Phospholipid−Model Membrane Interactions with Branched Polypeptide Conjugates of a Hepatitis a Virus Peptide Epitope

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, ¹¹⁰FWRGDLVFDFQV¹²¹ (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-(<SCP>dl</SCP>-Ala<INF>m</INF><INF></INF>-X<INF>i</INF><INF></INF>)] (i &lt; 1, m ≈ 3), where x = &slshcir; (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.