Low-density lipoprotein has an enormous capacity to bind (E)-4-hydroxynon-2-enal (HNE): detection and characterization of lysyl and histidyl adducts containing multiple molecules of HNE.
( E)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer's disease. Tritium- and deuterium-labeled HNE ( d 4-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of d 4-HNE and d 0-HNE with N-acetylhistidine, N-acetyl-Gly-Lys-OMe, or N-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry.
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106-7078, USA.
Suresh P
SP
Annangudi
Yijun
Y
Deng
Xiaorong
X
Gu
Wujuan
W
Zhang
John W
JW
Crabb
Robert G
RG
Salomon
D000111:Acetylcysteine / Q000737:chemistry / Q000378:metabolism; D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors
D000447:Aldehydes; D003432:Cross-Linking Reagents; D008077:Lipoproteins, LDL; D006639:Histidine; C027576:4-hydroxy-2-nonenal; D008239:Lysine; C023495:N-acetylhistidine; D000111:Acetylcysteine