Control of cholesteryl ester transfer protein activity by sequestration of lipid transfer inhibitor protein in an inactive complex

Abstract

Lipid transfer inhibitor protein (LTIP) is a physiologic regulator of cholesteryl ester transfer protein (CETP) function. We previously reported that LTIP activity is localized to LDL, consistent with its greater inhibitory activity on this lipoprotein. With a recently described immunoassay for LTIP, we investigated whether LTIP mass is similarly distributed. Plasma fractionated by gel filtration chromatography revealed two LTIP protein peaks, one coeluting with LDL, and another of approximately 470 kDa. The 470 kDa LTIP complex had a density of 1.134 g/ml, indicating approximately 50% lipid content, and contained apolipoprotein A-I. By mass spectrometry, partially purified 470 kDa LTIP also contains apolipoproteins C-II, D, E, J, and paraoxonase 1. Unlike LDL-associated LTIP, the 470 kDa LTIP complex does not inhibit CETP activity. In normolipidemic subjects, approximately 25% of LTIP is in the LDL-associated, active form. In hypercholesterolemia,this increases to 50%, suggesting that lipoprotein composition may influence the status of LTIP activity. Incubation (37 degrees C) of normolipidemic plasma increased active, LDL-associated LTIP up to 3-fold at the expense of the inactive pool. Paraoxon inhibited this shift by 50%. Overall, these studies show that LTIP activity is controlled by its reversible incorporation into an inactive complex. This may provide for short-term fine-tuning of lipoprotein remodeling mediated by CETP.

Fig. 1.

Distribution of lipid transfer inhibitor protein (LTIP) in plasma. Plasma (0.3 ml) was applied to tandem Superose 6 columns, and eluted fractions were assayed for LTIP mass and cholesterol. Fraction collection began 60 min after sample application. Inset: Western blot analysis of select column fractions and partially purified LTIP separated by 4–20% SDS-PAGE and reacted with anti-LTIP. Molecular mass standards are shown in kDa units. Data are representative of multiple experiments.

Fig. 2.

Agarose gel electrophoresis. Plasma was fractionated by sequential density ultracentrifugation to yield the three major lipoprotein fractions and the d > 1.21 g/ml lipoprotein-free fraction. Samples were subsequently electrophoresed on 1% agarose gels. Lipoproteins were visualized by staining with Fat Red 7B. The samples on other gels were transferred to polyvinylidene fluoride and reacted with anti-LTIP or preimmune antisera as indicated.

Fig. 3.

Apolipoprotein distribution in LTIP-containing fractions. Plasma (500 μl) was applied to tandem Superose 6 columns, and fractions were collected beginning 55 min after sample application. Apolipoprotein content was determined by Western blot. For apolipoprotein A-I, apolipoprotein C-I, and LTIP, samples were separated on 4–20% PAGE gels. For LCAT and phospholipid transfer protein (PLTP), 10% gels were used. Molecular mass standards are shown on the left-hand side of each blot, and are in kDa units. The HDL cholesterol peak was in fraction 23. HC, IgG heavy chain.

Fig. 4.

Characterization of 470 kDa LTIP isolated from HDL₃. A:HDL₃ (8.8 mg protein) was isolated from plasma by sequential ultracentrifugation and applied to tandem Superose 6 columns. Eluted fractions were assayed for LTIP mass and cholesterol. B:Fractions containing high LTIP and low cholesterol, equivalent to fractions 14–15 in Panel A, were pooled, and the protein constituents were fractionated by SDS-PAGE. Excised bands were digested with trypsin, and the resultant peptides were identified by liquid chromatography-tandem mass spectrometry as described in the Methods. Molecular mass standards are shown in kDa units. The 33 kDa band also contained apolipoprotein E, PON1, and apolipoprotein A-I. PON1, serum paraoxonase 1; SAA4, serum amyloid A4. Lane A = molecular mass standards; lane B = 470kDa LTIP fraction.

Fig. 5.

LTIP distribution in hypercholesterolemic plasma. Plasma (500 μl) was applied to tandem Superose 6 columns, and fractions were collected 60 min after sample application. LTIP mass, as determined by ELISA, and cholesterol were measured as described in the Methods. The hypercholesterolemic plasma sample contained 368 mg/dl cholesterol (LDL 271 mg/dl, HDL 78 mg/dl), and 97 mg/dl triglyceride. This LTIP profile was typical of that seen in three hypercholesterolemic subjects.

Fig. 6.

Incubation induced dissociation of LTIP from the inactive complex. Aliquots of normolipidemic plasma were incubated at 37°C for 0 or 24 h in the absence or presence of 1 mM paraoxon (prx), as indicated, then applied (500 μl) to tandem Superose 6 columns. LTIP mass and cholesterol of column fractions were measured. LDL and HDL designations at the top of the figure indicate the elution peaks for these lipoproteins. Data are representative of more than five experiments.