Chattopadhyay Kausik, Bhattacharyya Debasish, Banerjee Kalyan K
National Institute of Cholera and Enteric Diseases, Kolkata 700 010, India; Indian Institute of Chemical Biology, Kolkata 700 032, India.
Eur J Biochem. 2002 Sep;269(17):4351-8. doi: 10.1046/j.1432-1033.2002.03137.x.
Vibrio cholerae hemolysin (HlyA), a water-soluble protein with a native monomeric relative molecular mass of 65 000, forms transmembrane pentameric channels in target biomembranes. The HlyA binds to lipid vesicles nonspecifically and without saturation; however, self-assembly is triggered specifically by cholesterol. Here we show that the HlyA partitioned quantitatively to amphiphilic media irrespective of their compositions, indicating that the toxin had an amphiphilic surface. Asialofetuin, a beta1-galactosyl-terminated glycoprotein, which binds specifically to the HlyA in a lectin-glycoprotein type of interaction and inhibits carbohydrate-independent interaction of the toxin with lipid, reduced effective amphiphilicity of the toxin significantly. Resistance of the HlyA to proteases together with the tryptophan fluorescence emission spectrum suggested a compact structure for the toxin. Fluorescence energy transfer from the HlyA to dansyl-phosphatidylethanolamine required the presence of cholesterol in the lipid bilayer and was synchronous with oligomerization. Phospholipid bilayer without cholesterol caused a partial unfolding of the HlyA monomer as indicated by the transfer of tryptophan residues from the nonpolar core of the protein to a more polar region. These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipid-water interface precedes and promotes cholesterol-induced oligomerization to an insertion-competent configuration.
霍乱弧菌溶血素（HlyA）是一种水溶性蛋白，天然单体相对分子质量为65000，可在靶生物膜中形成跨膜五聚体通道。HlyA可非特异性且不饱和地结合脂质囊泡；然而，自组装由胆固醇特异性触发。在此我们表明，无论两亲介质的组成如何，HlyA都能定量分配到其中，这表明该毒素具有两亲性表面。去唾液酸胎球蛋白是一种β1-半乳糖基末端糖蛋白，它在凝集素-糖蛋白相互作用类型中特异性结合HlyA，并抑制毒素与脂质的非碳水化合物依赖性相互作用，显著降低了毒素的有效两亲性。HlyA对蛋白酶的抗性以及色氨酸荧光发射光谱表明该毒素具有紧密结构。从HlyA到丹磺酰磷脂酰乙醇胺的荧光能量转移需要脂质双层中存在胆固醇，并且与寡聚化同步。没有胆固醇的磷脂双层导致HlyA单体部分展开，这由色氨酸残基从蛋白质的非极性核心转移到更极性区域所表明。这些观察结果表明：（a）HlyA分配到脂质囊泡是由两亲性毒素减少与水的能量不利接触的趋势驱动的，并且不受囊泡组成的显著影响；（b）HlyA在脂质-水界面的部分展开先于并促进胆固醇诱导的寡聚化形成可插入的构象。
