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

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and plays multifunctional roles in the immune system. In the present study, a homolog of TRAIL from the Mongolian horse (named ecTRAIL) was identified and characterized. The 870-bp open reading frame encodes a polypeptide of 289 amino acid residues with a predicted molecular weight of 33.47 kDa and pI of 8.47. The genomic structure of ecTRAIL shares a five-exon/six-intron arrangement similar to its orthologs. Multiple alignments show that ecTRAIL is a type II transmembrane protein with a typical transmembrane region, three conserved cysteine residues (Cys56, Cys77, Cys238) and a TNF family signature sequence ([LV]-x-[LIVM]-x(3)-G-[LIVMF]-Y-[LIVMFY](2)-x(2)-[QEKHL]-[LIVMGT]-x-[LIVMFY]). Three-dimensional structure prediction based on the same template revealed that the positional arrangement of the key amino acid residues, Cys238 and Cys230 in ecTRAIL and human TRAIL, respectively, is significantly conserved. Evolutionary analysis suggests that ecTRAIL is most closely related to its ortholog from pigs, with an identity of 83.99%. The solubilizing small ubiquitin-related modifier (SUMO) tag fused recombinant protein SUMO-ecsTRAIL was successfully expressed in E. coli and exhibited binding activity and cytotoxicity to HeLa cells in a cross-species manner in vitro. These results provide a better understanding of TRAILs in mammals and indicate that ecTRAIL may play an important role in the immune response in horses.