Assay blanks containing these reagents were used when calculating CETP transfer activity

Assay blanks containing these reagents were used when calculating CETP transfer activity. == Chemical analyses == Protein was quantified by a modification of the Lowry et al. of TG toward HDL. For hamster CETP this process was not equimolar but resulted in VU 0240551 a net flow of lipid (TG) into HDL. When assayed for the ability to transfer lipid to an acceptor particle lacking CE and TG, monkey and hamster CETPs were most effective, although all CETP species were able to promote this one-way movement of neutral lipid. We conclude that CETPs from human, monkey, rabbit, and hamster are not functionally comparative. Most unique was hamster CETP, which strongly prefers TG as a substrate and promotes the net flow of lipid from VLDL to HDL. Keywords:triglyceride, unidirectional lipid transfer, thimerosal inhibition, TP2 antibody, very low density lipoprotein, high density lipoprotein Cholesteryl ester (CE) transfer activity was first observed in human plasma by Nichols and Smith (1). Zilversmit, Hughes, and Balmer (2) were the first to identify a plasma protein responsible for this activity, and subsequent study showed that this protein, now called cholesteryl ester transfer protein (CETP), is responsible for all CE and triglyceride (TG) transfer activity in human plasma (3,4). Although absent in the plasma of common laboratory animals such as mice and rats, genome sequencing has identified CETP homologs in more than 20 species. In species where CETP activity has been experimentally verified, this is typically based solely on the presence of CE transfer activity in plasma. Mutagenesis studies have shown that minor changes in the structure of human CETP can have profound effects on its ability to bind lipoproteins or selectively alter its ability to utilize TG and/or CE as a transfer substrate (5,6). Given this, it seems likely that CETP from common experimental models such as hamster and rabbit, which have only 80% identity to human CETP at the amino acid level, may have unique lipid transfer properties. The purpose of this study was to characterize the lipid transfer properties of CETP from three species (monkey, rabbit, and hamster) and compare them to human CETP. We have determined the relative preference of each CETP for CE and TG as a substrate and assessed their abilities to promote net TG and CE transfer between lipoproteins. Such processes are central to CETP’s role in facilitating lipoprotein metabolism. Additionally, because there is growing evidence that intracellular CETP influences cellular lipid metabolism (79), perhaps by facilitating the formation of lipid droplets (10), we investigated the capacity of all four CETP species to mediate net unidirectional lipid transfer. We show species-specific differences in the lipid transfer properties of CETPs in terms of their substrate preference and their ability to promote net lipid transfer between lipoproteins, and between lipoproteins and model membrane surfaces. == METHODS == == Materials == Mouse monoclonal antibody to human CETP (TP2) was purchased from the Ottawa Heart Institute (Ottawa, Ontario, Canada). [9,10-3H(n)]triolein and cholesteryl [1-14C]oleate were from PerkinElmer (Waltham, MA). Thimerosal and all buffers, salts, and detergents were purchased from Sigma (St. Louis, MO). == CETP preparations == Native plasma-derived human CETP (nHuman) was purified as previously described (3). A vector made up of human CETP cDNA (M30185.1) was purchased from Open Biosystems (Pittsburgh, PA) and subcloned into pCDNA3. The cDNAs for rabbit (Oryctolagus cuniculus,XM_002711536, 1,482 bp), monkey (Macaca fascicularis,M86343, 1,479 bp), and hamster (Cricetulus griseus,XM_003503614, 1,476 bp) CETPs were synthesized (GenScript, VU 0240551 Piscataway, NJ), subcloned into pCDNA3, and sequence verified. All constructs contained native start and stop codons including the coding sequence for a 17 amino acid signal peptide. pCDNA3 constructs (20 g/ VU 0240551 106cells) were transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies, Grand Island, NY). After overnight incubation in serum-containing medium, cells were washed and incubated in Opti-MEM (Life Technologies) for 48 h. Conditioned medium made up of secreted recombinant CETP (rCETP) was collected, centrifuged to remove cell debris, and supplemented with 0.05% BSA to stabilize CETP activity. == Liposome and lipoprotein preparation == Egg phosphatidylcholine (PC) (Avanti Polar Lipids, Inc., Rabbit polyclonal to ND2 Alabaster, AL) liposomes made up of 25 mol% cholesterol plus either 1 mol% cholesteryl oleate, 1 mol% triolein (Nu-Chek Prep, Inc., Elysian, MN), 0.5 mol% of both cholesteryl oleate and triolein, or no cholesteryl oleate or triolein, were prepared by cholate dialysis (3,11). Following extensive dialysis versus 10 mM Tris-HCl, 100 mM NaCl, and 0.02% EDTA (pH 7.4), multilamellar liposomes were VU 0240551 removed by centrifugation (35,600g, 60 min at 4C). Unilamellar liposomes were supplemented with 0.35% BSA and stored at 4C. Radiolabeled liposomes were prepared in the same way, with tracer quantities of3H-TG and14C-CE substituting for unlabeled TG and CE. Fresh plasma, obtained from the Cleveland Clinic Blood Lender, was fractionated by sequential density ultracentrifugation (12) to yield VLDL (d < 1.006 g/ml), LDL (1.019 < d > 1.063 g/ml), and HDL (1.063 < d > 1.21 g/ml). Doubled-labeled (3H-TG and14C-CE) lipoproteins were prepared by two methods. Plasma lipoproteins.