Cholesterol esterase action on human high density lipoproteins and inhibition studies: detection by MALDI-TOF MS.
The modification of lipoproteins by lipolytic enzymes such as cholesterol esterase (CEase) is assumed to play an important role in the pathogenesis of atherosclerosis. However, details of the activation and inhibition of CEase are still unknown. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to investigate the extracts of human lipoproteins after treatment with CEase and to monitor the effects of the inhibitor 2-(diethylamino)-6,7-dihydro-4H,5H-cyclopenta[4,5] thieno[2,3-d][1,3]oxazin-4-one (DOT-3). This approach has the advantage that all lipid classes can be independently detected; therefore, conclusions on the mechanism of the applied enzyme are possible. Besides the expected decrease of cholesteryl esters (CEs) in HDL, a significantly enhanced content of lysophosphatidylcholine (LPC) was also detected, confirming the broad substrate specificity of CEase. It was also demonstrated that DOT-3 significantly inhibited the CEase-catalyzed cleavage of CEs in HDL. Phospholipid (PL) vesicles prepared from phosphatidylcholine (PC) or PC and cholesteryl linoleate were treated with CEase, and the changes in lipid composition were investigated. From the analysis of the generated LPC species in HDL and in the isolated lipid mixtures, it is evident that CEase catalyzes the cleavage of the fatty acid residues in both the sn-1 and sn-2 positions of the PLs. These effects are obvious in the absence as well as in the presence of detergents.
Zschörnig
Olaf
O
University of Leipzig, Medical Faculty, Institute of Medical Physics and Biophysics, D-04107 Leipzig, Germany. zsco@medizin.uni-leipzig.de
Pietsch
Markus
M
Süss
Rosemarie
R
Schiller
Jürgen
J
Gütschow
Michael
M
D002384:Catalysis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D008074:Lipoproteins / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D002787:Sterol Esterase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D002787:Sterol Esterase