Translocation of human calcitonin in respiratory nasal epithelium is associated with self-assembly in lipid membrane

We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability similar to 10(-5) cm s(-1)). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [N-alpha-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 [N-alpha-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 [N-alpha-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.