RESUMEN

A peptide sequence, stearoyl-GESIKVAVS(NH2), related to a laminin fragment, has been synthesized. Formation of aggregates was controlled by titrating a sodium anilinonaphthalene sulphonate (ANS) solution with peptide and recording fluorescence intensity increases. The results show that this system experiences a sudden increase in fluorescence at peptide concentrations around 2.5 x 10(-4) mol/L. The interaction of this hydrophobic peptide with DPPC vesicles has been studied using fluorescence techniques. Its influence on the microviscosity of bilayers was determined by studying polarization/temperature dependence for ANS and diphenyl hexatriene (DPH) fluorescent probes. With both markers the presence of peptide promotes a clear increase in anisotropy values. This indicates a rigidifying effect. Leakage studies carried out with liposomes loaded with carboxyfluorescein (CF) indicate a stabilizing effect of the peptide on bilayers, in agreement with results obtained with fluorescent probes.

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RESUMEN

In this work we have studied the conjugation of the immunogenic peptide sequence (110-121) belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes by means of an amide bond between the vesicles and the synthetic peptide. The surface activity of the conjugate at air/water interface was determined. Moreover, the interaction of the conjugate with lipids was also studied recording the pressure increases produced after the injection of the liposome-peptide preparation under dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and stearylamine (SA) monolayers at different initial surface pressures. As expected, due to the negative net charge of the liposome-peptide complex, the higher interaction was found with positive charge monolayers (SA). However, the conjugate was also able to incorporate to zwitterionic and anionic lipids. This behaviour was also confirmed performing compression isotherms of monolayers of these lipids spread on subphases containing the conjugate. These results suggest that the coupling of VP3 (110-121) to liposomes does not influence its ability to interact with membrane lipids such as DPPC and DPPG. Then it can be assumed that its immunogenicity will be preserved or even increased after this modification. All these results are also useful in the preparation of liposome-based synthetic peptide vaccines.

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RESUMEN

The physicochemical interactions between an active peptide sequence derived from laminin and phospholipids have been studied. The main aim is to determine the suitability of this peptide fragment to be linked to liposome's with the purpose to develop targeting devices. Results indicate that this peptide is able to insert in lipid monolayers and also to form monomolecular layers at an air/water interface. Besides, miscibility studies carried out through compression isotherms of mixed monolayers [dipalmitoyl phosphatidylcholine (DPPC)/peptide], indicate a strong interaction at 60-80% DPPC molar composition. Studies carried out with lipid bilayers indicate that the interaction is restricted to the external face of the vesicles. Moreover, the presence of this peptide in the incubation media promotes a low level of carboxyfluorescein (CF) leakage and no fusion of vesicles. These results indicate that the association of this sequence to vesicles do not produce damage of the bilayer and can be used as potential targeting vector.

RESUMEN

A synthetic peptide with the sequence [Lys113]VP3(110--121): FWRKDLVFDFQV, corresponding to an epitope of the VP3 capsid protein of hepatitis A virus (HAV), was synthesized by solid phase and characterized. To obtain insight into its physicochemical properties and to understand its possible mechanism of action at the membrane level, interaction with DPPC or DPPC/DPPG (95/5) liposomes and lipid monolayers of DPPC, DPPG, SA, PS, PA and SM were studied by fluorescence spectroscopy and Langmuir--Blotgett films technique, respectively. Fluorescence studies showed that the peptide was in a hydrophobic environment when DPPC liposomes were used. The addition of a 5% of a charged lipid, DPPG, to the preparations changed the preference of the peptide towards a polar surrounding. However, the peptide had a high surface activity (nmol/L) and was able to incorporate into lipid monolayers. Interaction was higher with charged phospholipids than with neutral ones. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

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RESUMEN

Shorter analogues of a continuous epitope of hepatitis A virus, VP3(110-121) peptide, failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. To better understand the influence of the structural properties of this 12-mer peptide epitope on its biological activity, the interaction of smaller peptide analogues with phospholipid biomembrane models was investigated by a combination of spectroscopic and biophysical techniques. In this article we describe our findings concerning the surface activity and the interaction of peptides with simple mono- and bilayer membranes composed of a zwitterionic phospholipid (dipalmitoyl phosphatidylcholine, DPPC), an anionic phospholipid (dipalmitoyl phosphatidylglicerol, DPPG), or a DPPC/DPPG mixture. The results indicate that the net negative charge of the peptide is in some way responsible of the specific interactions between VP3(110-121) and membrane phospholipids, and necessary to induce beta-type conformations upon vesicle interaction.

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RESUMEN

To prepare liposomes containing a synthetic hepatitis A virus antigen (HAV) [VP3(110-121)] as a vaccine, the miscibility of this peptide (with negative net charge) with a neutral lipid [dipalmitoylphosphatidylcholine (DPPC)], a negatively charged lipid [dipalmitoylphosphatidylglycerol (DPPG)], and a positively charged lipid [Stearylamine (SA)] was studied through compression isotherms of monolayers. Mixtures with DPPC and SA showed a low degree of interaction with the peptide, the composition of the monolayer being stable through compression. For DPPG-containing monolayers larger positive deviations from ideality were found, and the peptide was squeezed out from the monolayer at a DPPG/VP3(110-121) mole fraction of 0.8/0.2. All this suggests that besides hydrophobic interactions between the peptide and the lipid, electrostatic forces also play a role; thus it seems that neutral and positively charged lipids would be more suitable for preparing stable liposomes with VP3(110-121). Copyright 2000 Academic Press.

RESUMEN

The use of synthetic peptides in the goal of developing a new, inexpensive vaccine against hepatitis A virus is one of the encouraging approaches followed by many laboratories. These peptides have to be well characterized, being their physicochemical properties one of the most relevant points to control. In that sense, one can consider the study of the peptide interaction with lipid monolayers by means of the Wilhelmy plate method, to gain insight into the possible mechanism of action at the membrane level. The peptide chosen corresponds to the lineal epitope of hepatitis A virus VP3(110-121). As far as the lipids used are concerned, they were selected according to the composition of hepatocytes and erythrocytes because these structures seem to play an important role in hepatitis proliferation and infection. The peptide was able to accommodate into lipid monolayers. Interaction was slightly lower in the hepatocyte model than in the erythrocyte model, probably due to the presence of cholesterol in the hepatocyte membrane. Copyright 1999 Academic Press.