Structure and Interfacial Properties of Human Apolipoprotein A-V

Abstract

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...

Specific Sequences in the N and C Termini of Apolipoprotein A-IV Modulate Its Conformation and Lipid Association

“…As the DMPC assay measures the end result of a complex process that involves both an initial apolipoprotein-lipid interaction and a subsequent lipid reorganization (see reaction scheme in Ref. 24), we also examined the lipid affinity of WT and ⌬333-376 apoA-IV using oil drop tensiometry (25). This method measures the rate at which apolipoproteins in solution bind to and lower the surface tension of the more hydrophobic triolein/water interface.…”

“…It has been postulated that its distinctively weak lipid binding behavior is due to: (a) the relative hydrophilicity and low amphipathic moment of its constitutive ␣-helices (29), (b) the fact that most of these helices are of the Y-type, which may not be capable of deeply penetrating lipid surfaces (30), and/or (c) the possibility that these helices are organized such that their hydrophobic regions are inaccessible for interaction with hydrophobic interfaces (2,23,25).…”

“…Based upon a series of fluorescence studies, Weinberg et al (2,23,25) proposed that apoA-IV exists as a loosely organized confluence/bundle of amphipathic ␣-helices in which the hydrophobic faces are oriented inwards toward the center of the bundle. Using a mutagenesis approach, we subsequently proposed that this bundle may exist as a single domain encompassing residues 40 -332 (14).…”

ApoAV Reduces Plasma Triglycerides by Inhibiting Very Low Density Lipoprotein-Triglyceride (VLDL-TG) Production and Stimulating Lipoprotein Lipase-mediated VLDL-TG Hydrolysis

“…Initially, apoAV may be secreted along with VLDL, and as lipolytic conversion of VLDL progresses and excess surface lipids are being transferred to HDL, apoAV may end up in HDL in a similar way. Structure predictions indicate that apoAV is a very hydrophobic, highly ␣-helical protein (7). At the protein level, apoAV appears most homologous (20 -28% amino acid identity) with exchangeable apolipoproteins apoAI, apoAIV, and apoE, prompting the study of lipid efflux and lecithin:cholesterol acyltransferase activation properties of apoAV (8).…”

“…Based on structural analysis, it has been proposed that, at the intracellular level, apoAV may affect hepatic VLDL production (7). Alternatively, apoAV may stimulate lipolytic conversion of TG-rich lipoproteins.…”