Genome-wide identification of glutathione S-transferase (GST) gene family and its regulating characteristics responding to sperm capacitation in Eriocheir sinensis.

Sperm capacitation is regarded to be a process of redox regulation. Within the oxidative regulatory system, glutathione S-transferases (GSTs) are important in the maintenance of the redox balance of reactive oxygen species (ROS). We identify 56 GST genes in the Chinese mitten crab Eriocheir sinensis genome, and categorize them into 13 classes, of which the largest is Mu. Chromosomal localization reveals an uneven distribution of EsGSTs across the E. sinensis genome, yet similar structures and conserved motifs occur within a class. Proteome analysis reveals EsGSTz2, EsGSTm2, and EsGSTd/e5 to be significantly up-regulated and EsGSTt to be down-regulated considerably after incubation with egg water. Capacitation also triggered the acetylation alteration in EsGSTs. Notably, six acetylation sites are identified in EsGSTm2, with notable deacetylation in K43, K132, and K123. Activity analysis reveals that deacetylation in both K43 and K123 can improve GST activity, whereas double-site mutations of K43R and K132R reduce enzyme activity. At pH 8.2, K123R has higher activity compared with other arginine mutants. Blocking of EsGSTm2 induces an imbalance in sperm oxidation levels and intracellular calcium homeostasis, and inhibits sperm acrosome reaction. This work extends our understanding of the GST regulation mechanisms during crustacean sperm capacitation.