Enhanced protein phosphorylation in Apostichopus japonicus intestine triggered by tussah immunoreactive substances might be involved in the regulation of immune-related signaling pathways

Highlights

• It is the first time to explore the phosphoproteomic profiles in sea cucumber by using tussah immunoreactive substances (TIS).

• Sixteen of the phosphoproteins exhibited differential phosphorylation in response to TIS.

• These phosphorylated proteins triggered by TIS might be involved in the regulation of immune-related signaling pathways.

Abstract

The sea cucumber Apostichopus japonicus is an economically important species owing to its high nutritive and medicinal value. In order to avoid the pollution resulting from the overuse of antibiotics in A. japonicus aquaculture, various immunostimulants have been used as an alternative to improve the efficiency of A. japonicus farming. Our previous proteomic investigation has shown that several proteins participating in the immune-related physiology of A. japonicus were differentially expressed in the intestinal tissue in response to tussah immunoreactive substances (TIS). This study further explored the immunostimulation mechanism of TIS in A. japonicus. Phosphoproteomics technology was used to investigate the effect of TIS on protein phosphorylation in the intestine of A. japonicus following feeding with a TIS-supplemented diet. A total of 213 unique phosphoproteins were detected from 225 unique phosphopeptides. KEGG pathway analysis showed that majority of the phosphoproteins are involved in endocytosis, carbon metabolism and spliceosome functional group. Sixteen of the phosphoproteins exhibited differential phosphorylation in response to TIS and 12 of these were found to associate with biological functions. Of these 12 phosphoproteins, eight exhibited enhanced phosphorylation while four displayed reduced phosphorylation. These 12 proteins were further analyzed and all were found to play a role in regulating some aspects of the immune system and the growth of sea cucumbers, especially in phagocytosis, energy metabolism and disease resistance. The findings of this study could therefore shed new light on the immune pathways of sea cucumber that are affected by TIS. This could help us to better understand the underlying mechanism linked to the immunoenhancement of A. japonicus in response to TIS, one that is associated with the change in protein phosphorylation.

Introduction

Phosphorylation plays an important role in a wide range of cellular functions and the phosphorylation of proteins is regarded as one of the most prevalent post-translational modifications (Lemeer and Heck, 2009). Phosphorylated proteins can be converted back to the non-phosphorylated forms by the removal of the phosphoryl group, a reaction catalyzed by protein phosphatases, which play a key role in signal transduction cascades (Pawson and Nash, 2000). Reversible phosphorylation has been studied extensively and is known to influence protein function by changing the activity, stability, and spatial organization of the protein as well as mediating protein-protein interactions (Cohen, 2000). The structural changes in the proteins introduced by phosphorylation can also recruit other proteins that contain structurally conserved domains to recognize and bind to specific motifs (Seet et al., 2006). Phosphorylation regulates protein function and cell signaling by triggering a change in the three-dimensional structure of the protein, which in turn can have a direct effect on the catalytic function of the protein through its activation or deactivation (Pawson and Scott, 1997). Phosphoproteomics approach plays an important role in the characterization of signaling pathways in a time and stimulus-dependent manner. Phosphoproteomics can enable the analysis of thousands of phosphoproteins in vivo (Schreiber et al., 2008; Choudhary and Mann, 2010). Therefore, through phosphoproteomics analysis, the events of phosphorylation crucial to the functional biology of sea cucumber might be determined.

In recent years, increased demand of sea cucumber by the consumer market has led to the over exploitation and reduction of the world natural population of sea cucumber, especially the over consumption of Apostichopus japonicus, and this has been the driving force behind the rapid development of sea cucumber mariculture (Purcell et al., 2013). However, the growing practice of sea cucumber mariculture has led to the spread of diseases among the population of cultured sea cucumbers because of inadequate prevention and treatment strategies (Li et al., 2016c). Furthermore, the indiscriminate use of antibiotics has led to serious environmental pollution. Thus, instead of antibiotics, immunostimulants, which also include immune proteins, are currently being used to prevent the spread of diseases among sea cucumber in aquaculture (Zhang et al., 2010). Immunostimulants confer a number of benefits for the animals that consume them, and these benefits include the enhancement of immunity, disease resistance and weight gain. Immune proteins in the pupae of Hyalophora cecropia were first detected in 1980 (Gotz et al., 1981), and this was followed by the isolation of tussah immunoreactive substances (TIS) from the pupal hemolymph of Chinese oak silkworm (Antheraea pernyi) in 1981 (Huang and Wang, 1981). TIS contains various biologically active materials such as antimicrobial peptide, lysozyme and lectin (Fan et al., 2009), and it has been used as a feed additive to improve the culture efficiency of multiple animals. The addition of TIS to the diet can significantly enhance the contents of many immune-related enzymes in broilers, including immunoglobulin G, immunoglobulin M, complement C3, and complement C4 (Li et al., 2016a). Diets that are supplemented with TIS can effectively promote the growth and development of weaned piglets and improve the immune ability of weaned piglets (Li et al., 2016b). TIS supplement has been shown to increase the resistance of shrimp to pathogens (Sun et al., 2017). The addition of TIS can also effectively prevent the occurrence of diseases and enhance the survival rate and body weight of sea cucumber (Ma et al., 2017). Our previous study has shown that several immune-related proteins in the intestine of sea cucumber can be up-regulated in response to TIS feeding (Mi et al., 2018), providing an impetus for further study into the mechanism of TIS-induced enhanced immunity in sea cucumber. Recently, some studies have reported the effects of posttranslational modification on some response mechanisms of sea cucumber, such as intestinal regeneration and aestivation (Chen et al., 2016; Sun et al., 2018). However, no study has been carried out at the posttranslational modification level to explain the enhanced immunity of sea cucumber induced by immunostimulants. Thus, in this study, in-depth phosphoproteomics analysis was used to investigate the changes in signal transduction in the intestines of sea cucumbers that had been fed with TIS. These identified phosphorylated proteins might reflect the ongoing response of sea cucumber to the oral administration of TIS, where these proteins might be involved in the immune-related signal transduction cascades, such as phagocytosis and metabolism. These data might provide new insight into the mechanism underlying the immune enhancement effect of TIS in sea cucumber.