Anti-epileptic Kunitz-like peptides discovered in the branching coral Acropora digitifera through transcriptomic analysis

Approximately 50 million people are suffering from epilepsy worldwide. Corals have been used for treating epilepsy in traditional Chinese medicine, but the mechanism of this treatment is unknown. In this study, we analyzed the transcriptome of the branching coral Acropora digitifera and obtained its Kyoto Encyclopedia of Genes and Genomes (KEGG), EuKaryotic Orthologous Groups (KOG) and Gene Ontology (GO) annotation. Combined with multiple sequence alignment and phylogenetic analysis, we discovered three polypeptides, we named them AdKuz1, AdKuz2 and AdKuz3, from A. digitifera that showed a close relationship to Kunitz-type peptides. Molecular docking and molecular dynamics simulation indicated that AdKuz1 to 3 could interact with GABA(A) receptor but AdKuz2-GABA(A) remained more stable than others. The biological experiments showed that AdKuz1 and AdKuz2 exhibited an anti-inflammatory effect by decreasing the aberrant level of nitric oxide (NO), IL-6, TNF-alpha and IL-1 beta induced by LPS in BV-2 cells. In addition, the pentylenetetrazol (PTZ)-induced epileptic effect on zebrafish was remarkably suppressed by AdKuzl and AdKuz2. AdKuz2 particularly showed superior anti-epileptic effects compared to the other two peptides. Furthermore, AdKuz2 significantly decreased the expression of c-fos and npas4a, which were up-regulated by PTZ treatment. In addition, AdKuz2 reduced the synthesis of glutamate and enhanced the biosynthesis of gamma-aminobutyric acid (GABA). In conclusion, the results indicated that AdKuz2 may affect the synthesis of glutamate and GABA and enhance the activity of the GABA(A) receptor to inhibit the symptoms of epilepsy. We believe, AdKuz2 could be a promising anti-epileptic agent and its mechanism of action should be further investigated.