Comparative investigation on the sizes and scavenger receptor binding of human native and modified lipoprotein particles with atomic force microscopy
The size and receptor-binding abilities of plasma lipoproteins are closely related with their structure/functions. Presently, the sizes of native lipoproteins have been measured by various methods including atomic force microscopy (AFM) whereas the sizes of modified lipoproteins are poorly determined and the receptor-binding ability of lipoproteins is less detected and compared at the nanoscale.
Here, AFM was utilized to detect/compare the size and scavenger receptor-binding properties of three native human lipoproteins including high-density lipoprotein, low-density lipoprotein (LDL), and very low-density lipoprotein, and two modified human lipoproteins including oxidized and acetylated LDL, as well as bovine serum albumin and their antibodies as negative and positive controls, respectively.
AFM detected that the sizes of these lipoproteins are close to the commonly known values and the previously-reported AFM-detected sizes and that native and modified LDL have different height/size. AFM also revealed that the CD36-binding abilities of the five lipoproteins are different from one another and from their SR-B1-binding abilities and that the anti-CD36/SR-B1 antibodies as positive controls have strong CD36/SR-B1-binding abilities.
The data provide important information on lipoproteins for better understanding their structures/functions. Moreover, the data certify that besides size measurement AFM also can visualize receptor-lipoprotein binding at the nanoscale, as well as antigen–antibody (scavenger receptors and their antibodies) binding.
KeywordsLipoproteins Atomic force microscopy (AFM) Scavenger receptors CD36 SR-B1
acetylated low-density lipoprotein
atomic force microscopy
bovine serum albumin
full width at half maximum
oxidized low-density lipoprotein
very low-density lipoprotein
Plasma lipoproteins, e.g. native low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), among others, play vital roles in transport and delivery of lipids, including cholesterol, triglycerides, and some lipid-soluble vitamins, to or from peripheral tissue cells. The lipoproteins, particularly their modified forms, e.g. oxidized LDL (oxLDL) and acetylated LDL (acLDL), have been associated with the pathophysiology of many disease states, including diabetes, atherosclerosis, cancer, and others . Therefore, it is meaningful and significant to investigate the structural and receptor-binding properties of these lipoproteins for better understanding their physiological functions or roles in diseases.
At present, multiple approaches are available to investigate the structural and receptor-binding properties of plasma lipoproteins, such as dynamic light scattering (DLS), nuclear magnetic resonance (NMR), cryo-electron microscopy (cryo-EM), etc. for detection of the structural properties, and immunoassay, fluorescence methods, surface plasmon resonance (SPR), etc. for detection of the receptor-binding properties. Currently, however, there are no ideal tools capable of detecting or even “seeing” both the structural and receptor-binding properties of plasma lipoproteins particularly under a physiological condition. Atomic force microscopy (AFM) may be a candidate of such tool.
After decades of development and applications, AFM has proved to be a promising and powerful technique for nanoscale detection of biological samples with a broad size range from micro-sized tissues/cells down to nano-sized molecules [2, 3, 4, 5, 6]. AFM can not only image individual biological molecules at the nanoscale but also directly “see” molecule–molecule (e.g. biotin-streptavidin, antigen–antibody, ligand-receptor, etc.) binding/interaction [7, 8, 9] also at the nanoscale even under physiological conditions.
Surprisingly, however, the AFM studies on plasma/circulating lipoproteins, an important type of natural lipid-protein complexes with a nanoscale size (generally 5–80 nm; chylomicrons have a size of up to 1 μm), are limited to the imaging and/or size measurement of native lipoproteins (mainly LDL and HDL) [10, 11, 12, 13, 14, 15, 16, 17], and even no AFM size measurement of modified lipoproteins (e.g. oxLDL or acLDL) was previously reported until now. Besides the size, the receptor-binding property is also correlated tightly with the structure and functions of lipoproteins. Unfortunately, however, AFM studies on this property of lipoproteins are also missing.
In this study, we utilized the powerful imaging function of AFM to investigate the sizes of multiple native/modified lipoproteins including HDL, LDL, oxLDL, acLDL, and VLDL. Thereafter, the abilities of these lipoproteins binding to CD36 or SR-B1 (two major type B scavenger receptors) were evaluated by AFM at the nanoscale. The data showed that AFM is able to detect/“see” both the structural and receptor-binding properties of plasma lipoproteins at the nanoscale under physiological conditions (e.g. in PBS).
Human LDL, oxLDL, acLDL, and HDL were purchased from Yiyuan Biotechnologies (Guangzhou, China). Human VLDL was from American Research Products (MA, USA). All lipoproteins were used immediately and stored at 4 °C for at most 3 weeks. Recombinant human CD36 and SR-B1, as well as anti-CD36 and anti-SR-B1 antibodies, were purchased from Abcam (Cambridge, MA). N,N-Diisopropylethylamine (DIPEA), aminopropyltriethoxysilane (APTES), and glutaraldehyde were from TCI (Shanghai, China; or Tokyo, Japan for APTES). Bovine serum albumin (BSA) was from Solarbio (Beijing, China).
Mica functionalization and sample preparation
The method for mica functionalization and sample preparation was modified from previous reports [16, 17, 18]. The first part of the procedure was performed in a clean and dry glass dessicator (2.5 l capacity, no dessicants, with a walve in the cover). Ultra pure argon was used to remove the air and moisture (~ 2 min) in the dessicator. The freshly cleaved mica sheets in clean petri dishes were immediately put inside following with argon purging again for ~ 2 min. Next, 30 μl of DIPEA and 50 μl of APTES were carefully pipetted into two containers in the dessicator. After argon purging for 0.5–1 min, the mica sheets were exposed to APTES vapor for ~ 2 h. After the APTES container was carefully removed, the dessicator was purged with argon and sealed, and the treated mica sheets were stored inside. Two days later, the mica sheets were removed from the dessicator and immediately incubated with 100 μl of 0.2% fresh glutaraldehyde solution in double distilled water for 10 min. After the mica sheets were rinsed with double distilled water, protein/lipoprotein samples were deposited immediately for 2 h, washed with PBS, incubated with l-glycine for ~ 15 min, washed again, and then subjected to AFM detection. For lipoprotein-receptor binding experiments, the functionalized mica sheets were rinsed, incubated with receptors (100 μl CD36 or SR-B1 at 0.1 μg/ml using BSA as a control) or heat-inactivated receptors/BSA (as controls), washed twice, incubated with l-glycine, washed again, and subsequently incubated with 100 μl lipoproteins at 0.1 μg/ml. After washing with PBS to remove excess lipoproteins, the samples were subjected to AFM imaging, or the samples were stained with Oil Red O and rinsed, and then the mica sheets bearing the samples were imaged.
All data are from at least three independent experiments. Statistical analyses were performed using one-way ANOVA to determine the statistically significant differences between different groups. A value of p < 0.05 was considered statistically significant.
Results and discussion
AFM imaging and size measurement of various lipoproteins at the nanoscale
AFM has been utilized to image and measure majorly LDL and HDL whereas AFM imaging and size measurement of VLDL and particularly modified LDL (e.g. acLDL and oxLDL) were less performed. Here, five types of lipoproteins including LDL, HDL, VLDL, acLDL, and oxLDL were imaged/measured and compared by AFM at the same time.
AFM-measured sizes of BSA, lipoproteins, receptors and their antibodies
Equivalent diameter of a sphere (nm)
2.2 ± 0.4
18.9 ± 4.3
8.4 ± 1.5
4.0 ± 0.9
22.2 ± 5.7
11.5 ± 2.4
8.9 ± 2.4
31.8 ± 8.6
19.5 ± 4.6
7.5 ± 1.5
34.4 ± 7.0
19.1 ± 3.2
10.8 ± 2.9
34.0 ± 6.0
21.8 ± 3.6
11.2 ± 2.2
95.9 ± 15.6
42.8 ± 7.1
2.6 ± 0.6
18.2 ± 4.5
8.6 ± 1.5
2.5 ± 0.5
18.4 ± 5.3
8.6 ± 1.7
3.0 ± 1.0
22.5 ± 3.9
10.5 ± 1.8
3.3 ± 0.8
20.8 ± 4.0
10.3 ± 1.7
The AFM-measured sizes of HDL, LDL, and VLDL in our study are close to the commonly known or previously reported AFM-measured values [10, 11, 12, 13, 14, 15, 16, 20, 21]. This result also implies that the individual particles imaged by AFM are single lipoproteins (not lipoprotein aggregates). The size relationship among native lipoproteins (i.e. HDL<LDL<VLDL) is well known. However, the size relationship between LDL and modified LDL (e.g. oxLDL and/or acLDL) remains unclear. For the first time, the sizes of LDL, oxLDL, and acLDL particles were compared here. Interestingly, the statistical analysis (Table 1) shows that when deposited on a substrate/mica oxLDL particles display a significantly smaller average height (7.5 ± 1.5 nm) than LDL particles (8.9 ± 2.4 nm) although they have a similar equivalent diameter of a sphere (19.1 ± 3.2 and 19.5 ± 4.6 nm for oxLDL and LDL, respectively; p > 0.05) whereas acLDL particles (height: 10.8 ± 2.9 nm) are significantly thicker and larger than LDL particles (the equivalent diameter of a sphere: 21.8 ± 3.6 and 19.5 ± 4.6 nm for acLDL and LDL, respectively; p < 0.001).
It is well known that oxidation can lead to loss of LDL lipids (generating biologically active products, e.g. peroxides, aldehydes, lyso-PC, oxysterols, etc.) and even hydrolysis/fragmentation of apoB-100 [22, 23] whereas acetylation generally modifies the amino acid residues (e.g. the ε-amino group of lysine) of LDL without alterations in lipid and protein composition of LDL . Therefore, compared with native LDL, the oxidation-induced partial loss of LDL components might contribute to the decrease in size/height of oxLDL whereas the increase in size/height of acLDL might result from the addition of acetyl groups and/or increased hydrophobicity of the acetylated surface.
AFM visualizes the receptor-lipoprotein binding at the nanoscale
Prior to investigation on the lipoprotein-receptor binding, CD36 and SR-B1 (Figs. 3a, 4a), as well as their antibodies (Figs. 3b, 4b), on GD-APTES-mica were imaged by AFM in PBS. The quantification data (Table 1) shows that the height and equivalent diameter of CD36/SR-B1 particles are ~ 2.6 and ~ 8.6 nm, respectively (p > 0.05 for CD36 vs. SR-B1), and that the height and equivalent diameter of their antibodies are ~ 3.0 and ~ 10.4 nm, respectively (p > 0.05 for anti-CD36 vs. anti-SR-B1). For the first time, scavenger receptor molecules (CD36 and SR-B1) were imaged and size-measured by AFM.
It implies that CD36/SR-B1 particles are significantly more flat and smaller than lipoprotein particles of all five types (Table 1; p < 0.001 for CD36/SR-B1 vs. each of the five lipoprotein types), based on which lipoprotein particles can be distinguished from receptor particles in the same field for direct visualization and quantification of receptor-lipoprotein binding. Even the anti-CD36/anti-SR-B1 antibodies with similar size (Table 1) could be identified from the CD36/SR-B1 monolayer based on an increased height due to the stacking/binding of antibody molecules onto the receptor monolayer (left panels of Fig. 3d and left panels of Fig. 4d). These data imply that utilizing AFM to directly visualize receptor-lipoprotein binding at the nanoscale is feasible and reliable.
Next, CD36 or SR-B1 molecules immobilized on GD-APTES-mica were incubated with each of the five types of lipoproteins in PBS. After removal of unbound lipoproteins, the samples were imaged by AFM in PBS (left panels of Figs. 3e–i, 4e–i). Clearly, two layers of particles were visualized in AFM topographical images, the bottom receptor monolayer with small particles and the upper lipoprotein layer with significantly larger and brighter particles. To make the topographical images more intuitive, pseudocolors were used to highlight the upper antibody/lipoprotein layers (green) by adjusting the bottom receptor monolayer to the red baseline (the Right panels of Fig. 3a–i and right panels of Fig. 4a–i). The pseudocolor Z scale bar was obtained by adjusting the Z scale bar of the control group (only CD36/SR-B1 monolayer on mica) to the situation (all particles were invisible), then all groups used the same pseudocolor Z scale bar. The size of each of the five lipoprotein types in the upper layer coincides with that of each lipoprotein directly deposited on substrate (Fig. 1), implying the reliability of the data and that the individual particles in the upper layer are single lipoproteins.
Obviously, all five types of lipoproteins could bind to CD36 or SR-B1 but to different extents. As a negative control, no BSA particles were found to bind onto the receptor layers (Figs. 3c, 4c). As a positive control, a large quantity of antibody (anti-CD36 or anti-SR-B1) was detected on the receptor layers (Figs. 3d, 4d). Statistical analyses further confirmed the observations and revealed that the receptor-binding abilities of the five lipoproteins are as follows: oxLDL>HDL/acLDL>LDL>VLDL for CD36 binding (Fig. 3j) and HDL>acLDL>oxLDL>LDL/VLDL for SR-B1 binding (Fig. 4j). Since the size/height of antibodies is smaller than that of all lipoprotein types the number of receptor-binding antibody particles in the topographical images (Figs. 3d, 4d) may visually be less abundant than that of CD36-binding oxLDL (Fig. 3g) or SR-B1-binding HDL (Fig. 4e).
Among the five lipoprotein types, oxLDL has an extremely (> 2-fold) stronger CD36-binding ability (Fig. 3g, j) whereas HDL has an extremely (> 3-fold) stronger SR-B1-binding ability (Fig. 4e, j) than the other lipoproteins. The results are consistent with the original characterizations of CD36 and SR-B1 as lipoprotein receptors for oxLDL  and HDL [28, 29], respectively, and also consistent with the well-known major roles of CD36 and SR-B1. The results further certify the effectiveness and reliability of the method.
For the first time, we determined the receptor-binding abilities of all five lipoprotein types. Unfortunately, we currently do not know the underlying mechanisms of causing the differences in receptor-binding ability among various lipoproteins. However, it may provide important information or evidence for elucidating the competition of recognizing cell-bound receptors among various lipoproteins. AFM detection of interaction forces between lipoproteins and free/cell-bound receptors may provide further important information although it is very challenging. More in-depth studies will be needed.
Taken together, the morphological and receptor-binding properties of three native (HDL, LDL, and VLDL) and two modified (oxLDL and acLDL) lipoproteins were detected and compared by AFM at the nanoscale under a physiological condition (in PBS). According to our knowledge, it is the first time that the sizes of modified lipoproteins and the receptor-binding abilities of lipoproteins were evaluated at the nanoscale by AFM. The data may provide important information for better understanding of the structures and functions of various lipoproteins. The data also certify the usefulness of AFM for obtaining more important information (e.g. the receptor-lipoprotein binding ability) besides the sizes of lipoproteins.
YC designed the research, analyzed and interpreted data, and wrote the manuscript; CG, KW, and QT carried out experiments, collected and analyzed data, and reviewed the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
Consent for publication
We give our consent for the manuscript to be published in Journal of Nanobiotechnology.
Ethics approval and consent to participate
Not applicable for this study.
This work was financially supported by the Natural Science Foundation of China (Grant Nos. 31260205 and 81560083) and the Natural Science Foundation of Jiangxi Province of China (Grant No. 20151BBG70035).
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