Epand R F, Macosko J C, Russell C J, Shin Y K, Epand R M
McMaster University Health Sciences Centre, 1200 Main Street West, Hamilton, ON, L8N 3Z5, Canada.
J Mol Biol. 1999 Feb 19;286(2):489-503. doi: 10.1006/jmbi.1998.2500.
To better understand the roles of different regions of influenza hemagglutinin in membrane fusion, we have studied the fusion properties of large unilamellar vesicles in the presence of constructs comprising the 127 amino acid ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 containing single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-terminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equimolar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 present, even at very low molar concentrations, whereas much lower rates were observed using the shorter constructs: SHA2, the fusion peptide, and the loop peptide. Concentrations of FHA2 which promoted extensive lipid mixing also induced leakage of aqueous contents. Marked effects of FHA2 were also observed with liposomes of egg phosphatidylcholine. All of the changes observed with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in promoting lipid mixing and demonstrate the contribution of other regions of the ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH dependence of fusion is not due solely to changes in the interactions between the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not required to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residues in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipid mixing activity of mutant forms of FHA2.
为了更好地理解流感血凝素不同区域在膜融合中的作用，我们研究了在包含HA2片段127个氨基酸胞外域（FHA2）的构建体以及含有单个氨基酸取代的FHA2突变形式、缺少N端融合肽的95个氨基酸截短形式FHA2（SHA2）、20个氨基酸的N端融合肽和对应于FHA2扭结环区域的10个氨基酸肽存在的情况下，大单层囊泡的融合特性。100nm脂质体由等摩尔比的二油酰磷脂酰乙醇胺、二油酰磷脂酰胆碱和胆固醇制成。在pH5时，即使在非常低的摩尔浓度下，存在FHA2时也观察到高脂质混合速率，而使用较短构建体（SHA2、融合肽和环肽）时观察到的速率要低得多。促进广泛脂质混合的FHA2浓度也会导致水相内容物泄漏。在卵磷脂脂质体中也观察到FHA2的显著作用。脂质体观察到的所有变化都高度依赖于pH，在pH7时仅发生可忽略不计的变化。结果证明了FHA2在促进脂质混合方面的强大作用，并证明了FHA2胞外域除融合肽外的其他区域对膜融合加速机制的贡献。结果还表明，融合的pH依赖性不仅仅是由于HA1和HA2亚基之间相互作用的变化。因此，考虑到FHA2已经处于其热稳定构象，不需要“弹簧加载能量”来实现两个膜的并置。扭结环区域中的酸性氨基酸残基似乎在pH依赖性融合过程中起特别重要的作用，FHA2突变形式的脂质混合活性明显丧失证明了这一点。
