Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve

Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

The C-terminal domain of apolipoprotein A-I is involved in ABCA1-driven phospholipid and cholesterol efflux

ABCA1, a member of the ATP-binding cassette family, mediates the efflux of cellular lipids to free apolipoproteins, mainly apoA-I. The role of the C-terminal domain of apoA-I in this process has been evaluated by measuring the efflux capacity of a truncated form (apoA-I-(1-192)) versus intact apoA-I in different cellular models. In stimulated J774 macrophages, cholesterol efflux to apoA-I-(1-192) was remarkably lower than that to the intact apoA-I. The truncated apoA-I, lacking an important lipid-binding domain, was also significantly less efficient in removing phospholipids from stimulated macrophages. No difference was detected with stimulated Tangier fibroblasts that do not express functional ABCA1. The C-terminal domain of apoA-I is clearly involved in ABCA1-driven lipid efflux. Independent of the interaction with the cell surface, it may be the decreased ability of the truncated apoA-I to recruit membrane phospholipids that impairs its capacity to promote cell cholesterol efflux.     

Brefeldin A inhibits cholesterol efflux without affecting the rate of cellular uptake and re-secretion of apolipoprotein A-I in adipocytes

A possible role of cellular uptake and re-secretion of apoA-I in the mechanism of cholesterol efflux induced by apoA-I was investigated using a novel experimental approach. Incubation of adipocytes with a recombinant human apoA-I containing a consensus PKA phosphorylation site, pka-ApoA-I, leads to the appearance of phosphorylated protein in the cell culture medium unambiguously proving cellular uptake and re-secretion of pka-ApoA-I. Phosphorylation of apoA-I is abolished by PKA inhibitors and enhanced by PKA activators demonstrating the specific involvement of PKA. Studies on the concentration dependence of pka-apoA-I phosphorylation and competition experiments with human apoA-I suggest that apolipoprotein uptake is a receptor mediated process. A possible role of apoA-I recycling in the mechanism of cholesterol efflux was investigated by determining the rates of apoA-I induced cholesterol efflux and apoA-I recycling in the presence and in the absence of Brefeldin A (BFA). The studies showed that BFA strongly inhibits cholesterol efflux without affecting the rate of apoA-I recycling. Since BFA affects vesicular trafficking of ABCA1, this study suggests that the interaction of apoA-I with ABCA1 does not mediate apolipoprotein uptake and re-secretion. This result suggests that lipidation of apoA-I and apolipoprotein uptake/re-secretion are independent processes.     

The extreme N-terminal region of human apolipoprotein A-I has a strong propensity to form amyloid fibrils

The N-terminal 1–83 residues of apolipoprotein A-I (apoA-I) have a strong propensity to form amyloid fibrils, in which the 46–59 segment was reported to aggregate to form amyloid-like fibrils. In this study, we demonstrated that a fragment peptide comprising the extreme N-terminal 1–43 residues strongly forms amyloid fibrils with a transition to β-sheet-rich structure, and that the G26R point mutation enhances the fibril formation of this segment. Our results suggest that in addition to the 46–59 segment, the extreme N-terminal region plays a crucial role in the development of amyloid fibrils by the N-terminal fragment of amyloidogenic apoA-I variants.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

Fructated apolipoprotein A-I showed severe structural modification and loss of beneficial functions in lipid-free and lipid-bound state with acceleration of atherosclerosis and senescence

Non-enzymatic glycation of serum apolipoproteins is a main feature of diabetes mellitus under hyperglycemia. Advanced glycation end products are implicated in the development of aging and metabolic syndrome, including premature atherosclerosis in diabetic subjects. ApoA-I is the principal protein constituent of HDL. In this study, glycated human apoA-I (gA-I) by fructation was characterized on functional and structural correlations in lipid-free and lipid-bound states.The gA-I showed more spontaneous multimeric band formation up to pentamer and exhibited slower elution profile with more degraded fragments from fast protein liquid chromatography. The gA-I showed modified secondary structure from fluorescence and circular dichroism analysis. Reconstituted high-density lipoprotein (rHDL) containing the gA-I had less content of phospholipid with a much smaller particle size than those of rHDL-containing nA-I (nA-I-rHDL). The rHDL containing gA-I (gA-I-rHDL) consisted of less molecular number of apoA-I than nA-I-rHDL with decreased α-helical content. Treatment of the gA-I-rHDL induced more atherogenic process in macrophage cell and premature senescence in human dermal fibroblast cell.Conclusively, fructose-mediated apoA-I glycation resulted in severe loss of several beneficial functions of apoA-I and HDL regarding anti-senescence and anti-atherosclerosis activities due to a lack of anti-oxidant activity with increased susceptibility of protein degradation and structural modification.     

Structure of apolipoprotein A-I N terminus on nascent high density lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.     

Nerve Regeneration and Cholesterol Reutilization Occur in the Absence of Apolipoproteins E and A-I in Mice

Abstract: Apolipoproteins have been implicated in the salvage and reutilization of myelin cholesterol during Wallerian degeneration and the subsequent nerve regeneration. Current evidence suggests that myelin cholesterol complexes with apolipoproteins E and A-I to form lipoproteins that are taken up via low-density lipoprotein receptors on myelinating Schwann cells. We recently reported, however, that apolipoprotein E is not required for nerve regeneration or reutilization of myelin cholesterol. We have now investigated nerve regeneration and the reutilization of cholesterol in mutant mice deficient in both apolipoproteins E and A-I. Morphologic examination of nerves 4 and 12 weeks after crush injury revealed that regeneration proceeded at a normal rate in the absence of these apolipoproteins. Autoradiography of regenerating nerves indicated that prelabeled myelin lipid was reutilized in the regenerating myelin. 3-Hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was down-regulated in the regenerating nerves, indicative of cholesterol uptake via lipoproteins. Prelabeled myelin cholesterol was present in lipoprotein fractions isolated from crushed nerves of mutant mice. These data suggest that there is considerable redundancy in the process of cholesterol reutilization within nerve, and that apolipoproteins other than apolipoproteins E and A-I may be involved in the recycling of myelin cholesterol.     

Optic Nerve Regeneration in Adult Fish and Apolipoprotein A-I

Fish optic nerves, unlike mammalian optic nerves, are endowed with a high capacity to regenerate. Injury to fish optic nerves causes pronounced changes in the composition of pulse-labeled substances derived from the surrounding non-neuronal cells. The most prominent of these injury-induced changes is in a 28-kilodalton (kDa) polypeptide whose level increases after injury, as revealed by one-dimensional gel electrophoresis and autoradiography. The present study identified as apolipoprotein A-I (apo-A-I) a polypeptide of 28 kDa in media conditioned by regenerating fish optic nerves. The level of this polypeptide increased after injury by approximately 35%. Apo-A-I was isolated by gel-permeation chromatography from delipidated high-density lipoproteins (HDL) that had been obtained from carp plasma by sequential ultracentrifugation. Further identification of the purified protein as apo-A-I was based on its molecular mass (28 kDa) as determined by gel electrophoresis, amino acid composition, and microheterogeneity studies. The isolated protein was further analyzed by immunoblots of two-dimensional gels and was found to contain six isoforms. Western blot analysis using antibodies directed against the isolated plasma protein showed that the 28-kDa polypeptide in the preparation of soluble substances derived from the fish optic nerves (conditioned media, CM) cross-reacted immunologically with the isolated fish plasma apo-A-I. Immunoblots of two-dimensional gels revealed the presence of three apo-A-I isoforms in the CM of regenerating fish optic nerves (pIs: 6.49, 6.64, and 6.73). At least some of the apo-A-I found in the CM is derived from the nerve, as was shown by pulse labeling with [35S]methionine, followed by immunoprecipitation. The apo-A-I immunoactive polypeptides in the CM of the fish optic nerve were found in high molecular-weight, putative HDL-like particles. Immunocytochemical staining revealed that apo-A-I immunoreactive sites were present in the fish optic nerves. Higher labeling was found in injured nerves (between the site of injury and the brain) than in non-injured nerves. The accumulation of apo-A-I in nerves that are capable of regenerating may be similar to that of apo-E in sciatic nerves of mammals (a regenerative system); in contrast, although its synthesis is increased, apo-A-I does not accumulate in avian optic nerves nor does apo-E in rat optic nerves (two nonregenerative systems).     

High level expression of human apolipoprotein A-I in transgenic rats raises total serum high density lipoprotein cholesterol and lowers rat apolipoprotein A-I

To examine the consequences of increased apolipoprotein A-I production on cholesterol and lipoprotein metabolism, we have produced two lines of transgenic rats; one expressing moderate and one very high levels of human apolipoprotein A-I. The rats were produced by microinjection of a 13 kbp DNA fragment containing the human apolipoprotein A-I gene plus 10 kbp of its 5′ flanking sequence and 1 kbp of its 3′ flanking sequence. Both lines of transgenic rats express human apolipoprotein A-I mRNA in liver and human apolipoprotein A-I in plasma. Sera from these rats contain significantly higher levels of total apolipoprotein A-I, high density lipoprotein cholesterol and phospholipid than sera from non-transgenic littermates. Transgenic rats expressing high levels of human apolipoprotein A-I have reduced levels of serum rat apolipoprotein A-I suggesting a mechanism exists to down-regulate apolipoprotein A-I production. These transgenic rats provide a unique animal model to examine the effects of increased apolipoprotein A-I production on lipid and lipoprotein metabolism.     

A 42 K protein of chick sciatic nerve is immunologically related to PO protein of peripheral nerve myelin

The major protein (PO) in PNS myelin is an integral membrane glycoprotein with a molecular weight of about 30 K. The level of PO protein in the developing sciatic nerve of the chicken was monitored by a solid-phase immunoassay and densitometry of Coomassie blue stained polyacrylamide gels. The most rapid rate of accumulation of PO protein occurred after 16 days of embryonic development. In addition to the 30 K PO protein, a number of higher molecular weight proteins could be distinctly detected by immunoblotting. Amongst these high molecular weight proteins, a species with an apparent molecular weight of 42 K was specifically immunostained with epitope-selected polyclonal antibodies against PO protein. This 42 K protein could be first detected after 16 days of embryonic development and increased rapidly following the pattern of myelination in the sciatic nerve. The enzyme endoglycosidase F, which specifically removes N-asparagine linked high mannose and complex carbohydrates from glycoproteins, converted the PO and 42 K proteins to lower molecular weight forms, which could be specifically immunostained by epitope selected polyclonal antibodies to the PO protein. Subcellular fractionation of the 17-day embryonic nerve demonstrated that the 42 K protein was enriched in myelin and microsomal subfractions relative to the total homogenate. These results indicate that the 42 K immuno-crossreactive protein might be chemically and functionally related to the PO protein of the PNS myelin.Abbreviations used CNP 2–3-cyclic-nucleotide 3-phosphodiesterase (EC 3.1.4.37) - EDTA ethylene(dinitrilo)tetraacetic acid - Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - NADPH Cyt. Red. NADPH-cytochrome C reductase (EC 1.6.2.4) - 5-NPase 5-ribonucleotide phosphohydrolase (EC 3.1.3.5) - PNS peripheral nervous system - SDS sodium dodecyl sulfate - TPPase thiamine pyrophosphatase (EC 3.1.6.X)     

The “beta-clasp” model of apolipoprotein A-I — A lipid-free solution structure determined by electron paramagnetic resonance spectroscopy

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. 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).     

Functional independence of a peptide with the sequence of human apolipoprotein A-I central region

Previous results [J. Biol. Chem. 276 (2001) 16978] indicated that an apolipoprotein A-I (apoAI) central region swings away from lipid contact in discoidal high density lipoproteins (HDL), but it is able to penetrate into the bilayer of lipid vesicles. In this work, we have studied the interaction with lipid membranes of a synthetic peptide with the sequence of apoAI region between residues 77 and 120 (AI 77-120). Like apoAI, AI 77-120 binds to phospholipid vesicles and shows selectivity for cholesterol-containing membranes. Moreover, AI 77-120 promotes cholesterol desorption from membranes in a similar fashion as apoAI and can stimulate cholesterol efflux from Chinese hamster ovary cells. AI 77-120 has a considerable alpha-helical content in water solution, and its secondary structure is not largely modified after binding to membranes. Both apoA-I and AI 77-120 are oligomeric in the lipid-bound state, suggesting that dimerization of the central domain could be required for the membrane binding activity of apoA-I in HDL.     

Tetrahydrocortisol-apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes

Tetrahydrocortisol stimulates DNA and protein biosynthesis in hepatocytes only when it enters the complex with apolipoprotein A-I. Tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex specifically interacts with eukaryotic DNA isolated from rat liver. In the process of interaction, rupture of hydrogen bonds between the pairs of nitrous bases occurs with the formation of single-stranded DNA structures. In such state DNA forms complexes with DNA-dependent RNA-polymerase. The most probable site of binding the tetrahydrocortisol–apolipoprotein A-I complex with DNA is the sequence of CC(GCC)_n type entering the structure of many genes, among them the structure of human apolipoprotein A-I gene. Oligonucleotide of this type has been synthesized. Association constant (K_ass) of it with tetrahydrocortisol–apolipoprotein A-I complex was shown to be 1.66×10⁶ M⁻¹. Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K_ass. It was assumed that in the GC-pairs of the given sequence tetrahydrocortisol itself participates in the formation of hydrogen bonds with cytosine, favoring their rupture with complementary base—guanine.     

Biophysical characterization of interaction between apolipoprotein A-I and bacterial lipopolysaccharide

We studied the effect of bacterial lipopolysaccharide (LPS)-apolipoprotein A-I (apo A-I) interaction on the structure and function of this protein. The micellization process of dimirystoil phosphatidylcholine liposomes (MLV-DMPC) by apo A-I in the presence of LPS was characterized. Apo A-I may interact with MLV-DMPC at the lipid transition temperature, forming micellar complexes. The kinetics of MLV-DMPC micellization was studied by turbidimetry. In the absence of LPS, a monoexponential decrease in turbidity is observed. Preincubation of apo A-I with LPS impairs the micellization reaction, resulting in biphasic kinetics. The amplitude of the fast phase decreases with increasing concentrations of LPS. In the absence or in the presence of low amounts of LPS (1∶0.1 protein:LPS weight ratio), two major micellization products-containing two and three apo A-I molecules per particle-were observed. However, in the presence of higher amounts of LPS (1∶1 protein:LPS weight ratio), particles mainly contained two apo A-I molecules. In contrast, a decrease in intrinsic fluorescence intensity of the protein was observed in the presence of an increasing LPS concentration. Finally, we studied the effect of LPS on the transition temperature (Tt) of MLV-DMPC without detecting changes in Tt. In conclusion, the changes found in the micellization process are likely to be mainly caused by changes in the apo A-I conformation by LPS interaction in solution.     

Steatosis induced by the accumulation of apolipoprotein A-I and elevated ROS levels in H-ras12V transgenic mice contributes to hepatic lesions

Hepatic steatosis is considered to have an important impact on liver tumorigenesis, despite a lack of clear experimental evidence. Histopathological analysis of H-ras12V transgenic mice showed liver lesions on a steatosis background had significantly higher incidence than on a non-steatosis background. Further investigation showed that apolipoprotein A-I was elevated and accumulated around fatty vacuoles. This elevated level of apolipoprotein A-I was coupled with an elevated level of H-ras12V protein and ROS. In conclusion, our results suggest that the expression of H-ras12V oncogene leads to elevated levels of ROS and apolipoprotein A-I that contribute to steatosis. The steatosis, in turn, promotes the development of hepatic lesions induced by H-ras12V oncogene.