Speciated human high-density lipoprotein protein proximity profiles

Abstract

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (∼58%), phospholipids (∼21%), total cholesterol (∼16%), triglycerides (∼5%), and free cholesterol (∼4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.

Figure 1

Main experimental steps and techniques used in the study.

Figure 2. Size exclusion chromatography of LpA-I and LpA-I/A-II

Chromatographic traces of LpA-I and LpA-I/A-II upon separation on two Superose 6 columns in tandem (top panel). Each main pool was divided into six individual subfractions based on the elution volume and was numbered 1–6. The individual chromatography profile of each subfraction is shown below the corresponding main pool. The distances between the largest average particle size and the smallest average particle size are shown by d₁ for LpA-I and d₂ for LpA-I/A-II respectively. The average size of the largest and the smallest particles are shown by vertical short dash and long dash lines respectively.

Figure 3. Major apolipoproteins in LpA-I and LpA-I/A-II subfractions as visualized by SDS-PAGE

4–30% SDS-PAGE of isolated LpA-I, LpA-I/A-II subfractions (separated by FPLC, Figure 2; adjusted to 1 mg/ml total protein). ApoA-I, apoA-II band locations as well as albumin contamination (prevalent in LpA-I fractions 6) are indicated. ApoC-I, apoC-II and apoC-III standards were included. Lanes with molecular weight standards are denoted as MWM. Gels were stained with Coomassie Blue.

Figure 4. SDS-PAGE of cross-linked LpA-I and LpA-I/A-II sub-fractions

Individual subfractions of LpA-I and LpA-I/A-II were cross-linked at 1:100 total protein to BS³ cross-linker ratio at 1 mg/ml protein concentration and were subjected to 4–15% SDS-PAGE. Band “b” on Lane 6 (Panel LpA-I) and band “m” on Lane 6 (Panel LpA-I/A-II) correspond to albumin contamination (~66kDa). The small amount of apoA-I that did not involve in intermolecular cross-linking is shown as “free apoA-I”. Labeling of fractions are as in Figures 2 and 3. Low Molecular Weight (LMW) markers are on Lane 7. Gels were stained with Coomassie Blue.

Figure 5. Native PAGE characterization of cross-linked LpA-I and LpA-I/A-II

Individual cross-linked LpA-I and LpA-I/A-II subfractions (at 1 mg/ml total protein concentration), were used in Figure 4 were subjected to 8–25% Native PAGE analysis. The locations of the hydro-dynamic diameters of the High Molecular Weight markers are indicated by arrows. Numbering of the sub-fractions is as in Figures 2, 3 and 4. Location of the LpA-I/A-II fraction 1, which is not clear on the gel, is marked with an arrow. Location of the internally cross-linked albumin in fraction 6 of LpA-I and LpA-I/A-II pools are marked by asterisks. Gels were stained with Coomassie Blue.

Figure 6. MALDI mass spectra of cross-linked LpA-I and LpA-I/A-II subfractions

Individual cross-linked subfractions (shown in Figs. 4 and 5) were subjected to MALDI-MS measurements in 10–200 kDa range. Possible combinations of apolipoproteins attributable to major peaks in the MALDI spectrum is indicated above the vertical dotted lines that connects similar M_r in each spectrum. The peak location corresponds to the albumin contamination is indicated as “albumin”.

Figure 7. Linear regression between theoretical masses for oligomeric apoA-I vs observed masses for cross-linked apoA-I

A. MALDI-MS of self associated lipid free apoA-I upon cross-linking with BS³ at 1:100 protein to BS³ molar ratio with the peak locations marked by solid lines and masses given in parentheses in Italics. Predicted M_r for non cross-linked oligomers are shown by dashed lines along with corresponding M_r in parentheses. B. Linear regression of predicted vs observed M_rs that are on panel A.

Figure 8. Main lipid and total protein compositional analysis of individual LpA-I and LpA-I/A-II subfractions

Total protein and lipid component analyses were carried out using WAKO chemical kits. The components are presented as mass percentages for individual subfractions. Subfraction labeling is as in Figures 2–6. Panels A, total protein; B, phospholipids; C, free cholesterol; D, total cholesterol; E, triglycerides.

Figure 9. Size exclusion chromatography profiles of LpA-I and LpA-I/A-II

LpA-I and LpA-I/A-II were separated on four sizing columns connected in tandem (three Superdex 200 columns followed by a Superose 6 column). LpA-I: black; LpA-I/A-II: grey.