Apolipoprotein A-II alters the proteome of human lipoproteins and enhances cholesterol efflux from ABCA1

Abstract

HDLs are a family of heterogeneous particles that vary in size, composition, and function. The structure of most HDLs is maintained by two scaffold proteins, apoA-I and apoA-II, but up to 95 other "accessory" proteins have been found associated with the particles. Recent evidence suggests that these accessory proteins are distributed across various subspecies and drive specific biological functions. Unfortunately, our understanding of the molecular composition of such subspecies is limited. To begin to address this issue, we separated human plasma and HDL isolated by ultracentrifugation (UC-HDL) into particles with apoA-I and no apoA-II (LpA-I) and those with both apoA-I and apoA-II (LpA-I/A-II). MS studies revealed distinct differences between the subfractions. LpA-I exhibited significantly more protein diversity than LpA-I/A-II when isolated directly from plasma. However, this difference was lost in UC-HDL. Most LpA-I/A-II accessory proteins were associated with lipid transport pathways, whereas those in LpA-I were associated with inflammatory response, hemostasis, immune response, metal ion binding, and protease inhibition. We found that the presence of apoA-II enhanced ABCA1-mediated efflux compared with LpA-I particles. This effect was independent of the accessory protein signature suggesting that apoA-II induces a structural change in apoA-I in HDLs.

Keywords: ATP binding cassette transporter A1; high density lipoprotein/structure; proteomics.

Fig. 1.

LpA-I and LpA-I/A-II separated from plasma. A: The 4–15% SDS-PAGE analysis of LpA-I and LpA-I/A-II particles separated from the plasma of four male donors. LpA-I particles are shown in lanes 2, 4, 7, and 9 and LpA-I/A-II particles are shown in lanes 3, 5, 8, and 10 with molecular mass markers in lanes 1 and 6. Each lane contains 10 μg of total protein stained with Coomassie blue. No reducing agent was present, thus the human apoA-II dimer band appears at ∼18 kDa. B, C: Western blots probed with antibodies for apoA-I and apoA-II, respectively. Three micrograms of total protein per lane were loaded onto an Any-KD^TM mini gel in sample buffer containing β mercaptoethanol to reduce apoA-II to monomeric form. The loading order was identical to (A).

Fig. 2.

LpA-I and LpA-I/A-II particle size analysis. A: A native PAGE gel with protein standards (lane 1), UC-HDL (lane 2), plasma LpA-I particles (lane 3), and plasma LpA-I/A-II particles (lane 4). B: A native gel containing LpA-I and LpA-I/A-II particles isolated from UC-HDL run in the same format. Both gels were stained with Coomassie blue. UC-HDL and LpA-I and LpA-I/A-II particles subfractionated from UC-HDL were analyzed by size exclusion chromatography. C: The UV trace of protein for UC-HDL, LpA-I and LpA-I/A-II particles. LpA-I and LpA-I/A-II particles were labeled with fluorescent rhodamine to track the phospholipid elution pattern. D: The UV and phospholipid trace for LpA-I particles. E: The UV and PL trace for LpA-I/A-II particles.

Fig. 3.

Particle protein and phospholipid composition. A: The total protein to PC mass ratio of UC-HDL and LpA-I and LpA-I/A-II subfractions isolated from plasma and UC-HDL. B: The apoA-I:PC mass ratio of UC-HDL and LpA-I and LpA-I/A-II subfractions isolated from plasma and UC-HDL. ApoA-I content was calculated by expressing apoA-I peptide spectral counts as a percentage of total protein spectral counts for each subfraction in each donor. Values were derived from the product of the percentage of apoA-I spectral counts with total protein as measured by Lowry protein assay. The bars represent the mean (±SD) for the four male donors and the letters indicate significant differences (P < 0.05) determined by a one-way ANOVA with a post hoc analysis using Tukey’s honestly significant difference test. C–E: The TC (C), FC (D), and CE (E) to PC mass ratios for LpA-I and LpA-I/A-II subfractions isolated from plasma. The bars represent the mean (±SD) for three male donors and asterisks indicate significant differences (P < 0.05) determined by a two-tailed paired Student’s t-test.

Fig. 4.

Proteins associated with apoA-I-containing particles (particles isolated by IAC) and UC-HDL. AI-LPs were obtained from a single plasma pass over the anti-apoA-I column (N = 2 donors) and compared directly to HDL isolated by sequential UC (N = 4 donors) using LC/MS.

Fig. 5.

Proteins associated with LpA-I and LpA-I/A-II particles isolated from plasma and UC-HDL. A: The unique proteins found in LpA-I and LpA-I/A-II particles isolated from plasma. B: The unique proteins found in LpA-I and LpA-I/A-II particles segregated from HDL isolated by UC. Proteomic data was obtained from plasma and UC-HDL acquired from four male donors.

Fig. 6.

Protein spectral count distribution across LpA-I and LpA-I/A-II particles. The protein distribution between LpA-I and LpA-I/A-II particles is shown for subfractions isolated from plasma (A) and UC-HDL (B). The proteins listed were identified in at least two out of the four donors. The bars show means and the asterisks denote statistically significant differences (P < 0.05) found between the subfractions determined by a two-tailed paired Student’s t-test.

Fig. 7.

GO analysis of LpA-I and LpA-I/A-II particle proteomes. A GO analysis was derived from the proteomic fingerprint of LpA-I and LpA-I/A-II particles isolated from plasma. Proteins were grouped by their GO function (listed in supplemental Table S4) and spectral counts were averaged within each population and each donor. Bars represent the average of the four male donors (±SD) and asterisks denote statistically significant differences (P < 0.05) found between the subfractions determined by a two-tailed paired Student’s t-test.

Fig. 8.

Cholesterol efflux capacity of LpA-I and LpA-I/A-II particles separated from plasma and UC-HDL. Cholesterol efflux was measured in RAW 264 mouse macrophages and particles were compared at equal phospholipid mass (20 μg/ml). Cholesterol efflux was quantified in the presence and absence of cAMP with the difference assumed to represent ABCA1-mediated cholesterol efflux. A: LpA-I and LpA-I/A-II isolated from plasma. B: Same subspecies separated from UC-HDL. Bars represent the average of four donors (±SD) and asterisks denote statistically significant differences (P < 0.05) found between the subfractions determined by a two-tailed paired Student’s t-test.

Fig. 9.

Cholesterol efflux capacity of rLpA-I and rLpA-I/A-II POPC particles and lipid-free plasma apoA-I and apoA-II. A: rLpA-I and rLpA-I/A-II POPC particles were present at equal PC mass (20 μg/ml) and cholesterol efflux capacity was quantified in the presence and absence of cAMP. B: Lipid-free plasma apoA-I, apoA-II, and a mixture of both at a molar ratio of 200:95 apoA-I:apoA-II were loaded at equal protein mass (10 μg/ml) and cholesterol efflux capacity was measured in the presence and absence of cAMP. The bars represent the average of three independent preparations (±SD) and the asterisks denote statistically significant differences (P < 0.05) found between the subfractions determined by a two-tailed paired Student’s t-test (A) and a one-way ANOVA with a post hoc analysis using Tukey’s honestly significant difference (B).

Fig. 10.

Limited proteolysis of LpA-I and LpA-I/A-II. LpA-I and LpA-I/A-II subfractions isolated from plasma and reconstituted discoidal particles were incubated with sequencing-grade trypsin for 0, 7.5, 15, and 30 min at 37°C. The reactions were quenched by addition of SDS sample buffer and boiling samples for 10 min at 100°C. The samples were frozen at −20°C until ready for analysis by SDS-PAGE. The values represent the relative density of the band corresponding to apoA-I expressed as a ratio to the density of the molecular mass markers at 20 and 25 kDa. A: Proteolytic digestion of apoA-I from LpA-I and LpA-I/A-II fractions isolated from plasma. A total of 8 μg of total protein was incubated with trypsin at a mass ratio of 20:1 protein:Trypsin. The points represent the average of three donors (±SD). B: The proteolytic digestion of apoA-I from reconstituted rLpA-I and rLpA-I/A-II particles. A total of 4 μg of total protein was incubated with trypsin at a mass ratio of 40:1 protein:Trypsin. Points represent the average of three independent preparations (±SD). Asterisks denote statistically significant differences (P < 0.05) found between the subfractions at a single time point as determined by a two-tailed paired Student’s t-test.