Lipid composition influences the shape of human low density lipoprotein in vitreous ice

Abstract

Earlier cryo-electron microscopic studies have indicated that the normal low density lipoprotein (N-LDL) has a discoid shape when its core is in the liquid-crystalline state. In the present study, we investigated whether the shape of LDL depends on the physical state and/or the lipid composition of the lipoprotein core. Using a custom-built freezing device, we vitrified NLDL samples from either above or below the phase-transition temperature of the core (42 and 24 degrees C, respectively). Cryo-electron microscopy revealed no differences between these samples and indicated a discoid shape of the N-LDL particle. In contrast, TG-enriched LDL (T-LDL) did not have discoid features and appeared to be quasi-spherical in preparations that were vitrified from either 42 or 24 degrees C. These results suggest that the shape of NLDL is discoid, regardless of the physical state of its core, whereas T-LDL is more spherical. Aspects that may influence the shape of LDL are discussed.

Figure 1

Custom built freezing device for the vitrification of samples from different temperatures. A pair of forceps (A), holding an EM grid with a lacey substrate (white arrow), is attached to a plunger (B). This plunger is connected to tubing that is pressurized with air. Air pressure in the tubing is adjusted with a control valve (not in the picture), usually set at 20 psi. Release of the pressure with a control switch (C) forces the plunger and the attached forceps down with high speed. The environment in the plexiglas box (D) can be warmed by a heating pad in the back of the box (rectangle, E). Temperature near the EM sample is measured with a thermocouple (F) that is connected to a thermostat (not in the picture). In the bottom of the box are two small containers (G) that are filled with water. Filter paper soaked with water is guided from the water containers to the top of the heating pad to allow efficient distribution of water vapor throughout the box. The front panel of the box hinges on the left (black arrows) and can be opened to allow attachment of the forceps to the plunger and application of sample to the EM grid (white arrow). After application of the sample, the front panel is closed and the environment is equilibrated at the desired temperature and high humidity. Subsequently, the bulk of the lipoprotein sample is blotted from the EM grid through a side entrance of the box (H), using a spatula covered with filter paper. Blotting of the sample results in the formation of thin films of lipoprotein solution in the holes of the lacey substrate on the EM grid. Upon formation of the thin films, the preparation is equilibrated at the set temperature for an additional 5 min. Subsequently, control switch (C) pressurizes the plunger (B), which forces the attached forceps through the opened shutter into the container of liquid propane (I). Propane in the central compartment of container (I) is kept in a liquid state by liquid nitrogen, present in the outer compartment of container (I).

Figure 2

DSC of N-LDL and T-LDL. Representative thermograms of N-LDL with a high phase-transition temperature (T_m = 31°C) and T-LDL with a low phase-transition temperature (T_m = 12°C) are shown.

Figure 3

Cryo-electron micrographs of N-LDL (A, B) and T-LDL (C, D), ultra-rapidly frozen from 24°C (A, C) and 42°C (B, D). Arrowheads indicate rectangular projections with two high-density bands, representing edge-on views of the LDL disc (–31). Magnification 300,000 x.

Figure 4

Frequency distribution of rectangular projections with two high-density bands in cryo-EM preparations of N-LDL and T-LDL, vitrified from 24°C or 42°C. The occurrence of rectangular projections in N-LDL preparations vitrified from 24°C or 42°C is not significantly different (t-test; P=0.105); “n” refers to the number of separate LDL preparations analyzed using cryo-EM.

Figure 5

Highly schematic representations of the liquid-crystalline LDL core (cross-sectional views). &dcl001; Phospholipids; &dcl002; Cholesteryl esters; &dcl003; Apo B. Free cholesterol and triacylglycerols have been omitted from the drawings for clarity A: Generalized representation of earlier models (–25). In these models, the LDL particle is spherical and the liquid-crystalline core is organized in concentric shells of cholesteryl esters. B: Generalized representation of new models (32,35,36), in which the liquid-crystalline core contains flat layers of cholesteryl esters.