Rotational and hinge dynamics of discoidal high density lipoproteins probed by interchain disulfide bond formation

To develop a detailed double belt model for discoidal HDL, we previously scored inter-helical salt bridges between all possible registries of two stacked antiparallel amphipathic helical rings of apolipoprotein (apo) A-I. The top score was the antiparallel apposition of helix 5 with 5 followed closely by appositions of helix 5 with 4 and helix 5 with 6. The rationale for the current study is that, for each of the optimal scores, a pair of identical residues can be identified in juxtaposition directly on the contact edge between the two antiparallel helical belts of apoA-I. Further, these residues are always in the ‘9th position’ in one of the eighteen 11-mer repeats that make up the lipid-associating domain of apoA-I. To illustrate our terminology, 129j (LL5/5) refers to the juxtaposition of the Cα atoms of G129 (in a ‘9th position’) in the pairwise helix 5 domains. We reasoned that if identical residues in the double belt juxtapositions were mutated to a cysteine and kept under reducing conditions during disc formation, we would have a precise method for determining registration in discoidal HDL by formation of a disulfide-linked apoA-I homodimer. Using this approach, we conclude that 129j (LL5/5) is the major rotamer orientation for double belt HDL and propose that the small ubiquitous gap between the pairwise helix 5 portions of the double belt in larger HDL discoidal particles is significantly dynamic to hinge off the disc edge under certain conditions, e.g., in smaller particles or perhaps following binding of the enzyme LCAT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Rotational and hinge dynamics of discoidal high density lipoproteins probed by interchain disulfide bond formation

To develop a detailed double belt model for discoidal HDL, we previously scored inter-helical salt bridges between all possible registries of two stacked antiparallel amphipathic helical rings of apolipoprotein (apo) A-I. The top score was the antiparallel apposition of helix 5 with 5 followed closely by appositions of helix 5 with 4 and helix 5 with 6. The rationale for the current study is that, for each of the optimal scores, a pair of identical residues can be identified in juxtaposition directly on the contact edge between the two antiparallel helical belts of apoA-I. Further, these residues are always in the '9th position' in one of the eighteen 11-mer repeats that make up the lipid-associating domain of apoA-I. To illustrate our terminology, 129j (LL5/5) refers to the juxtaposition of the Cα atoms of G129 (in a '9th position') in the pairwise helix 5 domains. We reasoned that if identical residues in the double belt juxtapositions were mutated to a cysteine and kept under reducing conditions during disc formation, we would have a precise method for determining registration in discoidal HDL by formation of a disulfide-linked apoA-I homodimer. Using this approach, we conclude that 129j (LL5/5) is the major rotamer orientation for double belt HDL and propose that the small ubiquitous gap between the pairwise helix 5 portions of the double belt in larger HDL discoidal particles is significantly dynamic to hinge off the disc edge under certain conditions, e.g., in smaller particles or perhaps following binding of the enzyme LCAT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

A robust all-atom model for LCAT generated by homology modeling

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A₂ (PLA₂) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA₂. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.     

Different Functional and Structural Characteristics between ApoA-I and ApoA-4 in Lipid-Free and Reconstituted HDL State: ApoA-4 Showed Less Anti-Atherogenic Activity

Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS₃-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties.     

Thermal Stability of Apolipoprotein A-I in High-Density Lipoproteins by Molecular Dynamics

Apolipoprotein (apo) A-I is an unusually flexible protein whose lipid-associated structure is poorly understood. Thermal denaturation, which is used to measure the global helix stability of high-density lipoprotein (HDL)-associated apoA-I, provides no information about local helix stability. Here we report the use of temperature jump molecular dynamics (MD) simulations to scan the per-residue helix stability of apoA-I in phospholipid-rich HDL. When three 20 ns MD simulations were performed at 500 K on each of two particles created by MD simulations at 310 K, bilayers remained intact but expanded by 40%, and total apoA-I helicity decreased from 95% to 72%. Of significance, the conformations of the overlapping N- and C-terminal domains of apoA-I in the particles were unusually mobile, exposing hydrocarbon regions of the phospholipid to solvent; a lack of buried interhelical salt bridges in the terminal domains correlated with increased mobility. Nondenaturing gradient gels show that 40% expansion of the phospholipid surface of 100:2 particles by addition of palmitoyloleoylphosphatidylcholine exceeds the threshold of particle stability. As a unifying hypothesis, we propose that the terminal domains of apoA-I are phospholipid concentration-sensitive molecular triggers for fusion/remodeling of HDL particles. Since HDL remodeling is necessary for cholesterol transport, our model for remodeling has substantial biomedical implications.     

Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 degrees C from 0.5 to 24 h. The products were analyzed by non-denaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 A A-II-POPCrHDL was a rearrangement into 78 A particles containing apoA-I and/or apoA-II, and 90 A particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 A A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 A to 105 A containing apoA-I and/or apoA-II, with main particles of 78 A, 88 A, and 98 A. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent V(m)ax and a 6-fold decrease in apparent K(m), while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent K(m); there was no significant change in apparent V(m)ax. Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism.     

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL₂ and HDL₃ were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K_d) measured for WT human apoA-I interactions with HDL₂ and HDL₃ are about 10 µM, indicating that the binding is low affinity. This K_d value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.Understanding the structure and function of HDL is significant because of the beneficial cardioprotective properties of this lipoprotein (1). The anti-atherogenic effects of HDL arise, in part, from its participation in the reverse cholesterol transport pathway where the principal HDL protein, apolipoprotein A-I (apoA-I), plays a central role (2). As a result, the structure-function relationships of apoA-I have been studied extensively (for reviews, see Refs. 3–5). Perhaps the most important characteristic of the apoA-I molecule is its ability to bind lipids; this interaction is mediated by the amphipathic α-helices present in the protein molecule (6). ApoA-I binds well to phospholipid (PL)-water interfaces and, under appropriate conditions, can solubilize the PL to create discoidal HDL particles (7, 8). The binding of apoA-I to a PL surface involves a two-step mechanism. First, α-helices in the C-terminal domain of the protein interact with the surface, and, second, the N-terminal helix bundle domain opens to allow more helix-lipid interactions to occur (5, 9). Although the binding of apoA-I to model PL particles has been studied extensively, the binding of apoA-I to HDL particles has not been investigated much because of the difficulty of separating free and bound apoA-I in this system. This lack of information about apoA-I/HDL interactions is significant because the cycling of apoA-I molecules on and off HDL particles occurs during the metabolism of HDL particles (10, 11), in particular to release apoA-I molecules into the preβ-HDL pool (10, 12). This recycling is consistent with the well-established ability of apolipoproteins, such as apoA-I, to exchange spontaneously between different populations of lipoprotein particles (13–16) and PL vesicles (17, 18). As a rule, any remodeling event that depletes HDL particles of PL induces particle fusion and dissociation of that fraction of the apoA-I molecules that is in a labile pool (19). At this stage, quantitative understanding of the kinetics of apoA-I interactions with HDL particles is unavailable.Here, we exploit surface plasmon resonance (SPR) to monitor in real time the association and dissociation reactions in the apoA-I/HDL system. SPR has been used to derive quantitative information about the binding of both lipoproteins (20) and apoE (21–23) to proteoglycans. As far as the application of SPR to the HDL system is concerned, the binding of several plasma remodeling factors to HDL immobilized on a sensor chip has been investigated successfully (24–26). Also, the conformation of apoA-I in HDL was explored by comparing the binding of HDL particles to anti-apoA-I monoclonal antibodies immobilized on an SPR chip (27). We have extended these approaches to study the binding of apoA-I to HDL particles. The results show that apoA-I can bind reversibly and with low affinity to HDL particles by a two-step mechanism.     

An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

Lecithin cholesterol acyltransferase (LCAT) plays a key role in the reverse cholesterol transport (RCT) process by converting cholesterol to cholesteryl ester to form mature HDL particles, which in turn deliver cholesterol back to the liver for excretion and catabolism. HDL levels in human plasma are negatively correlated with cardiovascular risk and HDL functions are believed to be more important in atheroprotection. This study investigates whether and how D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, influences LCAT activity in the completion of the RCT process. We demonstrated that the apparent rate constant value of the LCAT enzyme reaction gives a measure of LCAT activity and determined the effects of free metals and a reducing agent on LCAT activity, showing an inhibition hierarchy of Zn²⁺>Mg²⁺>Ca²⁺ and no inhibition with β-mercaptoethanol up to 10 mM. We reconstituted nano-disc particles using apoA-I or D-4F with phospholipids. These particles elicited good activity in vitro in the stimulation of cholesterol efflux from macrophages through the ATP-binding cassette transporter A1 (ABCA1). With these particles we studied the LCAT activity and demonstrated that D-4F did not activate LCAT in vitro. Furthermore, we have done in vivo experiments with apoE-null mice and demonstrated that D-4F (20 mg/kg body weight, once daily subcutaneously) increased LCAT activity and HDL level as well as apoA-I concentration at 72 hours post initial dosing. Finally, we have established a correlation between HDL concentration and LCAT activity in the D-4F treated mice.     

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1^−/−) mice were used. Apoa1^−/− mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1^−/− mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1^−/− mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.     

Single repeat deletion in ApoA-I blocks cholesterol esterification and results in rapid catabolism of delta6 and wild-type ApoA-I in transgenic mice

The deletion mutation Delta6 apolipoprotein A-I lacks residues 143-164 or repeat 6 in the mature apoA-I protein. In vitro studies show this mutation dramatically reduces the rate of lecithin:cholesterol acyltransferase (LCAT) catalyzed cholesterol esterification. The present study was initiated to investigate the effect of this mutation on in vivo high density lipoprotein (HDL) cholesterol esterification and metabolism. Transgenic mice expressing human Delta6 apoA-I (TgDelta6 +/+) were created and then crossed with apoA-I knockout mice (-/-) to generate mice expressing only human Delta6 apoA-I (TgDelta6 -/-). Human Delta6 apoA-I was associated with homogeneous sized alpha-HDL, when wild-type mouse apoA-I was present (in TgDelta6 +/+ and +/- mice). However, in the absence of endogenous mouse apoA-I, Delta6 apoA-I was found exclusively in cholesterol ester-poor HDL, and lipid-free HDL fractions. This observation coincides with the 6-fold lower cholesterol ester mass in TgDelta6 -/- mouse plasma compared with control. Structural studies show that despite the structural perturbation of a domain extending from repeat 5 to repeat 8 (137-178), Delta6 apoA-I binds to spherical unilamellar vesicles with only 2-fold less binding affinity. In summary, these data show a domain corresponding to apoA-I repeat 6 is responsible for providing an essential conformation for LCAT catalyzed generation of cholesterol esters. Deletion of apoA-I repeat 6 not only blocks normal levels of cholesterol esterification but also exerts a dominant inhibition on the ability of wild-type apoA-I to activate LCAT in vivo.     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

"Sticky" and "promiscuous", the yin and yang of apolipoprotein A-I termini in discoidal high-density lipoproteins: a combined computational-experimental approach

Apolipoprotein (apo) A-I-containing lipoproteins in the form of high-density lipoproteins (HDL) are inversely correlated with atherosclerosis. Because HDL is a soft form of condensed matter easily deformable by thermal fluctuations, the molecular mechanisms for HDL remodeling are not well understood. A promising approach to understanding HDL structure and dynamics is molecular dynamics (MD). In the present study, two computational strategies, MD simulated annealing (MDSA) and MD temperature jump, were combined with experimental particle reconstitution to explore molecular mechanisms for phospholipid- (PL-) rich HDL particle remodeling. The N-terminal domains of full-length apoA-I were shown to be "sticky", acting as a molecular latch largely driven by salt bridges, until, at a critical threshold of particle size, the associated domains released to expose extensive hydrocarbon regions of the PL to solvent. The "sticky" N-termini also associate with other apoA-I domains, perhaps being involved in N-terminal loops suggested by other laboratories. Alternatively, the overlapping helix 10 C-terminal domains of apoA-I were observed to be extremely mobile or "promiscuous", transiently exposing limited hydrocarbon regions of PL. Based upon these models and reconstitution studies, we propose that separation of the N-terminal domains, as particles exceed a critical size, triggers fusion between particles or between particles and membranes, while the C-terminal domains of apoA-I drive the exchange of polar lipids down concentration gradients between particles. This hypothesis has significant biological relevance since lipid exchange and particle remodeling are critically important processes during metabolism of HDL particles at every step in the antiatherogenic process of reverse cholesterol transport.     

Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements

The central region of apolipoprotein A-I (apoA-I), spanning residues 143--165, has been implicated in lecithin:cholesterol acyltransferase (LCAT) activation and also in high density lipoprotein (HDL) structural rearrangements. To examine the role of individual amino acids in these functions, we constructed, overexpressed, and purified two additional point mutants of apoA-I (P143R and R160L) and compared them with the previously studied V156E mutant. These mutants have been reported to occur naturally and to affect HDL cholesterol levels and cholesterol esterification in plasma. The P143R and R160L mutants were effectively expressed in Escherichia coli as fusion proteins and were isolated in at least 95% purity. In the lipid-free state, the mutants self-associated similarly to wild-type protein. All the mutants, including V156E, were able to lyse dimyristoylphosphatidylcholine liposomes. In the lipid-bound state, the major reconstituted HDL (rHDL) of the mutants had diameters similar to wild type (96--98 A). Circular dichroism and fluorescence methods revealed no major differences among the structures of the lipid-free or lipid-bound mutants and wild type. In contrast, the V156E mutant had exhibited significant structural, stability, and self-association differences compared with wild-type apoA-I in the lipid-free state, and formed rHDL particles with larger diameters. In this study, limited proteolytic digestion with chymotrypsin showed that the V156E mutant, in lipid-free form, has a distinct digestion pattern and surface exposure of the central region, compared with wild type and the other mutants. Reactivity of rHDL with LCAT was highest for wild type (100%), followed by P143R (39%) and R160L (0.6%). Tested for their ability to rearrange into 78-A particles, the rHDL of the two mutants (P143R and R160L) behaved normally, compared with the rHDL of V156E, which showed no rearrangement after the 24-h incubation with low density lipoprotein (LDL). Similarly, the rHDL of V156E was resistant to rearrangement in the presence of apoA-I or apoA-II. These results indicate that structural changes are absent or modest for the P143R and R160L mutants, especially in rHDL form; that these mutants have normal conformational adaptability; and that LCAT activation is obliterated for R160L.Thus, individual amino acid changes may have markedly different structural and functional consequences in the 143--165 region of apoA-I. The R160L mutation appears to have a direct effect in LCAT activation, while the P143R mutation results in only minor structural and functional effects. Also, the processes for LCAT activation and hinge mobility appear to be distinct even if the same region of apoA-I is involved. -- Cho, K-H., D. M. Durbin, and A. Jonas. Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements. J. Lipid Res. 2001. 42: 379--389.     

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.     

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer’s disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT^−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer’s-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.     

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.     

Characterization and properties of pre beta-HDL particles formed by ABCA1-mediated cellular lipid efflux to apoA-I

The contribution of ABCA1-mediated efflux of cellular phospholipid (PL) and cholesterol to human apolipoprotein A-I (apoA-I) to the formation of pre beta 1-HDL (or lipid-poor apoA-I) is not well defined. To explore this issue, we characterized the nascent HDL particles formed when lipid-free apoA-I was incubated with fibroblasts in which expression of the ABCA1 was upregulated. After a 2 h incubation, the extracellular medium contained small apoA-I/PL particles (pre beta 1-HDL; diameter = 7.5 +/- 0.4 nm). The pre beta 1-HDL (or lipid-poor apoA-I) particles contained a single apoA-I molecule and three to four PL molecules and one to two cholesterol molecules. An apoA-I variant lacking the C-terminal alpha-helix did not form such particles when incubated with the cell, indicating that this helix is critical for the formation of lipid-poor apoA-I particles. These pre beta 1-HDL particles were as effective as lipid-free apoA-I molecules in mediating both the efflux of cellular lipids via ABCA1 and the formation of larger, discoidal HDL particles. In conclusion, pre beta 1-HDL is both a product and a substrate in the ABCA1-mediated reaction to efflux cellular PL and cholesterol to apoA-I. A monomeric apoA-I molecule associated with three to four PL molecules (i.e., lipid-poor apoA-I) has similar properties to the lipid-free apoA-I molecule.     

Characterization of human apolipoprotein A-I expressed in Escherichia coli

Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)₆-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/l of culture medium. We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques. The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine. His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles. Lipid-bound native apoA-I and His-apoA-I showed very similar α-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I). The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes. In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values. Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I. The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture. In conclusion, we show that His-apoA-I expressed in E. coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux.     

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO⁻) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN⁻), yielding CNO⁻ and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on “lipid-poor” apoA-I forms via the nonenzymatic CNO⁻ pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.