On cell-penetrating peptides and cell-junction interactions

Sammanfattning: Cell-penetrating peptides have the remarkable ability to carry hydrophilic macromolecules over the cellular membrane in an energy and protein independent manner. To synthesise and characterise new variants of cell-penetrating peptides, mainly aimed at finding analogues with minimised side effects and efficient cellular delivery, was one of the two main objectives of this thesis. The other principal objective was to study cell-junction interactions by the protein vascular endothelial cadherin. In addition, a study where the two objectives were combined was aimed at investigating the possibility of utilising cell-penetrating peptides for trans-barrier delivery.In order to gain more information about the cell-junction interactions of vascular endothelial cadherin, monoclonal antibodies directed to the extracellular domain were tested for their activity. Three of the antibodies were able to increase paracellular permeability, inhibit VE-cadherin reorganisation, and block angiogenesis in vitro. In addition, epitope mapping of the antibodies located their binding sequences to EC1, EC3 and EC4 of VE-cadherin ectodomain. These results support the concept that VE-cadherin protein sequence consists of multiple biologically important domains.The aim of paper II was to compare transportan, developed by our group, with the first discovered cell-penetrating peptide, penetratin. Novel analogues were synthesised and tested for cellular uptake and also in molecular modelling. The transportan analogues were different chimeric constructs, elaborating with the two different parts of the peptide, while the penetratin analogues were modifications of the original sequence. Furthermore, the translocation ability of fluorescently labelled parent peptides, transportan and penetratin, were quantitatively measured. The results imply that the two peptides and their analogues do not enter the cells by the same mechanism.Since it was clear that the mastoparan part of transportan was necessary for cellular penetration while the galanin part was more adaptable, several shorter analogues were synthesised in order to define the essential sequence for transportan penetration. In order to reduce the unwanted side effects, the biological effects of transportan and its deletion analogues were studied by GTPase activity measurements. It was concluded that the deletion of six amino acids from the N-terminus did not significantly impair the cell penetration, while truncation of the C-terminus or in the middle of the peptide decreased or even abolished the cellular uptake of transportan.pVEC is a new type of cell-penetrating peptides derived from the murine sequence of VE-cadherin. In this study we show that pVEC is a non-toxic, efficient cell-penetrating peptide that can be used for cellular delivery of both PNA and large proteins, such as streptavidin. The uptake of pVEC was quantified, by direct fluorescence labelling in both mouse and human endothelial cell lines. Furthermore, no significant effect could be detected on the parent protein VE-cadherins cell-junction clustering.In paper V it is shown that transportan and the analogue, transportan 10, enter and pass across a human colon cancer Caco-2 epithelial cell layer. However, the peptides decreased the trans-epithelial electric resistance of the barrier model, but not the dextran passage to the same extent. Taken together, these data demonstrate that transportan and transportan 10 are conveyed over the epithelial cell layer, mainly by the transcellular pathway, but at higher peptide concentration, the paracellular pathway may contribute to the passage. To conclude, cell-penetrating peptides may be a new possible strategy for drug delivery over a tight junction barrier, such as the blood-brain barrier.