Differential effects of lecithin and cholesterol on the immunoreactivity and conformation of apolipoprotein A-I in high density lipoproteins

The effects of different acylglycerides on the conformation and charge of apolipoprotein A-I (apoA-I) have been investigated in reconstituted high density lipoproteins (LpA-I). Various amounts of diacylglycerol (DG) and triacylglycerol (TG) were incorporated into sonicated spherical LpA-I particles containing 2 molecules of apoA-I and 80 molecules of phospholipid. Inclusion of 30 molecules of TG into the LpA-I particle increases the net negative charge of apoA-I ( ؊ 8.5 to ؊ 9.3 mV), but has little effect on the amount and thermodynamic stability of the ␣ helices in apoA-I. Incorporation of 30 molecules of DG into the lipoprotein complex promotes a small increase in the ␣ -helix content and stability, but greatly increases the net negative charge of apoA-I ( ؊ 8.5 to ؊ 11.2 mV). Inclusion of DG increases the immunoreactivity of two epitopes in the N terminus of apoA-I, but decreases the exposure of a domain closer to the C terminus (residues 148-186) of the apoprotein. In contrast, TG increases the exposure of epitopes over the entire apoA-I molecule; TG increases the immunoreactivity of epitopes for 13 different monoclonal antibodies to apoA-I. Incubations with purified lecithin:cholesterol acyltransferase show that cholesterol esterification is stimulated by DG, but inhibited by TG. The data show that TG and DG have different effects on apoA-I structure and function and this suggests that the TG-to-DG ratio in HDL may directly affect the metabolism of this lipoprotein class. -Braschi, S.

Section: Effect Of Dg On Apoa-i Tertiary Structure In Lpa-i Particlessupporting

confidence: 71%

Effect of acylglyceride content on the structure and function of reconstituted high density lipoprotein particles

Braschi

Coffill²,

Neville

et al. 2001

“…We located a PLTP-binding site between amino acids 27-141, and the most effective competitor for PLTP binding to apoA-I in an ELISA assay was a monoclonal anti-apoA-I antibody whose epitope is located between amino acids 27-48. The immunoreactivity of these antibodies is influenced by phospholipid and cholesterol (53,54), which are both transferred by PLTP (8,55). The antibodies with C-terminal or the most N-terminal epitopes had no effect on PLTP binding.…”

Section: Discussionmentioning

confidence: 96%

Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I

Pussinen

Jauhiainen

Metso

et al. 1998

The interaction of plasma phospholipid transfer protein (PLTP) with HDL has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-mediated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule. To study the PLTP/apolipoprotein interaction we used a solid phase ligand binding assay, the ELISA technique, and apoA-I and apoA-II affinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a concentration-dependent manner, and the binding could be displaced by preincubating the PLTP sample with purified apolipoproteins. To determine which portion of apoA-I is recognized by PLTP, we coated ELISA plates with either recombinant full-length apoA-I or three shortened apoA-I forms sequentially truncated from the C-terminus. To characterize the PLTP binding ability of the C-terminal region of apoA-I, we used both C-terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. To further confirm the identity of the binding region, we probed the interaction with a polyclonal and several monoclonal anti-apoA-I antibodies. The antibodies that inhibited the interaction between PLTP and apoA-I were directed towards apoA-I epitopes localized between amino acids 27-141. The polyclonal antibody, R33, and the monoclonal antibody A-I-1 (epitope between amino acids 27-48) were most effective and reduced PLTP binding by 70%. These results show that PLTP binds to both apoA-I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.

“…It also leads to reduced protease accessibility suggesting a major conformational change upon association with lipids (30,63). Studies from our laboratory using mAbs as conformational probes have also indicated that apoA-I undergoes major conformational changes upon lipid binding (53,54,64). In the case of discoidal complexes, specific domains are affected with increased phospholipid or cholesterol content, i.e., changes in epitope accessibility are mostly observed in the central (residues and N-terminal domain (residues 2-8).…”

Section: Mode Of Interaction With Lipidsmentioning

confidence: 99%

“…This property is also shared by other apolipoproteins and suggests a very similar mechanism of association with lipids for all exchangeable apolipoproteins. ApoA-I immunoreactivity toward mAbs has also been shown to be altered by the presence of lipids (53,54), which indicates important conformational changes affecting most domains upon association with lipids.…”

Section: Mode Of Interaction With Lipidsmentioning

confidence: 99%

Apolipoprotein A-I: structure–function relationships

Frank

Marcel

2000

266

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic ␣ -helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent ␣ -helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal ␣ -helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I ␣ -helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates.No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism. -Frank, P. G., and Y. L. Marcel. Apolipoprotein A-I: structure-function relationships.