Proteomic analysis by iTRAQ in red claw crayfish, Cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection

Abstract

To elucidate proteomic changes of Hpt cells from red claw crayfish, Cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (iTRAQ) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (WSSV) infection. Protein database search revealed 594 protein hits by Mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. Generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, DNA replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small GTPases, G-protein-alpha stimulatory subunit, proteins bearing PDZ- or 14-3-3-domains that help holding together and organize signaling complexes, casein kinase I and proteins of the MAP-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response RNA-binding protein 1. Taken together, these protein information shed new light on the host cellular response against WSSV infection in a crustacean cell culture.

Keywords: Crayfish hematopoietic tissue cells; Proteomics; White spot syndrome virus; iTRAQ.

Fig. 1

GO analysis of total number of proteins involved in biological processes identified in crayfish Hpt cells post WSSV infection.

Fig. 2

Validation of proteomic data using RT-qPCR. Relative mRNA levels (y axis) of WSSV treated hpt cells at 1 hpi and 12 hpi where UV treated WSSV was used as control. Selected genes validated were: a) Tudor staphylococcal nuclease; b) Thymidylate synthase; c) MAPKK; d) MAPK-14; e) 14-3-3 epsilon; f) Ras opposite; g) Transglutaminase; h) Calponin – 3.

Fig. S1

Illustration of experimental design in iTRAQ experiment. The infected and mock infected samples were collected in duplicates, pooled and divided into two technical replicates. Samples were lysed and digested with trypsin. The proteins from the mock-infected samples (1 hpi with 113/117; 12 hpi with 115/119) and infected (1 hpi with 114/118; 12 hpi with 116/121) were labeled with iTRAQ reagents. These labeled peptides were pooled, subjected to strong cation exchange chromatography fractionation, and each fraction was analyzed using LC MS/MS.