Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue.

The study provides a comprehensive analysis of the major cytoskeletal proteins present in the equine hoof lamellae, a structure pivotal to the horse’s mobility and prone to a debilitating disease called laminitis. Using mass spectrometry and other techniques, two novel keratins, K42 and K124, were identified as the most abundant cytoskeletal proteins in equine hoof lamellae.

Objective and methodology

• The purpose of this research was to identify and quantify the cytoskeletal proteins in the equine hoof lamellae, a tissue that connects the horse’s distal phalanx (last bone of the fingers and toes) to the cornified hoof wall. This area has significant biomechanical importance to horses but is also commonly affected by a disease called laminitis.
• Protein was extracted from the mid-dorsal epidermal (outer layer of skin) and dermal lamellae (inner layer of skin) from the front feet of 5 Standardbred geldings and 1 Thoroughbred stallion.
• The researchers used mass spectrometry-based spectral counting techniques, PAGE (polyacrylamide gel electrophoresis), and immunoblotting to identify and quantify cytoskeletal proteins. They also used indirect immunofluorescence for the cellular localization of two specific keratins, K14 and K124.

Findings

• Mass spectrometry analysis identified a range of proteins including 3 actin microfilament proteins, 30 keratin proteins, 6 tubulin microtubule proteins, as well as other proteins including vimentin, desmin, peripherin, and internexin.
• Two novel keratins, K42 and K124, were found to be the most abundant cytoskeletal proteins in equine hoof lamellae, constituting 22.0 ± 3.2% and 23.3 ± 4.2% of the cytoskeletal proteins respectively.
• Immunoreactivity to K14 was found in the basal cell layer, while that to K124 was found in basal and suprabasal cells in the secondary epidermal lamellae.
• The most abundant proteins identified on 1- and 2-dimensional polyacrylamide gels were K124, K42, K14, K5, and α(1)-actin. This corroborates the results of previous studies.

Conclusion

• The study accomplished its objective of identifying and quantifying the cytoskeletal proteins in the equine hoof lamellae, which is crucial for understanding the equine hoof’s structure and function. Notably, it discovered two keratins (K42 and K124) to be the most abundant cytoskeletal proteins in this structure.
• The results provide foundational data that could be used in further research into the pathogenesis of laminitis and potential treatment options for the disease.