
Lipids

Cite this

Pasha, S. ; Mahalka, A.K. ; Reis, A. ; Pitt, A.R. ; Kinnunen, P.K.J. ; Spickett, C.M. / Mass spectrometry analysis of modified bee venom phospholipase A2 in correlation with increased activity. In: Free Radical Biology and Medicine. 2012 ; Vol. 53, No. Supplement 1. pp. S213-S214.
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abstract = "Oxidative stress and inflammatory conditions result in the formation of carbonyl-containing lipid derivatives. These reactive carbonyl groups can interact with amine groups on proteins via the formation of Schiff base adducts, modifications that can lead to changes to the proteins structure and influence overall activity. Phospholipase A2 (PLA2) modified with 1-palmitoyl-2-(9'oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) forms Schiff bases that trigger an increase in protein activity, possibly through changes in enzyme conformation, protein-protein interaction, or peptide-protein heteroligomerisation (Code et al., 2010). However, the site of carbonyl modification that is responsible for the change in PLA2 activity remains unknown, so further studies were required to address this question. In this study, PLA2 from Apis mellifera modified with physiological concentrations of PoxnoPC (15μM) was analysed by mass spectrometry. Fragmentation data was used to identify the nature and sites of modification in the modified PLA2. No modifications containing PoxnoPC were found in the protein; however, modifications containing 9-oxononanoic acid (ON) were found on a number of lysine residues including Lys14 and Lys66. These residues are located in the vicinity of the catalytic site, the calcium binding site, and the carboxyl terminal end loop of the PLA2 structure, suggesting that PLA2 catalysed the breakdown of PoxnoPC to ON, although it is not known whether this occurred before or after adduct formation. The modifications of lysine residues found could account for the generation of highly reactive protofibril intermediates that increase the enzyme's overall activity (Code et al., 2010) which supports the need for detailed quantitative mass spectrometry and 3D mapping studies to complement these findings. References: Code, C. et al., 2010. Biochim Biophys Acta 1798 (8); 1593-1600. Acknowledgments: AR was supported by a Marie Curie Intra-European Fellowship within the 7th European Community Framework Program (IEF 255076). This work is supported by the EPSRC (EP/I017887) and Aston University.",
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Mass spectrometry analysis of modified bee venom phospholipase A2 in correlation with increased activity. / Pasha, S.; Mahalka, A.K.; Reis, A.; Pitt, A.R.; Kinnunen, P.K.J.; Spickett, C.M.

In: Free Radical Biology and Medicine, Vol. 53, No. Supplement 1, 0578, 02.09.2012, p. S213-S214.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Mass spectrometry analysis of modified bee venom phospholipase A2 in correlation with increased activity

AU - Pasha, S.

AU - Mahalka, A.K.

AU - Reis, A.

AU - Pitt, A.R.

AU - Kinnunen, P.K.J.

AU - Spickett, C.M.

PY - 2012/9/2

Y1 - 2012/9/2

N2 - Oxidative stress and inflammatory conditions result in the formation of carbonyl-containing lipid derivatives. These reactive carbonyl groups can interact with amine groups on proteins via the formation of Schiff base adducts, modifications that can lead to changes to the proteins structure and influence overall activity. Phospholipase A2 (PLA2) modified with 1-palmitoyl-2-(9'oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) forms Schiff bases that trigger an increase in protein activity, possibly through changes in enzyme conformation, protein-protein interaction, or peptide-protein heteroligomerisation (Code et al., 2010). However, the site of carbonyl modification that is responsible for the change in PLA2 activity remains unknown, so further studies were required to address this question. In this study, PLA2 from Apis mellifera modified with physiological concentrations of PoxnoPC (15μM) was analysed by mass spectrometry. Fragmentation data was used to identify the nature and sites of modification in the modified PLA2. No modifications containing PoxnoPC were found in the protein; however, modifications containing 9-oxononanoic acid (ON) were found on a number of lysine residues including Lys14 and Lys66. These residues are located in the vicinity of the catalytic site, the calcium binding site, and the carboxyl terminal end loop of the PLA2 structure, suggesting that PLA2 catalysed the breakdown of PoxnoPC to ON, although it is not known whether this occurred before or after adduct formation. The modifications of lysine residues found could account for the generation of highly reactive protofibril intermediates that increase the enzyme's overall activity (Code et al., 2010) which supports the need for detailed quantitative mass spectrometry and 3D mapping studies to complement these findings. References: Code, C. et al., 2010. Biochim Biophys Acta 1798 (8); 1593-1600. Acknowledgments: AR was supported by a Marie Curie Intra-European Fellowship within the 7th European Community Framework Program (IEF 255076). This work is supported by the EPSRC (EP/I017887) and Aston University.

AB - Oxidative stress and inflammatory conditions result in the formation of carbonyl-containing lipid derivatives. These reactive carbonyl groups can interact with amine groups on proteins via the formation of Schiff base adducts, modifications that can lead to changes to the proteins structure and influence overall activity. Phospholipase A2 (PLA2) modified with 1-palmitoyl-2-(9'oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) forms Schiff bases that trigger an increase in protein activity, possibly through changes in enzyme conformation, protein-protein interaction, or peptide-protein heteroligomerisation (Code et al., 2010). However, the site of carbonyl modification that is responsible for the change in PLA2 activity remains unknown, so further studies were required to address this question. In this study, PLA2 from Apis mellifera modified with physiological concentrations of PoxnoPC (15μM) was analysed by mass spectrometry. Fragmentation data was used to identify the nature and sites of modification in the modified PLA2. No modifications containing PoxnoPC were found in the protein; however, modifications containing 9-oxononanoic acid (ON) were found on a number of lysine residues including Lys14 and Lys66. These residues are located in the vicinity of the catalytic site, the calcium binding site, and the carboxyl terminal end loop of the PLA2 structure, suggesting that PLA2 catalysed the breakdown of PoxnoPC to ON, although it is not known whether this occurred before or after adduct formation. The modifications of lysine residues found could account for the generation of highly reactive protofibril intermediates that increase the enzyme's overall activity (Code et al., 2010) which supports the need for detailed quantitative mass spectrometry and 3D mapping studies to complement these findings. References: Code, C. et al., 2010. Biochim Biophys Acta 1798 (8); 1593-1600. Acknowledgments: AR was supported by a Marie Curie Intra-European Fellowship within the 7th European Community Framework Program (IEF 255076). This work is supported by the EPSRC (EP/I017887) and Aston University.

U2 - 10.1016/j.freeradbiomed.2012.08.447

DO - 10.1016/j.freeradbiomed.2012.08.447

M3 - Meeting abstract

VL - 53

SP - S213-S214

JO - Free Radical Biology and Medicine

T2 - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

IS - Supplement 1

M1 - 0578

ER -