Mapping of the Histatin Domain Involved in Cell Migration

Objectives: 1. To confirm the wound healing properties of histatin in a fysiologically more relevant skin equivalent model. 2. To map the minimal domain on histatin that is required for its cell-activating properties.

Methods: Histatin-1, cyclic histatin-1 and truncated histatins were synthesised by solid phase peptide synthesis using Fmoc chemistry. In vitro wound-closure properties of histatin and histatin-variants were evaluated using scratch-assays. In addition, the effect of histatin-1 was measured in epidermal skin equivalent, as follows: full-thickness wounds were created by freeze burning. After 6 days culturing in the presence or absence of histatin-1, re-epithelialization was analyzed by microscopy on paraffin sections. Effect of histatin on cell-spreading was examined by immunofluorescence microscopy. Effect of histatin on cell-proliferation was examined by determining the number of cells, after two day incubation with histatin.

Results: Histatin enhanced re-epithelialization in an epidermal skin equivalent closely resembling normal human skin. The peptide did not stimulate cell-proliferation, but induced cell-spreading and -migration, two key initiating steps in re-epithelialization. Using a stepwise-truncation method, we mapped the minimal domain of the peptide required for activity. N-to-C terminal cyclization of Histatin-1 potentiated the molar activity approximately 1,000-fold, indicating that the recognition of histatin by its cognate receptor required a specific spatial conformation of the peptide.

Conclusion: Our results emphasize the importance of histatin in human saliva for tissue protection and recovery, and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents.