Beckstead Jennifer A, Wong Kasuen, Gupta Vinita, Wan Chung-Ping L, Cook Victoria R, Weinberg Richard B, Weers Paul M M, Ryan Robert O
Center for Prevention of Obesity, Diabetes, and Cardiovascular Disease, Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
J Biol Chem. 2007 May 25;282(21):15484-9. doi: 10.1074/jbc.M611797200. Epub 2007 Mar 31.
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 approximately 50% alpha-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-alpha-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.
人载脂蛋白A-V（apoA-V）是血浆三酰甘油（TG）水平的有效调节剂。为了探究这个由343个氨基酸组成的蛋白质的不同区域，制备了四种单色氨酸apoA-V变体。在293-296位残基的四脯氨酸序列远端的325位带有色氨酸的变体，在与脂质结合时，最大荧光发射波长出现了11纳米的蓝移。为了评估该C末端片段的结构和功能作用，构建了一个包含1-292位氨基酸的截短型apoA-V。全长apoA-V和apoA-V-(1-292)的远紫外圆二色光谱相似，α-螺旋含量约为50%。在盐酸胍变性实验中，全长和截短的apoA-V均产生了与两个结构域存在一致的双相图谱。截短影响了较低稳定性组分（而非较高稳定性组分）的变性图谱。与全长apoA-V相比，截短的apoA-V溶解l-α-二肉豆蔻酰磷脂酰胆碱磷脂囊泡的能力减弱，而对应于缺失的C末端片段的肽表现出明显增强的动力学。这些数据支持了这样一种观点，即apoA-V采用折叠蛋白结构不需要C末端区域，但该区域具有调节apoA-V脂质结合活性的功能；因此，这一观点可能与apoA-V影响血浆TG水平的机制相关。
