Thiol-Associated Antioxidant Activity of Recombinant Mussel Foot Protein Mfp6-1 Supports Cutaneous Wound Repair in a Murine Model

Mussel foot proteins (Mfps) are renowned for their underwater adhesion, whereas their biotechnological potential for cutaneous wound repair remains largely underexplored. In this study, we identified and characterized a cysteine-rich mussel foot protein, Mfp6-1, from Mytilus coruscus and investigated its therapeutic potential for wound healing. Sequence analysis showed that Mfp6-1 is enriched in cysteine (11.0%) and tyrosine (~16.5%). We successfully expressed recombinant Mfp6-1 (rMfp6-1) in E. coli. Structural prediction based on the mature peptide sequence suggested that rMfp6-1 adopts a relatively compact fold containing several short β-structural elements. In vitro assays demonstrated that rMfp6-1 possesses antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and alkylation experiments suggested that cysteine residues contribute importantly to this activity. Dithio-bis-nitrobenzoic acid (DTNB)-based thiol quantification further demonstrated that rMfp6-1 contained abundant accessible free sulfhydryl groups, supporting an important contribution of cysteine-derived thiols to its antioxidant activity. Experiments on a full-thickness mouse wound model showed that rMfp6-1 treatment resulted in significantly faster wound contraction. Morphological analysis further revealed that rMfp6-1 optimizes the healing microenvironment by promoting collagen accumulation and re-epithelialization. Additionally, the treatment was found to trigger vascular endothelial growth factor (VEGF)-mediated angiogenesis, thereby improving the overall quality of the regenerated tissue. Furthermore, rMfp6-1 treatment significantly reduced interleukin-6 (IL-6) expression, suggesting that its antioxidant capacity creates a permissive microenvironment for tissue regeneration by suppressing excessive inflammation. These findings indicate that recombinant rMfp6-1 is a promising bioactive candidate for wound-healing applications.