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.

Rational design of aggregation-resistant bioactive peptides: Reengineering human calcitonin

“…3c). This finding can be related to longer-term receptor activation, as it is the case for sCT, perhaps associated with an increased facility of the CT variants to interact with membrane lipids (42,43). Such an observation could have important repercussions in the use of these reengineered variants as therapeutics.…”

Converting the Highly Amyloidogenic Human Calcitonin into a Powerful Fibril Inhibitor by Three-dimensional Structure Homology with a Non-amyloidogenic Analogue

“…6). This finding can be related to longer term receptor activation as is the case for sCT and perhaps is associated with an increased facility of phCT and the phCT-hCT (1:1) mixture to interact with membrane lipids (53,54). Such an observation could have important consequences in the use of phCT, alone or mixed with wild-type hCT, as therapeutics.…”

Cell-penetrating peptides: tools for intracellular delivery of therapeutics

“…In this review, we describe the most popular cell-penetrating peptides and their use as transfection agents and, where possible, try to correlate their transfection characteristics with their physicochemical properties. Among them, we will mainly focus on peptides derived from protein transduction domains or their kin for classification reasons (transportan [8], penetratin [9], Tat [10] and VP22 [11]), amphipathic peptides (MAP [12], KALA [13], ppTG20 [14], proline-rich peptides [15], MPG-derived peptides [16] and Pep-1 [17]) and three peptides which cannot be fit into the classes cited above (loligomers [18], arginine-rich peptides [19] and calcitonin-derived peptides [20]). The sequences of the leader compounds of each family are reported in table 1.…”