Translocation of Human Calcitonin in Respiratory Nasal Epithelium Is Associated with Self-Assembly in Lipid Membrane
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- 作者：Maria Christiane Schmidt ; Barbara Rothen-Rutishauser ; Beate Rist ; Annette Beck-Sickinger ; Heidi Wunderli-Allenspach ; Werner Rubas ; Wolfgang Sadé ; e ; and Hans Peter Merkle
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：November 24, 1998
- 年：1998
- 卷：37
- 期：47
- 页码：16582 - 16590
- 全文大小：222K
- 年卷期：v.37,no.47(November 24, 1998)
- ISSN：1520-4995
文摘
We studied the mechanisms involved in the translocation of human calcitonin (hCT) throughexcised bovine nasal mucosa (net mucosal-to-serosal permeability ~10-5 cm s-1). To determine structuralrequirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments,the C-terminal fragment [N
-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) weresynthesized. 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 microscopyshowed punctated fluorescence indicating vesicular internalization of both hCT and [N-fl]hCT(9-32).In contrast, the N-terminal fragment lacking the 
-sheet forming C-terminus (25-32) was not internalized.Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCTadopts -sheet conformation. In a concentrated aqueous solution, -sheet formation induces hCT self-assembly and fibrillation. High partitioning of hCT into lipid bilayer membranes was reflected by anapparent partition coefficient log D(pH 7.4) = 2.5 (liposome-buffer equilibrium dialysis). We proposethat the high lipid partitioning and -sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [N-fl]hCT(9-32) in lipid membranes. Vesicular internalization is suggested to beassociated with self-assembly induced perturbation of the lipid bilayer. Condensed hCT self-assembliesmay explain the high capacity of net mucosal-to-serosal hCT permeation, which compares favorably withthe low transport capacity of receptor-mediated endocytosis.
-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) weresynthesized. 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 microscopyshowed punctated fluorescence indicating vesicular internalization of both hCT and [N-fl]hCT(9-32).In contrast, the N-terminal fragment lacking the -sheet forming C-terminus (25-32) was not internalized.Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCTadopts -sheet conformation. In a concentrated aqueous solution, -sheet formation induces hCT self-assembly and fibrillation. High partitioning of hCT into lipid bilayer membranes was reflected by anapparent partition coefficient log D(pH 7.4) = 2.5 (liposome-buffer equilibrium dialysis). We proposethat the high lipid partitioning and -sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [N-fl]hCT(9-32) in lipid membranes. Vesicular internalization is suggested to beassociated with self-assembly induced perturbation of the lipid bilayer. Condensed hCT self-assembliesmay explain the high capacity of net mucosal-to-serosal hCT permeation, which compares favorably withthe low transport capacity of receptor-mediated endocytosis.