Large-scale lysine crotonylation analysis reveals its potential role in spermiogenesis in the Chinese mitten crab Eriocheir sinensis

Abstract Lysine crotonylation (Kcr) is a recently-discovered type of post-translational modification. Although Kcr has been reported in many species, little is known about this process in crustaceans. In this study, pan anti-lysine crotonylation antibody enrichment and high-resolution liquid chromatogram-mass spectrometry analysis were employed to characterize Kcr in testis of the Chinese mitten crab Eriocheir sinensis testis. Overall, 2799 Kcr sites were identified on 908 proteins with 14 conserved motifs. Bioinformatics analysis showed that Kcr was predominant on proteins found in cytoplasm, mitochondria and nucleus, and those involved in ribosome, proteasome, carbon metabolism and protein processing in endoplasmic reticulum. In total, 83 up-regulated and 12 down-regulated non-histone crotonylated sites were identified during spermiogenesis. These differentially expressed proteins were enriched in protein processing in endoplasmic reticulum pathway during formation of acrosome. In contrast, histone Kcr associated with mammalian spermatogenesis. These results provide foundational knowledge on the role of non-histone Kcr in spermiogenesis of E. sinensis. Significance Lysine crotonylation (Kcr) is a recently-identified post-translational modification, and histone Kcr was found to associate with mammalian spermatogenesis. However, crotonylation of non-histone proteins has not been reported in spermatogenesis regulation. Further, there is no information on crotonylation in crustaceans. This study was the first large-scale Kcr proteome characterization in crustaceans. A total of 2799 Kcr sites on 908 proteins with 14 conserved motifs were identified from Eriocheir sinensis testis. Of which, 83 up-regulated and 12 down-regulated non-histone crotonylated sites were identified during spermiogenesis. Our results provide the basic information for further functional validation of Kcr proteins and revealed new roles of Kcr in spermiogenesis of E. sinensis.