Several features of apoA-V, including extremely low plasma concentration, lack of correlation with plasma cholesterol levels despite its association with HDL, and insolubility at neutral pH in the absence of lipid, are unlike those of other exchangeable apolipoproteins. Current and future studies of apoA-V will help to shed light on the molecular basis whereby this protein functions to modulate plasma lipid homeostasis.

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...

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic α-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.Hypertriglyceridemia (HTG) 1 is strongly and positively correlated with susceptibility to atherosclerosis (1). Furthermore, HTG is a risk factor for development of the metabolic syndrome and the accompanying insulin resistance, hypertension, obesity, and inflammation. On this basis, there is considerable interest in understanding factors that regulate plasma triglyceride (TG) levels. The discovery of a new apolipoprotein in 2001, termed apolipoprotein † This work was supported by grants from the National Institutes of Health to R.O.R. (HL 073061) (apo) A-V, has sparked an intensive research effort (2,3). The impact of apoA-V on plasma TG levels is vividly illustrated by the seminal report of Pennacchio et al. (4). Using genetically modified mice, these authors showed that plasma TG concentrations in human APOA5 transgenic mice were 3-fold lower than those of control littermates. At the same time, apoA-V gene-disrupted mice exhibited a 4-fold increase in plasma TG concentration.Human apoA-V is synthesized in the liver as a 366-amino acid preprotein. Following cleavage of a 23-amino acid signal peptide, mature apoA-V, consisting of 343 residues, can be detected in plasma (5). Comparative sequence analysis indicates apoA-V is a member of the class of exchangeable apolipoproteins and predicts a hydrophobic protein with a significant amount of α-helical secondary structure (6). This prediction...