Lipid Transfer Inhibitor Protein Research Articles

Differential Interaction of the Human Cholesteryl Ester Transfer Protein with Plasma High Density Lipoproteins (HDLs) from Humans, Control Mice, and Transgenic Mice to Human HDL Apolipoproteins

Plasma high density lipoproteins (HDLs) from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoprotein A-II (HuAIITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with the human cholesteryl ester transfer protein (CETP) was studied. Whereas cholesteryl ester transfer rates were gradually enhanced by the addition of moderate amounts of HDL from the different sources, striking differences appeared when HDL levels kept increasing beyond a maximal transfer value. Indeed, while a plateau value corresponding to maximal CETP activity was maintained when raising the concentration of HuAITg HDL and HuAIAIITg HDL, inhibitions could be observed with the highest levels of human, control mouse, and HuAIITg mouse HDL. The concentration-dependent inhibition of CETP activity could be reproduced by the addition of delipidated HDL apolipoproteins from control mice, but it was abolished by a 1-h preheating treatment at 56 degrees C. In contrast, no significant inhibition of CETP activity was observed with the delipidated protein moiety of HuAITg HDL, and cholesteryl ester transfer rates remained unchanged before and after a 1-h, 56 degrees C preheating step. Finally, the CETP-mediated transfer of radiolabeled cholesteryl esters from human low density lipoprotein to human HDL was significantly higher in the presence of lipoprotein-deficient plasma from HuAITg mice than in the presence of lipoprotein-deficient plasma from control mice. Interestingly, cholesteryl ester transfer rates measured with both control and HuAITg lipoprotein-deficient plasmas became remarkably similar following a 1-h, 56 degrees C preheating treatment. It is concluded that human, control mouse, and HuAIITg mouse HDL contain a heat-labile lipid transfer inhibitory activity that is absent from HDL of HuAITg and HuAIAIITg mice. Alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitory activities.

Enhanced detection of lipid transfer inhibitor protein activity by an assay involving only low density lipoprotein.

Lipid transfer inhibitor protein (LTIP) activity has been typically quantitated by its ability to suppress lipid transfer protein-mediated lipid movement between low density lipoprotein (LDL) and high density lipoprotein (HDL). In an attempt to establish an LTIP activity assay that is more sensitive, we have exploited the reported preference of the inhibitor protein to interact with LDL. A lipid transfer assay was established that involves LDL as both the donor and the acceptor; LDL in one of these two pools was biotinylated to facilitate its removal with immobilized avidin. Compared to the standard LDL to HDL assay, LTIP inhibited lipid transfer from radiolabeled LDL to biotin-LDL 7-fold more. In the absence of LTIP, lipid transfer activity was the same in both assays. An added benefit of this assay was the near linearity (up to 85%) of the inhibitory response, in contrast to the highly curvilinear response of LTIP in LDL to HDL transfer assays. The high sensitivity of the LDL to biotin-LDL transfer assay in measuring LTIP activity could not be duplicated by other transfer assays including assays containing only HDL (HDL to biotin-HDL), assays between liposomes and LDL, or assays between LDL and HDL where the concentration of lipoproteins was reduced 10-fold. Thus, LTIP activity is most effectively measured in homologous lipid transfer assays involving only LDL (and its biotin derivative). This increased sensitivity to LTIP suggests that the inhibitor binds more avidly to the LDL surface than does lipid transfer protein.

Regulation of lipid transfer between lipoproteins by an endogenous plasma protein: selective inhibition among lipoprotein classes.

Lipid transfer protein (LTP) remodels plasma lipoproteins by promoting mass transfers of cholesteryl ester (CE) and triglyceride between lipoproteins. We have investigated the capacity of an additional plasma protein, lipid transfer inhibitor protein (LTIP) to modify the functional activity of LTP in a complex mixture of lipoproteins. Transfer assays containing isolated LTP, LTIP, and the three major lipoprotein classes, and assays with intact human plasma supplemented with exogenous LTIP were used. In both assays, the inhibition of CE transfer by LTIP varied markedly depending on the lipoproteins involved and was dependent on LTIP concentration. Inhibition of lipid transfer between a given pair of lipoproteins was similar. However, between lipoprotein pairs the extent of inhibition was very different, varying up to 7-fold. Inhibition followed the order of very low density lipoprotein (VLDL)-low density lipoprotein (LDL) transfers > LDL-high density lipoprotein (HDL) transfers > VLDL-HDL transfers. Consistent with the preferential inhibition of transfer events involving LDL, LTIP was shown by gel filtration studies to associate primarily with LDL in plasma. The addition of LTIP to native plasma stimulated the LTP-mediated efflux of CE from HDL to VLDL; this occurred at the expense of LDL CE depletion. Thus, LTIP alters the pattern of lipid transfer reactions in plasma by uniquely affecting the individual transfer events mediated by LTP. By preferentially diminishing transfer events involving LDL, especially those between VLDL and LDL, LTIP enhances the ability of LTP to remove CE from HDL, and thus alters HDL metabolism.

Determination of lipid transfer inhibitor protein activity in human lipoprotein-deficient plasma.

Lipid transfer protein (LTP) activity is modulated by a distinct plasma protein termed lipid transfer inhibitor protein (LTIP). The objective of this study was to establish an assay for LTIP that could be used to quantify its activity in lipoprotein-deficient plasma. A straightforward heating protocol (56 degrees C for 60 minutes) was found to inactivate more than 90% of LTIP activity. The responses of individual lipoprotein-deficient plasma samples to this heating procedure were unique. Among normolipidemic donors, inactivation of LTIP caused a 230% to 600% increase in LTP activity. Essentially all measurable transfer activity in native and heated samples was inhibited by an antibody to LTP. Whole-plasma samples from these donors were spiked with radiolabeled lipoproteins to measure the rates of lipid transfer among the major lipoprotein classes. In general, plasma lipid transfer rates were negatively correlated with LTIP activity in these samples. However, the decrease in lipid transfers from very-low-density lipoprotein (VLDL) to low-density lipoprotein (LDL) and from LDL to VLDL was from 2.4- to 5.1-fold greater than in the transfers from VLDL to high-density lipoprotein (HDL) or from HDL to VLDL. In these samples, the molecular weight of HDL2 was negatively correlated with LTIP activity. Thus, LTIP activities among normolipidemic individuals were observed to vary severalfold; compared with other lipoprotein transfers, higher LTIP activities were associated with a relative reduction in LDL-VLDL lipid transfer events.

Inhibition of lipid transfer by a unique high density lipoprotein subclass containing an inhibitor protein.

We have isolated from human plasma a unique subclass of the high density lipoproteins (HDL) which contains a potent lipid transfer inhibitor protein (LTIP) that inhibited cholesteryl ester, triglyceride, and phospholipid transfer mediated by the lipid transfer protein, LTP-I, and phospholipid transfer mediated by the phospholipid transfer protein, LTP-II. This HDL subclass not only inhibited cholesteryl ester transfer from HDL to LDL or VLDL, but also inhibited cholesteryl ester transfer from HDL to HDL. The inhibitor protein was isolated by sequential chromatography of human whole plasma on dextran sulfate-cellulose, phenyl-Sepharose, and chromatofocusing chromatography. Isolated LTIP had the following characteristics: an apparent molecular weight of 29,000 +/- 1,000, (n = 10) by sodium dodecyl sulfate gel electrophoresis, and an isoelectric point of 4.6 as determined by chromatofocusing. LTIP remained functional following delipidation with organic solvents. Antibody to LTIP was produced, and an immunoaffinity column of the anti-LTIP was prepared. Passage of human, rat, or pig whole plasma over the anti-LTIP column enhanced cholesteryl ester transfer activity in human (17%), pig (200%), and rat plasma (125%). The HDL subclass containing LTIP was isolated from whole human HDL (d 1.063-1.21 g/ml) by immunoaffinity chromatography. The isolated LTIP-HDL complex was shown to: i) contain about 60% protein and 40% lipid, ii) have alpha and pre-beta electrophoretic mobility, iii) have particle size distribution somewhat smaller than whole HDL, about 100,000 daltons, as determined by gradient gel electrophoresis, and iv) contain only a small amount of apoA-I (less than 5%) and a trace amount of apoA-II. Assay of ultracentrifugally obtained lipoprotein fractions revealed that approximately 85% of the total functional LTIP activity was in the d 1.063-1.21 g/ml HDL fraction. Furthermore, immunoblot analysis of whole plasma by nondenaturing gradient gel electrophoresis revealed that LTIP was found predominantly in particles in the size range of HDL. This unique HDL subclass may play an important role in the regulation of plasma lipid transfer and metabolism.

Regulation of plasma lipid transfer by the high-density lipoproteins.

Mechanisms that regulate the activity of the plasma cholesteryl ester transfer protein (lipid protein transfer-I) are poorly understood. We have examined the role of endogenous regulators of the lipid transfer process, in particular the plasma high-density lipoproteins (HDLs), in both humans and two species previously reported to be devoid of cholesteryl ester transfer activity, the rat and the pig. Chromatography of d greater than 1.21 g/ml plasma on phenyl-Sepharose either enhanced (human) lipid transfer activity or unmasked (rat and pig) a nearly completely suppressed lipid transfer activity. The HDLs (either native or delipidated) were shown to be a rich source of a plasma lipid transfer inhibitor protein. Partial purification of the HDL-derived lipid transfer inhibitor protein was achieved by sequential chromatography on phenyl- and DEAE-Sepharose. Additional analysis of plasma from patients with very low (Tangier disease), normal, or very high levels of HDL cholesterol (hyperalphalipoproteinemia) revealed a significant inverse correlation between plasma cholesteryl ester transfer activity and the subjects HDL cholesterol level. These results suggest that circulating levels of the plasma high density lipoproteins may be an important determinant of plasma cholesteryl ester transfer activity, through the function of the HDL-derived lipid transfer inhibitor protein.