Identification, cellular localization, and function analysis of a novel toll-like receptor (PtToll4) in Portunus trituberculatus

Toll-like receptor (TLR) is one of the most important pattern recognition receptors in both invertebrate and vertebrate animals. In previous study, three different kinds of TLR cDNAs (PtToll1-3) have been cloned from Portunus trituberculatus. In this study, a novel PtToll4 cDNA sequence was identified in this crab. The PtToll4 ORF is predicted to encode 894 peptides with an initiating signal peptide, an extracellular LRRs domain, a transmembrane region, and an intracellular toll-interleukin-1 receptor domain. Based on the sequence and phylogenetic analysis, PtToll4 distinctly clustered with almost all crustacean tolls, which mostly clustered with some shrimp TLRs and Eriocheir sinensis Toll 2 (EsToll 2). The real-time quantitative polymerase chain reaction analysis showed that the transcript of PtToll4 was constitutively expressed in tissues. In hemocytes, the transcript of PtToll4 in large granulocytes is approximately threefold higher than in small granulocytes and hyalocytes. Moreover, the expression of PtToll4 could be moderately and shortly upregulated by Vibrio alginolyticus or lipopolysaccharide challenge in the primary cultured hemocytes. In HEK 293T cell model, over-expressed PtToll4 was not able to activate the mammal NF-κB, compared with a positive plasmid. In the Drosophila S2 cell model, over-expressed PtToll4 distributed mainly in cell membrane and could affect the activation of some Drosophila antimicrobial peptides promoters. The RNA interference experiment showed that the expressions of PtToll4 was significantly inhibited by its specific double-stranded RNA in the crab hemocytes and accompanied by the suppressed expressions of the anti-lipopolysaccharide factor 4 (ALF4), ALF7, hyastatin3, crustin1, and crustin3.