Cajal Y, Rabanal F, Alsina M A, Reig F
Peptide Department, CID-CSIC, Barcelona, Spain.
Biopolymers. 1996 May;38(5):607-18. doi: 10.1002/(sici)1097-0282(199605)38:5<607::aid-bip6>3.0.co;2-w.
The interaction of the immunogenic peptide of human hepatitis B virus (HBV) preS(120-145), including B and T epitopes, with phospholipid vesicles has been studied by fluorescence techniques and CD. In addition, interaction of three lipopeptides derived from preS(120-145) containing stearoyl, cholanoyl, and tripalmitoyl-S-glyceryl-cysteine (Pam3C) SS moieties with dipalmitoylphosphatidylcholine (DPPC) has been investigated by polarization fluorescence spectroscopy. Fluorescence experiments showed an increase in fluorescence intensity and a blue shift of the maximum emission wavelength upon interaction of preS(120-145) with DPPC vesicles below the transition temperature (Tc), indicating that the tryptophan moiety enters a more hydrophobic environment. Moreover, fluorescence polarization experiments showed that the peptide decreased the membrane fluidity at the hydrophobic core, increasing the Tc of the lipid and decreasing the amplitude of the change of fluorescence polarization associated with the cooperative melting of 1,6-diphenyl-1,3,5-hexatriene labeled vesicles. The absence of leakage of vesicle-entrapped carboxyfluorescein indicates that the peptide did not promote vesicle lysis. Besides, the three lipopeptides derived from preS(120-145) showed a more pronounced rigidifying effect at the hydrophobic core of the bilayer, with a significative increase in the Tc. Stearoyl- and cholanoyl-preS(120-145) restricted the motion of lipids also at the polar surface, whereas Pam3CSS-preS(120-145) did not alter the polar head group order. Finally, CD studies in 2,2,2-trifluoroethanol or in presence of vesicles suggested that the bound peptide adopted amphiphilic alpha-helical and beta-sheet structures, with an important contribution of the beta-turn. It is concluded that preS(120-145) can interact with the lipid membrane through the formation of an amphipathic structure combination of beta-sheet and alpha-helix aligned parallel to the membrane surface, involving the N-terminal residues, and penetrating only a short distance into the hydrophobic core. The C-terminal part, with a combination of beta-turn and beta-sheet structure, remains at the outer part of the bilayer, being potentially accessible to immunocompetent cells. Furthermore, coupling of an hydrophobic moiety to the N-terminal part of the peptide favors anchoring to the membrane, probably facilitating interaction of the peptide with the immunoglobulin receptor. These results are in agreement with the induction of immune response by preS(120-145) and with the enhanced immunogenicity found in general for lipid-conjugated immunopeptides.
利用荧光技术和圆二色光谱（CD）研究了包含B和T表位的人乙型肝炎病毒（HBV）前S（120 - 145）免疫原性肽与磷脂囊泡的相互作用。此外，通过偏振荧光光谱研究了源自前S（120 - 145）的三种脂肽（分别含有硬脂酰、胆酰和三棕榈酰 - S - 甘油基 - 半胱氨酸（Pam3C）SS基团）与二棕榈酰磷脂酰胆碱（DPPC）的相互作用。荧光实验表明，在前S（120 - 145）与低于转变温度（Tc）的DPPC囊泡相互作用时，荧光强度增加且最大发射波长发生蓝移，这表明色氨酸部分进入了更疏水的环境。此外，荧光偏振实验表明，该肽降低了疏水核心处的膜流动性，提高了脂质的Tc，并降低了与1,6 - 二苯基 - 1,3,5 - 己三烯标记囊泡协同熔化相关的荧光偏振变化幅度。囊泡包裹的羧基荧光素没有泄漏，这表明该肽没有促进囊泡裂解。此外，源自前S（120 - 145）的三种脂肽在双层膜的疏水核心处表现出更显著的硬化作用，Tc有显著增加。硬脂酰 - 和胆酰 - 前S（120 - 145）在极性表面也限制了脂质的运动，而Pam3CSS - 前S（120 - 145）没有改变极性头部基团的有序性。最后，在2,2,2 - 三氟乙醇中或在囊泡存在下的CD研究表明，结合的肽采用两亲性α - 螺旋和β - 折叠结构，β - 转角起重要作用。得出的结论是，前S（120 - 145）可以通过形成与膜表面平行排列的β - 折叠和α - 螺旋的两亲结构组合与脂质膜相互作用，涉及N端残基，并且仅短距离穿透疏水核心。C端部分具有β - 转角和β - 折叠结构的组合，保留在双层膜的外部，可能被免疫活性细胞所接触。此外，将疏水部分与肽的N端部分偶联有利于锚定到膜上，可能促进肽与免疫球蛋白受体的相互作用。这些结果与前S（120 - 145）诱导免疫反应以及脂质偶联免疫肽普遍具有增强的免疫原性一致。
