C-Terminal Hemocyanin from Hemocytes of Penaeus vannamei Interacts with ERK1/2 and Undergoes Serine Phosphorylation

Abstract

To understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up-regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (pI), whereas the N-terminal fragments had less acidic pI. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co-immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin.

Transcriptome Analysis of Pacific White Shrimp (Litopenaeus vannamei) Hepatopancreas in Response to Taura Syndrome Virus (TSV) Experimental Infection

“…Hemocyanins have the defense-related functions that are mediated through phenoloxidase activity. Several previous studies reported that hemocyanins in shrimp were greatly over expressed during WSSV infection[56]–[58]. Similarly, several other previously reported differentially expressed genes, such as heat shock protein, lysozyme and fatty acid-binding protein, strongly up-regulated in shrimp challenged with WSSV[59]–[61].…”

The proteomic response of cheliped myofibril tissue in the eurythermal porcelain crabPetrolisthes cinctipesto heat shock following acclimation to daily temperature fluctuations

“…An almost identical pattern of hemocyanin isoforms was observed in hemocytes of the Pacific white shrimp Penaeus vannamei upon virus infection (Chongsatja et al, 2007). Subsequent work showed that isoforms similar to those with low molecular mass in cluster II are peptides cleaved off the N-terminal hemocyanin sequence in white shrimp (Havanapan et al, 2009). Hemocyanin isoforms of intermediate mass (spots 62, 138 and 179) may represent peptides cleaved off the C-terminus and may be phosphorylated by the ERK1/2 MAP kinase signaling pathway (see below for link to inflammation), explaining the relatively low pI of two of these isoforms (Havanapan et al, 2009).…”

“…Subsequent work showed that isoforms similar to those with low molecular mass in cluster II are peptides cleaved off the N-terminal hemocyanin sequence in white shrimp (Havanapan et al, 2009). Hemocyanin isoforms of intermediate mass (spots 62, 138 and 179) may represent peptides cleaved off the C-terminus and may be phosphorylated by the ERK1/2 MAP kinase signaling pathway (see below for link to inflammation), explaining the relatively low pI of two of these isoforms (Havanapan et al, 2009). Only one of these three isoforms increased with heat shock after 10-30°C (spot 138).…”

Identification and characterization of glycosylation sites on Litopenaeus vannamei hemocyanin

“…Our previous studies showed that the Ig‐like domain of Penaeus vannamei hemocyanin could react with anti‐human Ig and was also significantly upregulated during Taura syndrome virus infection . All these suggest that the Ig‐like domain of hemocyanin possessed immunology function.…”