Identification, characterization and bioactivity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand from Equus caballus.

The study is focused on the identification and characterization of a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) from the Mongolian horse. The research further explores its molecular configuration, evolutionary origin, and bioactivity against cancer cells, which could potentially contribute to improvements in equine immune responses.

Research Methodology and Findings

• The researchers identified and characterized an 870-bp open reading frame encoding a polypeptide of 289 amino acid residues, termed as ecTRAIL, in the Monglian horse. This molecule has a predicted molecular weight of 33.47 kDa and a pI (isoelectric point) of 8.47.
• The genomic structure of this ecTRAIL shares a five-exon/six-intron arrangement that is similar to its orthologs. This means that the gene’s structure involving coding (exon) and non-coding (intron) sequences mirrors that of its analogous genes from other animals.
• Further analysis revealed that ecTRAIL is a type II transmembrane protein, implying it spans the cell membrane, with its N-terminus inside the cell and C-terminus outside. This protein contains three conserved cysteine residues (Cys56, Cys77, Cys238) and exhibits a unique TNF family signature sequence.
• Three-dimensional structure prediction showed that the positional arrangement of key amino acid residues, particularly Cys238 in ecTRAIL and Cys230 in human TRAIL, were significantly conserved. This suggests that despite they come from different species, these TRAIL proteins share similar structural characteristics.
• Evolutionary analysis suggests a close relationship between the horse’s TRAIL and pig TRAIL, with an identity of 83.99%, indicating that these TRAILs might have evolved from a common ancestral gene.
• The solubilizing small ubiquitin-related modifier (SUMO) tag fused recombinant protein SUMO-ecsTRAIL was successfully expressed in E. coli. The study also demonstrated that SUMO-ecsTRAIL bound effectively to human cancer cells (HeLa cells) and induced cytotoxicity in vitro, indicating its potential therapeutic applications.

Implication of The Research

• The outcomes of this research underscored the importance of ecTRAIL in the immune response of horses. its cytotoxicity against cancer cells could potentially contribute to advancements in equine healthcare.
• Moreover, the successful expression of the SUMO-ecsTRAIL in E. coli, and its interaction with human cancer cells, could pave the way for the development of horse-derived therapeutic molecules with cytotoxic effects on cancer cells.