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Identification of the sites in opsin modified by photoactivated azido[125I]iodobenzene.

ABSTRACT: Opsin labelled with photoactivated 1-azido-4-[125I]iodobenzene was proteolysed in situ with Staphylococcus aureus V8 proteinase to yield two radioactive membrane-bound fragments. These were separated, cleaved with CNBr and the resultant peptides sequenced in order to locate the radiolabelled residues. In the whole molecule, there was clear evidence for modification of at least 20 sites, identified as derivatives of cysteine, tryptophan, tyrosine, histidine and lysine residues. The probe primary reacted, therefore, with nucleophilic substituents. The positions of the modified sites relative to the confines of the phospholipid bilayer were consistent with all other studies on the disposition of the polypeptide chain. The location of these sites substantiated an earlier suggestion that not all the transmembrane segments should be regarded as continuous regular alpha-helices.

SUBMITTER: Davison MD

PROVIDER: S-EPMC1146852 | biostudies-other | 1986 Jun

REPOSITORIES: biostudies-other

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Project description:Apolipoprotein E (apoE), an antiatherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins, in addition to playing a role in promoting macrophage cholesterol efflux in atherosclerotic lesions. The objective of this study is to examine the effect of acrolein modification on the structure and function of rat apoE and to determine the sites and nature of modification by mass spectrometry. Acrolein is a highly reactive aldehyde, which is generated endogenously as one of the products of lipid peroxidation and is present in the environment in pollutants such as tobacco smoke and heated oils. In initial studies, acrolein-modified apoE was identified by immunoprecipitation using an acrolein-lysine specific antibody in the plasma of 10-week old male rats that were exposed to filtered air (FA) or low doses of environmental tobacco smoke (ETS). While both groups displayed acrolein-modified apoE in the lipoprotein fraction, the ETS group had higher levels in the lipid-free fraction compared with the FA group. This observation provided the rationale to further investigate the effect of acrolein modification on rat apoE at a molecular level. Treatment of recombinant rat apoE with a 10-fold molar excess of acrolein resulted in (i) a significant decrease in lipid-binding and cholesterol efflux abilities, (ii) impairment in the LDLr- and heparin-binding capabilities, and (iii) significant alterations in the overall stability of the protein. The disruption in the functional abilities is attributed directly or indirectly to acrolein modification yielding an aldimine adduct at K149 and K155 (+38); a propanal adduct at K135 and K138 (+56); an N(?)-(3-methylpyridinium)lysine (MP-lysine) at K64, K67, and K254 (+76), and an N(?)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) derivative at position K68 (+94), as determined by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). The loss of function may also be attributed to alterations in the overall fold of the protein as noted by changes in the guanidine HCl-induced unfolding pattern and to protein cross-linking. Overall, disruption of the structural and functional integrity of apoE by oxidative modification of essential lysine residues by acrolein is expected to affect its role in maintaining plasma cholesterol homeostasis and lead to dysregulation in lipid metabolism.

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Identification of the sites in opsin modified by photoactivated azido[125I]iodobenzene.

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