2 research outputs found
Cholesterol Determines and Limits rHDL Formation from Human Plasma Apolipoprotein A‑II and Phospholipid Membranes
Apolipoprotein (apo) A-II, the second most abundant protein
after
apo A-I of human plasma high-density lipoproteins (HDL), is the most
lipophilic of the exchangeable apolipoproteins. The rate of microsolubilization
of dimyristoylphosphatidylcholine (DMPC) membranes by apo A-I to give
rHDL increases as the level of membrane free cholesterol (FC) increases
up to 20 mol % when the level of reaction decreases to nil. Given
its greater lipophilicity, we tested the hypothesis that apo A-II
and its reduced and carboxymethylated monomer (rcm apo A-II) would
form rHDL at a membrane FC content of >20 mol %. According to turbidimetric
titrations, the DMPC/apo A-II stoichiometry is 65/1 (moles to moles).
At this stoichiometry, apo A-II forms rHDL from DMPC and FC. Contrary
to our hypothesis, apo A-II, like apo A-I, reacts poorly with DMPC
containing ≥20 mol % FC. The rate of formation of rHDL from
rcm apo A-II and DMPC at all FC mole percentages is faster than that
of apo A-II but nil at 20 mol % FC. In parallel reactions, monomeric
and dimeric apo A-II form large FC-rich rHDL coexisting with smaller
FC-poor rHDL; increasing the FC mole percentage increases the number
and size of FC-rich rHDL. On the basis of the compositions of coexisting
large and small rHDL, the free energy of transfer of FC from the smallest
to the largest particle is approximately −1.2 kJ. On the basis
of our data, we propose a model in which apo A-I and apo A-II bind
to DMPC via surface defects that disappear at 20 mol % FC. These data
suggest apo A-II-containing HDL formed intrahepatically are likely
cholesterol-rich compared to the smaller intracellular lipid-poor
apo A-I HDL
Free Cholesterol Determines Reassembled High-Density Lipoprotein Phospholipid Phase Structure and Stability
Reassembled high-density lipoproteins
(rHDL) of various sizes and
compositions containing apo A-I or apo A-II as their sole protein,
dimyristoylphosphatidylcholine (DMPC), and various amounts of free
cholesterol (FC) have been isolated and analyzed by differential scanning
calorimetry (DSC) and by circular dichroism to determine their stability
and the temperature dependence of their helical content. Our data
show that the multiple rHDL species obtained at each FC mole percent
usually do not have the same FC mole percent as the starting mixture
and that the size of the multiple species increases in a quantized
way with their respective FC mole percent. DSC studies reveal multiple
phases or domains that can be classified as virtual DMPC, which contains
a small amount of DMPC that slightly reduces the melting temperature
(<i>T</i><sub>m</sub>), a boundary phase that is adjacent
to the apo A-I or apo A-II that circumscribes the discoidal rHDL,
and a mixed FC/DMPC phase that has a <i>T</i><sub>m</sub> that increases with FC mole percent. Only the large rHDL contain
virtual DMPC, whereas all contain boundary phase and various amounts
of the mixed FC/DMPC phase according to increasing size and FC mole
percent. As reported by others, FC stabilizes the rHDL. For rHDL (apo
A-II) compared to rHDL (apo A-I), this occurs in spite of the reduced
number of helical regions that mediate binding to the DMPC surface.
This effect is attributed to the very high lipophilicity of apo A-II
and the reduction in the polarity of the interface between DMPC and
the aqueous phase with an increasing FC mole percent, an effect that
is expected to increase the strength of the hydrophobic associations
with the nonpolar face of the amphipathic helices of apo A-II. These
data are relevant to the differential effects of FC and apolipoprotein
species on intracellular and plasma membrane nascent HDL assembly
and subsequent remodeling by plasma proteins