title: Translocation of human calcitonin in respiratory nasal epithelium is associated with self-assembly in lipid membrane.
abstract: We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability approximately 10(-)5 cm s-1). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [Nalpha-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) were synthesized. In presence of the endocytosis inhibitor cytochalasin D mucosal-to-serosal and serosal-to-mucosal hCT permeabilities were equal. Pathway visualization by confocal laser scanning microscopy showed punctated fluorescence indicating vesicular internalization of both hCT and [Nalpha-fl]hCT(9-32). In contrast, the N-terminal fragment lacking the beta-sheet forming C-terminus (25-32) was not internalized. Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCT adopts beta-sheet conformation. In a concentrated aqueous solution, beta-sheet formation induces hCT self-assembly and fibrillation. High partitioning of hCT into lipid bilayer membranes was reflected by an apparent partition coefficient log D(pH 7.4) = 2.5 (liposome-buffer equilibrium dialysis). We propose that the high lipid partitioning and beta-sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [Nalpha-fl]hCT(9-32) in lipid membranes. Vesicular internalization is suggested to be associated with self-assembly induced perturbation of the lipid bilayer. Condensed hCT self-assemblies may explain the high capacity of net mucosal-to-serosal hCT permeation, which compares favorably with the low transport capacity of receptor-mediated endocytosis.
authors.affiliation: Department of Pharmacy, Swiss Federal Institute of Technology Zurich (ETH), Switzerland.
authors.forename: M C
authors.initials: MC
authors.lastname: Schmidt
authors.forename: B
authors.initials: B
authors.lastname: Rothen-Rutishauser
authors.forename: B
authors.initials: B
authors.lastname: Rist
authors.forename: A
authors.initials: A
authors.lastname: Beck-Sickinger
authors.forename: H
authors.initials: H
authors.lastname: Wunderli-Allenspach
authors.forename: W
authors.initials: W
authors.lastname: Rubas
authors.forename: W
authors.initials: W
authors.lastname: Sadée
authors.forename: H P
authors.initials: HP
authors.lastname: Merkle
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002116:Calcitonin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002463:Cell Membrane Permeability; D002942:Circular Dichroism; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D008081:Liposomes; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; D015060:1,2-Dipalmitoylphosphatidylcholine; D002116:Calcitonin; C030345:1,2-dipalmitoylphosphatidylglycerol