Lipid-Binding Studies of Human Apolipoprotein A-I and Its Terminally Truncated Mutants

Fang, Yiling; Gursky, Olga; Atkinson, David

doi:10.1021/bi0354031

Cited by 53 publications

(86 citation statements)

References 31 publications

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“…More interestingly, the overall secondary structure of the constructs containing the α-helical domain and the C-sheet domain remains unchanged when these proteins form reconstituted particles with DMPC. This observation is in contrast to apoA-I, whose α-helical content increases upon phospholipid binding (43). These differences indicate structural inelasticity in apoB, which is likely to be a result of protein tertiary folding.…”

Section: Discussionmentioning

confidence: 71%

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

et al. 2006

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Apolipoprotein B (ApoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted α-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogenous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous to lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.Apolipoprotein B (apoB) is the only known nonexchangeable apolipoprotein in humans. It is the major protein component of low density lipoprotein (LDL), a major risk factor for atherosclerosis. Two forms of apoB are synthesized in humans, B48 from in small intestine and B100 in the liver (1). The production of B48 is the result of a tissue specific mRNA processing occurring in the small intestine (2,3). The translation of B48 and B100 in the endoplasmic reticulum (ER) is directly coupled with the assembly of two major classes of lipoproteins: chylomicrons and very low density lipoproteins (VLDL) (4).Although an apoB structure at atomic resolution is still unavailable, it is generally accepted that the full length apoB has an βα1-β1-α2-β2-α3 pentapartite domain organization, in which α represents predominantly α-helical structures and β corresponds to β-sheets (5). According to this model, apoB is depicted as a belt wrapped around a lipoprotein particle (6). Evidence from electron microscopy indicates that the N-terminus of apoB, largely the βα1 domain, is globularly folded and protrudes away from the LDL particle (7,8). These findings are consistent

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Section: Discussionmentioning

confidence: 71%

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

et al. 2006

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“…The characteristics (density, weight ratio, size, composition and the total helix length per particle) of the two types of disks formed by the peptide are similar to those formed by the intact apoA-I under similar conditions [32,39]. This suggests that the C-terminal peptide can mimic the phospholipid interaction of intact apoA-I, also supporting the high lipid affinity of this peptide.…”

Section: Lipid Interactionmentioning

confidence: 88%

“…For each initial lipid:peptide w/w ratio sample, the actual ratio of each fraction containing complexes was calculated from the measured lipid and peptide concentrations. apoA-I/DMPC complexes, simple mixtures and peak fractions after density gradient separation, were visualized by electron microscopy using the negative staining technique [32]. Samples were observed on a CM12 electron microscope (Philips Electron Optics, Eindhoven, the Netherlands).…”

Section: Density Gradient Ultracentrifugationmentioning

confidence: 99%

Conformation and Lipid Binding of a C-Terminal (198−243) Peptide of Human Apolipoprotein A-I

Zhu

Atkinson

2007

Biochemistry

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Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on apoA-I demonstrated that this N-terminal region is unstructured and folds into ~ 60% α-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46 residue peptide, apoA-I, encompassing putative helix repeats 10, 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far ultraviolet circular dichroism spectra show that apoA-I is also unfolded in aqueous solution. However, self-association induces ~ 50% α-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid mimicking detergents, above their CMC, ~ 60% α-helix was induced in the peptide. In contrast, SDS, an anionic lipid mimicking detergent, induced helical folding in the peptide at a concentration of ~ 0.003% (1 00 μM), ~ 70 fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solublize dimyristoyl phosphatidyl choline (DMPC) liposomes and fold into ~ 60% α-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy, demonstrated that the peptide binds to DMPC with high affinity to form at least two sizes of relatively homogenous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide w/w, diameter and density) of both complexes are similar to those of plasma A-I/DMPC formed under similar conditions: small discoidal complexes (~ 3:1 w/w, ~ 110Å and ~ 1.10g/cm 3 ) formed at initial 1:1 w/w ratio and larger discoidal complexes ( ~ 4.6:1 w/w, ~ 165 Å and ~ 1.085g/cm 3 ) formed at initial 4:1 w/w ratio. The cross-linking of the peptide on the two sizes of disks is consistent with the calculated peptide numbers per particle, which result in sufficient helix to surround the lipid bilayer twice. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this Cterminal peptide model can mimic the interaction with phospholipid of plasma apoA-I to form two sizes of homogenous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.The plasma concentration of high-density lipoprotein (HDL) is an inverse marker of potential cardiovascular disease. The well-documented anti-atherogenic function of HDL is related to its critical role in reverse cholesterol transport, such as mediation of cholestero...

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“…The structure of the N-and C-terminal regions have been studied extensively and shown to be mainly non-helical in the lipid-free state but take on some alpha-helical character upon lipid binding (17)(18)(19)(20)(21). Although the N-and C-terminal domains both appear to be involved in maintaining the stability of the lipid-free conformation and binding to lipids, these domains also appear to have distinct functions (5,22,23).Functionally, the C-terminus has been implicated in the initiation of binding to ABCA1 and/ or lipid surfaces (18,24), while the N-terminus has been linked to LCAT activation and HDL maturation (25). Studies show that the Δ43 mutant form of apoA-I exhibits similar lipid binding ability to full length apoA-I (21).…”

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confidence: 99%

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Conformational Adaptation of Apolipoprotein A-I to Discretely Sized Phospholipid Complexes

et al. 2007

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The conformational constraints for apoA-I bound to recombinant phospholipid complexes (rHDL) were attained from a combination of chemical cross-linking and mass spectrometry. Molecular distances were then used to refine models of lipid-bound apoA-I on both 80 and 96Å diameter rHDL particles. To obtain molecular constraints on the protein bound to phospholipid complexes, three different lysine-selective homo-bifunctional cross-linkers with increasing spacer arm lengths (i.e., 7.7, 12.0, and 16.1 Å) were reacted with purified, homogeneous recombinant 1-palmitoyl-2-oleoylsn-glycerol-3-phosphocholine (POPC) apoA-I rHDL complexes of each diameter. Cross-linked dimeric apoA-I products were separated from monomeric apoprotein using 12% SDS PAGE, then subjected to in-gel trypsin digest, and identified by MS/MS sequencing. These studies aid in the refinement of our previously published molecular model of 2 apoA-I molecules bound to ~150 molecules of POPC and suggest that the protein hydrophobic interactions at the N-and C-terminal domains decrease as the number of phospholipid molecules or "lipidation state" of apoA-I increases. Thus, it appears that these incremental changes in the interaction between the N-and C-terminal ends of apoA-I stabilize its tertiary conformation in the lipid-free state as well as allowing it to unfold and sequester discrete amounts of phospholipid molecules.Apolipoprotein A-I (apoA-I) is a 28 kDa protein synthesized by the liver and intestine and is responsible for modulating the formation, metabolism, and catabolism of high density lipoprotein cholesterol. HDL has been known for decades to be a negative risk factor for predicting the development of coronary artery disease in humans, but the specific mechanism (s) responsible for its protective role in cholesterol metabolism continues to be studied and elucidated (1-3).ApoA-I shares a number of similarities to other members of the apoprotein super gene family, as well as possessing a unique set of properties related to its unique role in lipid metabolism (4). Totally soluble in aqueous solution, apoA-I monomers exist in a lipid-free state, but also avidly bind to each other, as well as lipid surfaces (5). Although apoA-I readily binds lipid surfaces, the formation of small apoA-I containing phospholipid and cholesterol particles, an important step in HDL metabolism, does not occur to a significant extent in the absence of the ABCA1 transporter. During the formation of "nascent" HDL, monomers of lipid-free apoA-I bind to ABCA1 which adds phospholipid and cholesterol to yield a particle containing two 1To whom correspondence should be addressed: Dept. of Pathology, Section on Lipid Sciences, Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-2147; Fax 336-716-6279; E-mail:msthomas@wfubmc.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 25. Published in final edited form as:Biochemistry. -I (3,6,7). In this form, the conformation of ...

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Lipid-Binding Studies of Human Apolipoprotein A-I and Its Terminally Truncated Mutants

Cited by 53 publications

References 31 publications

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

Conformation and Lipid Binding of a C-Terminal (198−243) Peptide of Human Apolipoprotein A-I

Conformational Adaptation of Apolipoprotein A-I to Discretely Sized Phospholipid Complexes

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