The C Terminus of Apolipoprotein A-V Modulates Lipid-binding Activity

Abstract

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...

Effect of apolipoprotein A-V on plasma triglyceride, lipoprotein size, and composition in genetically engineered mice

“…This is consistent with the previous observations that the protein is extremely hydrophobic and readily binds lipids (37,38). Truncation studies with apoA-V further revealed that the C-terminal region of the molecule (amino acids 293-343) has lipid binding activity (38). The tight association of human apoA-V with HDL particles is in contrast with a recent report by Dichlberger et al (35) with the chicken apoA-V homolog, in which a significant fraction of apoA-V was found in the d .…”

“…This is consistent with the previous observations that the protein is extremely hydrophobic and readily binds lipids (37,38). Truncation studies with apoA-V further revealed that the C-terminal region of the molecule (amino acids 293-343) has lipid binding activity (38).…”

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

“…On the other hand, this mutant showed no detectable changes in the capacity to bind LRP1 cluster II or to activate LPL. The C-terminal region of apoA-V (residues 296-343) has been previously shown to modulate its lipid-binding activity ( 19,59 ). The apparent contradiction with our current results suggests that residues 296-334 play a particularly significant role in this regard.…”

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I

“…ApoA-I and other native and mutant apolipoproteins spontaneously solubilizes MLV and LUV of DMPC to form discoidal rHDL [15][16][17][18][19][20]. This process is not unique to DMPC, since apoA-I can also spontaneously solubilize bilayers of sphingomyelin [21], binary mixtures of saturated phosphatidylcholines [22], and phospholipid and cholesterol mixtures that represent the composition of isolated nascent HDL [3].…”

“…At a second high capacity site which is created by the phospholipid translocase activity of ABCA1, apoA-I through lipid-protein interactions associates with membrane domains that are involved in the assembly of nascent HDL particles. ApoA-I [14,[15][16][17] and other apolipoproteins [18][19][20] can spontaneously solubilize bilayer membranes of DMPC and other phospholipid mixtures [3,21,22] to form discoidal rHDL. In one model for ABCA1, apoA-I via a microsolubilization step simultaneously removes phospholipid and cholesterol from ABCA1 created membrane domains to form nascent HDL by the same mechanism by which apoA-I mediates the solubilization of DMPC MLV to form rHDL.…”