Phospholipid-model membrane interactions with branched polypeptide conjugates of a Hepatitis A virus peptide epitope
Phospholipid-model membrane interactions with branched polypeptide conjugates of a Hepatitis A virus peptide epitope
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DOI：
10.1021/bc9900385
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发表时间：
2000-01-01
影响因子：
4.7
通讯作者：
Hudecz, F
中科院分区：
文献类型：
--
作者：
Nagy, IB;Alsina, MA;Hudecz, F
为了建立结构特性（电荷、组成和构象）与膜穿透能力之间的相关性，研究了表位肽-载体构建体与磷脂模型膜的相互作用。为此，我们将来自甲型肝炎病毒的VP 3衣壳蛋白的线性表位肽（110）FWRGDLVFDFQV（121）（110-121）与具有不同化学特性的基于多聚赖氨酸的分支多肽缀合。通过酰胺-硫醇异双功能试剂3-（2-吡啶基二硫代）丙酸N-羟基琥珀酰亚胺酯将N-末端延长一个Cys残基的表位肽[C（110-121）]连接到聚[Lys-（DL-Ala（m）-X-i）]（i < 1，m约为3）上，其中x = 0（AK）、Ser（SAK）或Glu（EAK）。这些聚合物-[C（110-121）]共轭物与磷脂单分子膜的相互作用。使用DPPC和DPPC/PG（95/5摩尔/摩尔）混合物研究了双层。用1，6-二苯基-1，3，5-己三烯（DPH）和苯胺基萘磺酸钠（ANS）两种荧光探针检测了偶联物诱导脂质体流动性的变化。比较了缀合物与模型膜的结合，并评价了聚合物组分对这些相互作用的贡献。我们发现，具有聚阴离子/EAK-[C（110-121）]或聚阳离子/SAK-[C（110-121）]、AK-[C（110-121）]/特征的缀合物能够在空气/水界面形成单分子层，其稳定性顺序为：EAK-[C（110-121）] > SAK-[C（110 - 121）] > AK-[C（110-121）]。从磷脂单层渗透研究中获得的数据表明，EAK-[C（110-121）]的结合物插入比AK-[C（110 -121）]或SAK-[C（110-121）]更明显。DPPC/PG脂质体极性表面（ANS）或疏水核心（DPH）内荧光探针的荧光强度和偏振变化表明，所有三种缀合物均与双层外表面相互作用。仅使用聚阴离子化合物/EAK-[C（110-121）]时，转变温度显著升高，证明了显著渗透。总之，我们发现缀合物与磷脂模型膜的结合/渗透取决于构建体的电荷性质。考虑到连接到分支多肽的VP 3表位肽的方向和数量几乎相同，我们可以得出结论，载体的结构特征（氨基酸组成、电荷和表面活性）对缀合物-磷脂膜相互作用具有显著影响。这些观察结果表明，用于表位附着的聚合物载体的选择可能会显着影响缀合物的膜活性，并为免疫原性肽向细胞的充分呈现提供指导方针。
To establish correlation between structural properties (charge, composition, and conformation) and membrane penetration capability, the interaction of epitope peptide-carrier constructs with phospholipid model membranes was studied. For this we have conjugated a linear epitope peptide, (110)FWRGDLVFDFQV(121) (110-121), from VP3 capside protein of the Hepatitis A virus with polylysine-based branched polypeptides with different chemical characteristics. The epitope peptide elongated by one Cys residue at the N-terminal [C(110-121)] was attached to poly[Lys-(DL-Ala(m)-X-i)] (i < 1, m approximate to 3), where x = 0 (AK), Ser (SAK), or Glu (EAK) by the amide-thiol heterobifunctional reagent, 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester. The interaction of these polymer-[C(110-121)] conjugates with phospholipid monolayers and. bilayers was studied using DPPC and DPPC/PG (95/5 mol/mol) mixture. Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions were evaluated. We found that conjugates with polyanionic/EAK-[C(110-121)] or polycationic/SAK-[C(110-121)], AK-[C(110-121)]/character were capable to form monomolecular layers at the air/water interface with structure dependent stability in the following order: EAK-[C(110-121)] > SAK-[C(110-121)] > AK-[C(110-121)]. Data obtained from penetration studies into phospholipid monolayers indicated that conjugate insertion is more pronounced for EAK-[C(110-121)] than for AK-[C(110-121)] or SAK-[C(110-121)]. Changes in the fluorescence intensity and in polarization of fluorescent probes either at the polar surface (ANS) or within the hydrophobic core (DPH) of the DPPC/PG liposomes suggested that all three conjugates interact with the outer surface of the bilayer, Marked penetration was documented by a significant increase of the transition temperature only with the polyanionic compound/EAK-[C(110-121)]. Taken together, we found that the binding/penetration of conjugates to phospholipid model membranes is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptides were almost identical, we can conclude that the structural characteristics (amino;acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction. These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate presentation of immunogenic peptides to the cells.