A simple , rapid , and sensitive fluorescence assay for microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP’s lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists. This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening. —Athar, H. , J. Iqbal, X-C. Jiang, and M. M. Hussain. A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein. J. Lipid Res. 2004. 45: 764–772. Supplementary key words lipoprotein assembly • cholesteryl esters • phospholipids • triacylglycerol • apolipoprotein B Microsomal triglyceride transfer protein (MTP) is a dedicated chaperone that is required for the assembly of apolipoprotein B (apoB) lipoproteins [for reviews, see refs. (1–6)]. It is believed that MTP transfers lipids to nascent apoB in the endoplasmic reticulum and renders it secretion-competent by forming primordial lipoprotein particles [for reviews, see refs. (1–9)]. The importance of MTP’s lipid transfer activity in apoB secretion has been established by three independent approaches. First, mutations in MTP have been correlated with the absence of apoB lipoproteins in abetalipoproteinemia (10, 11). Second, antagonists that inhibit MTP’s lipid transfer activity in vitro have been shown to decrease apoB secretion in vivo (12–14). Third, the coexpression of MTP with apoB has been demonstrated to facilitate apoB secretion in cells that do not express apoB and MTP (15, 16). In addition to its lipid transfer activity, MTP is known to physically interact with apoB [for reviews, see refs. (1, 2)]. Recently, MTP was implicated in the import of triacylglycerols (TAGs) from cytosol to the lumen of the endoplasmic reticulum (17–19). Thus, it appears that MTP is a multifunctional protein (2) that plays a crucial role in the transport of TAG within intracellular organelles and in its secretion out of the cells. MTP was identified and purified by Wetterau and Zilversmit (20, 21) based on a radioisotope assay. In this assay, radiolabeled TAGs are incorporated into donor vesicles consisting of phosphatidylcholine (PC) and cardiolipin. These vesicles are incubated with acceptor vesicles in the presence of MTP. After 1–3 h of incubation, the cardiolipin-containing donor vesicles are allowed to bind to DE52 and removed by centrifugation. Radioactivity remaining in the supernatant is quantified by scintillation counting. This procedure is labor-intensive and time-consuming. Negatively charged lipids, such as cardiolipin, are known to inhibit the lipid transfer activity of MTP (22). Because of the multiple steps involved in this procedure, it is difficult to automate. Thus, it might be advantageous to have a simple, one-step procedure to measure MTP activity. Several pharmaceutical companies have identified antagonists that inhibit MTP activity (12–14, 23). A general approach taken by these companies was to identify compounds that decrease apoB secretion by HepG2 cells and then to determine their ability to inhibit MTP activity [for reviews, see refs. (3, 23)]. The primary screening involvAbbreviations: apoB, apolipoprotein B; CE, cholesteryl ester; PC, phosphatidylcholine; PL, phospholipid; MTP, microsomal triglyceride transfer protein; TAG, triacylglycerol. 1 To whom correspondence should be addressed. e-mail: mahmood.hussain@downstate.edu Manuscript received 5 September 2003, in revised form 16 December 2003, and in re-revised form 20 January 2004. Published, JLR Papers in Press, February 1, 2004. DOI 10.1194/jlr.D300026-JLR200 by gest, on S etem er 0, 2019 w w w . j.org D ow nladed fom