Protein phosphorylation in hemocytes of Fenneropenaeus chinensis in response to white spot syndrome virus infection

Abstract

Protein phosphorylation and dephosphorylation are the most common and important regulatory mechanisms in signal transduction, which play a vital role in immune defense response. Our previous study has found the level of tyrosine phosphorylation was significantly changed in the hemocytes of Fenneropenaeus chinensis upon white spot syndrome virus (WSSV) infection. In order to explore the relationship between protein phosphorylation and WSSV infection, the quantitative phosphoproteomics was employed to identify differential phosphorylated proteins in hemocytes of F. chinensis before and after WSSV infection, and elucidate the role of key differential phosphorylated proteins in WSSV infection process. The results showed that a total of 147 differential phosphorylated proteins were identified in the hemocytes, including 64 phosphorylated proteins and 83 dephosphorylated proteins, which were mostly enriched in pyruvate metabolism, TCA cycle, glycolysis, and ribosomal biosynthesis. Functional analysis of differential phosphorylated proteins showed that they were involved in cell apoptosis, cell phagocytosis, cell metabolism and antiviral infection. A total of 236 differential phosphorylation sites were found, including 91 modified sites in the phosphorylation proteins and 145 modified sites in the dephosphorylation proteins. Motif analysis showed that these phosphorylation sites could activate mitogen-activated protein kinase, P70 S6 kinase and other kinases in hemocytes. Moveover, the phosphorylation levels of eukaryotic protein initiation factor 4E binding proteins and histone H3 were further determined by ELISA and Western blotting, which both exhibited a significant increase post WSSV infection and reach their peak levels at 6 and 12 h, respectively. Moreover, we found that lactate, a metabolite closely related to pyruvate metabolism, TCA cycle and glycolysis, was significantly increased in the hemocytes after WSSV infection. This study revealed the protein phosphorylation response in hemocytes of F. chinensis to WSSV infection, which help to clarify the response characteristics and virus resistance mechanism of hemocytes in F. chinensis, and also facilitate further understanding of the interaction between WSSV and shrimp hemocytes.