Sterol Carrier Protein-2-Facilitated Intermembrane Transfer of Cholesterol- and Phospholipid-Derived Hydroperoxides

Abstract

Sterol carrier protein-2 (SCP-2) facilitates cholesterol (Ch) and phospholipid (PL) transfer/exchange between membranes and appears to play a key role in intracellular lipid trafficking. Whether SCP-2 can also facilitate lipid hydroperoxide (LOOH) transfer between membranes and thereby potentially enhance dissemination of peroxidative damage was examined in this study. Transfer kinetics of photochemically generated cholesterol hydroperoxide (ChOOH) species (5alpha-OOH, 6alpha/6beta-OOH, 7alpha/7beta-OOH) and phospholipid hydroperoxide (PLOOH) families (PCOOH, PEOOH, PSOOH) were determined, using HPLC with electrochemical detection for peroxide analysis. LOOH donor/acceptor pairs employed in transfer experiments included (i) all liposomes (e.g., agglutinable SUVs/ nonagglutinable LUVs); (ii) photoperoxidized erythrocyte ghosts/SUVs or vice versa; and (iii) SUVs/mitochondria. In a SUV/ghost system at 37 degrees C, the rate constant for total ChOOH spontaneous transfer was approximately 8 times greater than that for unoxidized Ch. Purified bovine liver and human recombinant SCP-2 exhibited an identical ability to stimulate overall ChOOH transfer, 0.5 unit/mL (based on [(14)C]Ch transfer) increasing the first-order rate constant (k) approximately 7-fold. SCP-2-enhanced translocation of individual ChOOHs increased with increasing hydrophilicity in the following order: 6beta-OOH < 6alpha-OOH < 5alpha-OOH < 7alpha/7beta-OOH. Likewise, SCP-2 stimulated PCOOH, PEOOH, or PSOOH transfer approximately 6-fold, but the net k was 1/5 that of 5alpha-OOH and 1/10 that of 7alpha/7beta-OOH. Donor membrane properties favoring SCP-2-enhanced LOOH transfer included (i) increasing PL unsaturation and (ii) increasing net negative charge imposed by phosphatidylserine. Cytotoxic relevance was demonstrated by showing that SCP-2 accelerates 7alpha-OOH transfer from SUVs to isolated mitochondria and that this enhances peroxide-induced loss of the mitochondrial membrane potential. On the basis of these findings, we postulate that SCP-2, by trafficking ChOOHs and PLOOHs in addition to parent lipids, might exacerbate cell injury under oxidative stress conditions.

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

“…This is close to the calculated mass (3676.13 Da) for a biotinylated AviTag extension proteolytically cleaved at lysine 122, the penultimate C-terminal residue of human SCP-2 [11,25]. Using Ellman's reagent for thiol determination [37], we also showed that a bSCP-2 preparation contained 0.85 ± 0.05 mol cysteine/mol protein, which agrees with evidence [14,38] that there is a single accessible cysteine residue in SCP-2. Collectively, the properties described for bSCP-2 confirmed that it was the protein we expected.…”

“…The low level of CHAPS (0.001%, w/v) used in running buffers also proved important for minimizing lipid losses on plastic tubing and other surfaces. In addition, EDTA and DFO were included to prevent any copper-or iron-catalyzed oxidation of bSCP-2 or lipid analytes [14].…”

“…The transfer activities of recombinant SCP-2 and bSCP-2 were determined by measuring the kinetics of [ 14 C]cholesterol translocation from small unilamellar vesicles (50 nm SUVs) to red cell ghost membranes [14,27,28]. Departure from SUV donors is the rate limiting step in the transfer process [27].…”

“…Assay mixtures (2.0 ml starting volume) typically contained 0.6 mM total ghost lipid, 0.06 mM total SUV lipid, and 5-10 μM SCP-2 or bSCP-2 in PBS/0.1 mM EDTA/0.1 mM DFO (pH 7.4). The chelators were included to minimize any metal ion-catalyzed lipid or protein oxidation [14,27,28]. At various times during transfer incubation at 37 °C, a 0.1 ml sample was removed, mixed with ice-cold PBS/EDTA/DFO, and centrifuged (16000 × g, 10 min), after which 0.1 ml of the SUV-containing supernatant was analyzed by scintillation counting.…”

“…More recently, it was shown that SCP-2 can also enhance intermembrane transfer of peroxidized cholesterol and phospholipids, thereby expanding the damaging prooxidant range of these species [14,15]. With regard to mechanism of transfer enhancement, it appears that SCP-2 actually binds and transports donor membrane lipids rather than facilitates their departure without physically interacting with them [7].…”