Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of ␣-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion.Lipoprotein metabolism is regulated by the plasma apolipoproteins, a family of surface-active lipid binding proteins. The smaller, exchangeable members of this family evolved from a single primordial gene to control various processes in intravascular lipid transport (1). The largest member of the family, apoB, 1 evolved from ancient lipid transport proteins involved in oogenesis (2) to play a central role in the intracellular assembly of triglyceride-rich lipoproteins in the intestine and liver. ApoA-V is the most recently described member of the plasma apolipoprotein family. Unlike all other apolipoproteins, which were identified in human plasma, apoA-V was discovered by comparative sequence analysis of the human and murine genomes as a gene on chromosome 11, downstream of the A-I/C-III/A-IV gene cluster, displaying homology to apoA-IV (3). Concurrently and independently, apoA-V was identified as a gene up-regulated in the early phase of hepatic regeneration in the rat (4). Mature human apoA-V is a 39-kDa protein with 343 residues and 27% sequence identity with human A-IV.ApoA-V was found to have a powerful effect on plasma triglycerides. Overexpression of human apoA-V in transgenic mice (3) or by use of adenoviral vectors (5) lowers plasma triglyceride levels, whereas inactivation of the apoA-V gene by homologous recombination causes a 4-fold increase in plasma VLDL triglycerides (3). Several single nucleotide polymorphisms in the a...

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.

Human apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To probe different regions of this 343-amino-acid protein, four single Trp apoA-V variants were prepared. The variant with a Trp at position 325, distal to the tetraproline sequence at residues 293-296, displayed an 11-nm blue shift in wavelength of maximum fluorescence emission upon lipid association. To evaluate the structural and functional role of this C-terminal segment, a truncated apoA-V comprising amino acids 1-292 was generated. Far UV circular dichroism spectra of full-length apoA-V and apoA-V-(1-292) were similar, with ϳ50% ␣-helix content. In guanidine HCl denaturation experiments, both full-length and truncated apoA-V yielded biphasic profiles consistent with the presence of two structural domains. The denaturation profile of the lower stability component (but not the higher stability component) was affected by truncation. Truncated apoA-V displayed an attenuated ability to solubilize L-␣-dimyristoylphosphatidylcholine phospholipid vesicles compared with full-length apoA-V, whereas a peptide corresponding to the deleted C-terminal segment displayed markedly enhanced kinetics. The data support the concept that the C-terminal region is not required for apoA-V to adopt a folded protein structure, yet functions to modulate apoA-V lipid-binding activity; therefore, this concept may be relevant to the mechanism whereby apoA-V influences plasma TG levels.In 2001, a new member of the exchangeable apolipoprotein family was independently discovered by comparative genomics (1) and as an mRNA that is up-regulated in rat liver following partial hepatectomy (2). In humans, the mature protein, termed apolipoprotein (apo) 2 A-V, comprises 343 amino acids (3).Northern blot analysis of various tissues revealed that apoA-V mRNA expression is restricted to hepatocytes (1). To evaluate its function, Pennacchio et al.(1) generated transgenic mice that overexpressed apoA-V as well as gene-disrupted mice that lacked apoA-V. The transgenic mice displayed a 3-fold lower plasma triacylglycerol (TG) level compared with control littermates. By contrast, apoA-V gene knock-out mice revealed a 4-fold higher plasma TG content compared with controls. Levels of very low density lipoprotein (VLDL) particles were increased in homozygous knock-out mice and decreased in transgenic mice compared with controls. Van der Vliet et al. (4) confirmed the effect of apoA-V on plasma TG levels using an adenovirus construct to overexpress apoA-V in mice. ApoA-Voverexpressing mice displayed markedly decreased plasma TG levels that were the result of lower VLDL levels. Interestingly, changes in plasma TG concentration were directly opposite of those reported for apoC-III knock-out and transgenic mice (5, 6). Although apoA-V knock-out mice displayed a 4-fold increase in plasma TG, apoC-III gene-disrupted animals showed a 30% decrease. The mechanism whereby apoA-V influences plasma TG levels is unknown but may be related to an ability to influence TG-rich l...

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Cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II) are known to differentially facilitate retinoid metabolism by several membrane-associated enzymes. The mechanism of ligand transfer to phospholipid small unilamellar vesicles was compared in order to determine whether differences in ligand trafficking properties could underlie these functional differences. Unidirectional transfer of retinol from the CRBPs to membranes was monitored by following the increase in intrinsic protein fluorescence that occurs upon ligand dissociation. The results showed that ligand transfer of retinol from CRBP-I was >5-fold faster than transfer from CRBP-II. For both proteins, transfer of the other naturally occurring retinoid, retinaldehyde, was 4 -5-fold faster than transfer of retinol. Rates of ligand transfer from CRBP-I to small unilamellar vesicles increased with increasing concentration of acceptor membrane and with the incorporation of the anionic lipids cardiolipin or phosphatidylserine into membranes. In contrast, transfer from CRBP-II was unaffected by either membrane concentration or composition. Preincubation of anionic vesicles with CRBP-I was able to prevent cytochrome c, a peripheral membrane protein, from binding, whereas CRBP-II was ineffective. In addition, monolayer exclusion experiments demonstrated differences in the rate and magnitude of the CRBP interactions with phospholipid membranes. These results suggest that the mechanisms of ligand transfer from CRBP-I and CRBP-II to membranes are markedly different as follows: transfer from CRBP-I may involve and require effective collisional interactions with membranes, whereas a diffusional process primarily mediates transfer from CRBP-II. These differences may help account for their distinct functional roles in the modulation of intracellular retinoid metabolism.

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer ␣-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the ␣-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic ␣-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r ‫؍‬ 0.790, p ‫؍‬ 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.

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Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/ water interface apolipoproteins A-IV and B-17 displayed wide area-tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/ water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 ؋ 10 ؊ 3 sec ؊ 1 , whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 ؋ 10 ؊ 3 sec ؊ 1 . Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure. We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles.