Conversion of lipid transfer inhibitor protein (apolipoprotein F) to its active form depends on LDL composition

Abstract

Lipid transfer inhibitor protein (LTIP) exists in both active and inactive forms. Incubation (37°C) of plasma causes LTIP to transfer from a 470 kDa inactive complex to LDL where it is active. Here, we investigate the mechanisms underlying this movement. Inhibiting LCAT or cholesteryl ester transfer protein (CETP) reduced incubation-induced LTIP translocation by 40-50%. Blocking both LCAT and CETP completely prevented LTIP movement. Under appropriate conditions, either factor alone could drive maximum LTIP transfer to LDL. These data suggest that chemical modification of LDL, the 470 kDa complex, or both facilitate LTIP movement. To test this, LDL and the 470 kDa fraction were separately premodified by CETP and/or LCAT activity. Modification of the 470 kDa fraction had no effect on subsequent LTIP movement to native LDL. Premodification of LDL, however, induced spontaneous LTIP movement from the native 470 kDa particle to LDL. This transfer depended on the extent of LDL modification and correlated negatively with changes in the LDL phospholipid + cholesterol-to-cholesteryl ester + triglyceride ratio. We conclude that LTIP translocation is dependent on LDL lipid composition, not on its release from the inactive complex. Compositional changes that reduce the surface-to-core lipid ratio of LDL promote LTIP binding and activation.

Fig. 1.

Effect of LCAT and CETP inhibition on LTIP movement to LDL in plasma. Plasma (750 µl) was preincubated overnight at 4°C with or without anti-CETP monoclonal antibody (27.5 µg). This blocked CETP activity by ∼70%. These treated samples, ± 1 mM paraoxon to block LCAT activity, were subsequently incubated at 37°C for 24 h to induce LTIP movement. Control samples were held at 4°C. A 500 µl aliquot of each sample was then fractionated by FPLC gel filtration (see Methods) and eluted fractions were assayed in triplicate for LTIP by immunoassay. A and B show the LTIP elution profiles. The recovery of LTIP in 37°C incubated samples was 92–105% of 4°C controls. A also shows the distribution of cholesterol in control plasma. The elution position of lipoproteins was not altered by these incubation conditions. Data are representative of three similar experiments.

Fig. 2.

Effect of VLDL and CETP on LTIP movement in plasma. A: Plasma ± VLDL (600 µg cholesterol/ml plasma) was incubated at 37°C for 8 h. ^† P < 0.05 compared with native plasma incubated for the same time. Panel B: Plasma was incubated with or without partially purified CETP for 8 h at the indicated temperature. The CETP addition increased plasma CETP activity 3-fold. Because partially purified CETP contains low levels of LTIP, the increase in LDL-associated LTIP in 4°C samples due to CETP addition has been subtracted from the data shown. * P < 0.01 compared with native plasma at 37°C. Data are representative of at least two similar experiments.

Fig. 3.

Effect of LCAT inhibition on the time course of LTIP movement to LDL in plasma. Plasma without (squares) or with (circles) 1 mM paraoxon to inhibit LCAT was incubated for the indicated times. Inset: ratio of free cholesterol (FC) to total cholesterol (TC) in samples incubated at 37°C for the indicted time. * P < 0.01 compared with native plasma. Data are representative of three time course experiments.

Fig. 4.

Effect of VLDL removal on incubation-induced association of LTIP with LDL. Plasma was centrifuged at density 1.019 g/ml to generate VLDL (d < 1.019 g/ml) and VLDL-free (d > 1.019 g/ml) plasma fractions. A: The d > 1.019 g/ml plasma fraction was combined with the d < 1.019 g/ml fraction, to reconstitute plasma, or with buffer alone. Samples were incubated at 37°C for the indicated times. B: One milliliter aliquots of the d > 1.019 g/ml fraction were incubated ± 45.9 µg anti-CETP for 1 h at 25°C. Subsequently, samples received 1 mM paraoxon as indicated and were incubated at 4°C or 37°C for 16 h. Negative symbols indicate that CETP or LCAT activities were inhibited by blocking antibody or paraoxon, respectively. Plus symbols designate that the indicated activities are present at endogenous levels. * P < 0.01 compared with native plasma at 4°C. ns, not significant.

Fig. 5.

Reconstitution of LTIP movement activity. A: Plasma was sequentially fractioned by ultracentrifugation to generate VLDL (d < 1.019 g/ml) and VLDL-free plasma (d > 1.019 g/ml), or VLDL+LDL (d < 1.063 g/ml) and HDL + protein (d > 1.063 g/ml) fractions. Dialyzed samples were recombined as indicated at equivalent original plasma volumes. Samples were incubated at 4°C or 37°C for 24 h. ^† P < 0.05, * P < 0.01 compared with native plasma at 37°C. ns, not significant. B: LDL (220 µg cholesterol), the d > 1.21 g/ml lipoprotein-free fraction of plasma (0.25 ml) and the 470 kDa fraction of LTIP (200 µg protein) were combined and incubated at 4°C or 37°C for 24 h. * P < 0.01 compared with the corresponding 4°C sample. ns, not significant. Data are representative of two-three similar experiments.

Fig. 6.

Influence of LDL composition on LTIP association with LDL. A: The d < 1.063 g/ml (VLDL+LDL) fraction of plasma was recombined with a plasma equivalent volume of d > 1.21 g/ml (lipoprotein-free fraction) of plasma ± 1 mM paraoxon and incubated at 4°C or 37°C for 22 h. Under these conditions, LDL was either unmodified (4°C), modified by CETP (37°C + paraoxon) or modified by CETP and LCAT activities (37°C, no paraoxon). LDL was isolated from these mixtures by ultracentrifugation. Native or modified LDL (160 (1×) or 320 (2×) µg protein) was then combined with native 470 kDa LTIP fraction (200 µg protein) and 500 µl of lipoprotein-free plasma (d > 1.21 fraction). All samples received anti-CETP (21 µg) and 1 mM paraoxon so that CETP and LCAT were inactive during this phase of the experiment. Samples were incubated at 37°C for 6 h. ^† P < 0.05, * P < 0.01 compared with unmodified LDL or as indicated. B: The percent LTIP on LDL measured in A is plotted versus the ratio of free cholesterol (FC) + phospholipid (PL) divided by cholesteryl ester (CE) + triglyceride (TG) in LDL. The reverse x axis places native LDL on the left side of the graph. Correlation coefficients for both regression lines are ≥ 0.98. C: The percent LTIP on LDL after incubation of whole plasma as described in Table 3 is plotted versus the FC + PL-to-CE + TG ratio of the LDL isolated from those incubations. Correlation coefficient for the regression line is 0.99, P = 0.02. Data are representative of three similar experiments. D: The percent LTIP on LDL in normolipidemic subjects and those with borderline high cholesterol or TG is plotted versus LDL composition. Values on the y axis have been normalized by dividing the %LTIP on LDL by the LDL cholesterol (LDLc) content of each sample. Correlation coefficient for the regression line is 0.87, P = 0.025. Of the seven plasma samples analyzed, six are plotted here. The remaining sample, perhaps due to its very low HDL and LTIP content, did not fit this correlation.