Translocation of Human Calcitonin in Respiratory Nasal Epithelium Is Associated with Self-Assembly in Lipid Membrane

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Abstract
We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability ∼10-5 cm s-1). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [Nα-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 [Nα-fl]hCT(9−32). In contrast, the N-terminal fragment lacking the β-sheet forming C-terminus (25−32) was not internalized. Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCT adopts β-sheet conformation. In a concentrated aqueous solution, β-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 β-sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [Nα-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.