title: Solid-phase synthesis and application of double-fluorescent-labeled lipopeptides, containing a CTL-epitope from the measles fusion protein.
abstract: The mechanism which enables lipopeptides to induce cytotoxicity is not known. By preparing fluorescent-labeled lipopeptides one might unravel the mechanism of their entry into the cell and their intracellular pathway. A method of preparing double-fluorescent-labeled peptides by solid-phase chemistry is described. As model peptides we have chosen analogs of the sequence RRYPDAVYL, which occurs in the measles fusion protein (F438-446) and is an epitope for cytotoxic T lymphocytes. The peptides Pal-K(TMR)KKKRRYPDAVK(FL)L (7) and Pal-K(FL)KKKRRYPDAVK(TMR)L (8), in which Pal is palmitoyl and K(TMR) and K(FL) are Nepsilon-carboxytetramethylrhodamine- and Nepsilon-carboxyfluorescein-labeled lysyl residues, respectively, were prepared and obtained in approximately 30% yield after purification by high-performance liquid chromatography. The fluorescence of fluorescein and tetramethylrhodamine in lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L (7) was quenched to 98-99% due to intramolecular interaction of the labels. On incubation with trypsin (i.e. cleavage at the KKKRR-site) the fluorescence of both labels was restored. The intracellular routing of lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L was studied with human melanoma cell line, Mel/J, which was transfected with human leukocyte antigen B*2705. It appeared that the double-fluorescent-labeled lipopeptide was able to induce antigen-specific cytotoxicity. Furthermore, preliminary confocal microscopical studies indicated that this lipopeptide is observed intracellularly.
authors.affiliation: RIVM, National Institute of Public Health and the Environment, Laboratory for Vaccine Research, Bilthoven, The Netherlands. Peter.Hoogerhout@rivm.nl
authors.forename: P
authors.initials: P
authors.lastname: Hoogerhout
authors.forename: K J
authors.initials: KJ
authors.lastname: Stittelaar
authors.forename: H F
authors.initials: HF
authors.lastname: Brugghe
authors.forename: J A
authors.initials: JA
authors.lastname: Timmermans
authors.forename: G J
authors.initials: GJ
authors.lastname: ten Hove
authors.forename: W
authors.initials: W
authors.lastname: Jiskoot
authors.forename: J H
authors.initials: JH
authors.lastname: Hoekman
authors.forename: P J
authors.initials: PJ
authors.lastname: Roholl
mesh_terms: D002851:Chromatography, High Pressure Liquid; D000939:Epitopes; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008074:Lipoproteins / Q000138:chemical synthesis* / Q000276:immunology; D008856:Microscopy, Fluorescence; D010169:Palmitic Acids / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000276:immunology; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000939:Epitopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008074:Lipoproteins; D010169:Palmitic Acids; D010446:Peptide Fragments; D012235:Rhodamines; D014760:Viral Fusion Proteins; D014764:Viral Proteins; C005358:tetramethylrhodamine