Engineered Caf1 constructs displaying laminin-332 motifs enhance keratinocyte migration and restore adhesion in LAMA3-deficient cells

Effective wound healing relies on robust re-epithelialization, a process critically supported by Laminin-332, a key extracellular matrix (ECM) protein. Its deficiency, as seen in conditions like Junctional Epidermolysis Bullosa (JEB), severely impairs epithelial repair. Direct therapeutic application of full-length laminin or its fragments faces challenges due to poor stability and high production costs. To overcome these limitations, researchers are exploring biomaterial scaffolds. The Capsular Antigen F1 (Caf1) offers a promising, stable, and customizable protein scaffold for presenting bioactive motifs, providing a practical alternative for therapeutic delivery.

Researchers identified key Laminin-332-derived peptide motifs via literature review and engineered them into Caf1 constructs, expressed and purified from Escherichia coli. In vitro assessments used normal human epidermal keratinocytes and LAMA3-knockdown cells cultured on Caf1-coated plates. They evaluated keratinocyte migration, single-cell motility, adhesion, and pSMAD2 signaling. For translational relevance, an ex vivo human skin wound model was employed: wounded skin explants were treated with Caf1 constructs and cultured at the air-liquid interface for 7 days. Re-epithelialization and epithelial thickness were subsequently assessed using haematoxylin and eosin staining.

The study successfully expressed and purified Caf1 proteins displaying Laminin-332-derived motifs from E. coli. Among the various engineered constructs, Caf1-J3 and Caf1-J4 emerged as leading candidates, demonstrating significant therapeutic potential. In vitro, both Caf1-J3 and Caf1-J4 significantly enhanced keratinocyte migration and single-cell motility.

Combined application of Caf1-J3 and Caf1-J4 further accelerated wound closure, suggesting synergistic or complementary effects between these motifs. Crucially, in LAMA3-deficient keratinocytes, these Caf1 coatings effectively restored adhesion, addressing a critical deficit in laminin-deficient conditions. The findings indicate that these engineered constructs can effectively mimic the biological functions of native Laminin-332 in promoting epithelial repair, even under compromised conditions.