Abstract 409: Site-Specific Solvent Exposure of Major Apolipoproteins in Human High-Density Lipoprotein Complexes of Different Composition and Diameter

Abstract

It is believed that the interactions of high density lipoproteins (HDL) with different plasma enzymes are partly determined by the site specific solvent exposure of apolipoproteins in HDL. Ultra-centrifugally isolated human HDL was separated into subpopulations containing both major apolipoproteins apoA-I and apoA-II (termed LpA-I/A-II) and only apoA-I (termed LpA-I) using thiopropyl sepharose chromatography. LpA-I/A-II and LpA-I were further separated into three sub-fractions based on the particle diameter utilizing fast performance liquid chromatography. Individual sub-fractions were subjected to partial acetylation; the extent of acetylation that represents the solvent exposure was quantified by mass spectrometry (MS). LpA-I of ∼85 Å, demonstrated solvent exposure variations for Lys residues in apoA-I in the range of 9-55%. Lys 133 (helix 5) and Lys 226 (helix 10) showed the highest solvent exposure of 54-55%. Surprisingly Lys residues 238 and 239 showed the lowest exposure of 9-10%. The rest of the Lys residues located in N-term through helix 10 ranged from 12-40% demonstrating distinctive differences among specific sites of exposure. The presence of apoA-II in comparable diameter LpA-I/A-II showed similar solvent exposure for apoA-I, implying the non-perturbing nature of apoA-II on apoA-I conformation. Limited proteolysis experiments resulted in similar fragment patterns for apoA-I in LpA-I and LpA-I/A-II, agreeing with mass spectrometry observations. Furthermore, proteolysis demonstrated that apoA-II in HDL is less susceptible to proteolysis compared to apoA-I in HDL. Confirming this observation further, the extent of acetylation of individual Lys residues of apoA-II in LpA-I/A-II demonstrated overall low solvent exposure (9-14%) by MS. Subtle solvent exposure differences were seen among particles of different diameter (75 Å vs 110 [[Unable to Display Character: Ǻ]]). These novel findings bring out conformational details of major apolipoprotein in different HDL particles. Assessment of site specific solvent exposure of minor HDL apolipoproteins including apoE, apoD, apoCs, apoM and determination of their specific preference for LpA-I vs LpA-I/A-II are currently underway implementing high throughput mass spectrometry techniques.