Antennapedia Peptide (43-58), acid

Antp(43-58) is one of many peptides with basic and aromatic residues capable of crossing cell membranes efficiently in a receptor-independent manner. The basic-aromatic motif is responsible for peptide binding to the negatively charged surface of membrane bilayers. However, the mechanism of membrane penetration is unclear. We use high-resolution (1)H solution NMR methods to establish the location of the Antp(43-58) peptide bound to membrane bicelles composed of DMPC, DMPG, and DHPC, and compare it to the location of an Antp(43-58) variant which is not able to cross cell membranes. Two critical tryptophans are substituted with phenylalanine in this variant (W48F and W56F). Additional (31)P and (2)H NMR measurements of membrane bicelles are used to probe the changes in orientation of the lipid headgroups and the changes in the mobility or segmental order of the lipid acyl chains upon peptide binding. We find that Trp48 and Trp56 of Antp(43-58) insert into the hydrophobic core of the membrane and that this induces a change in the orientation of the negatively charged DMPG headgroups. The depth of insertion and the change in lipid orientation are concentration-dependent and argue for an electroporation-like mechanism for membrane penetration.

The peptides comprising the sequence of HIV-1 Tat protein (positions 48-60), Antennapedia (positions 43-58), and HIV-1 Rev protein (positions 34-50) are known to be cell-permeating. In this study, we examined how the distribution of Fab fragments in rats is affected by conjugation with these peptides. Fab fragment was iodinated by a chloramine-T method and then chemically conjugated with cell-permeating peptide. The complex of 125I-Fab and cell-permeating peptide was administered to male rats intravenously at a dose of 1 mg/kg, and whole-body autoradiography was performed at 4 and 24 h after administration. The patterns of distribution of 125I-Fab exhibited remarkable variation depending on the cell-permeating peptide used. In particular, at 4 h, high concentrations of radioactivity were observed in the spleen, adrenal gland, renal medulla, and liver with Rev peptide-Fab complex, in the liver and spleen with Tat peptide-Fab complex, and in the spleen, adrenal gland, and liver with Antennapedia peptide-Fab complex. Even at 24 h, high concentrations of radioactivity were still observed in the spleen and renal medulla of rat with Rev peptide-Fab complex, and in the spleen and renal cortex of rat with Antennapedia peptide-Fab complex. These findings demonstrate that the patterns of distribution of peptide-125I-Fab complexes can be modulated by selection of cell-penetrating peptides. Moreover, the patterns of retention of peptide-125I-Fab complexes in internal organs also differed at 24 h after administration. These findings provide valuable information for the development of novel antibody pharmaceuticals and therapeutic systems.

The third helix of antennapedia homeodomain pAntp-(43-58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp-(43-58) and [Pro50] pAntp-(43-58) has been analyzed by different biophysical methods. pAntp-(43-58) adopts an amphipathic helical structure in 30% (by vol.) hexafluoroisopropanol, in perfluoro-tert-butanol and in the presence of SDS micelles. CD spectra indicate that the conformation of [Pro50]pAntp-(43-58) in contrast to pAntp-(43-58) is independent of the media used. 1H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2(7) type conformation. These conformations became the predominant structure when Gln50 was replaced by Pro50. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: alpha-helix and essentially extended structures. Consecutive CH alpha (i)/ CH alpha (i + 1) NOEs are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with phi and psi angles in the beta-sheet and gamma-turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 microM). Similar interaction was observed with reverse-phase micelles made of bis(2-ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 31P-NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.