Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus

Toll receptors are cell-surface receptors acting as pattern recognition receptors (PRRs) that are involved in the signaling pathway for innate immunity activation and are genetically conserved from insects to mammals. Tolls from penaeid shrimp are found in white leg shrimp Litopenaeus vannamei (lToll) and black tiger shrimp Penaeus monodon (PmToll). However, the molecular ligand-recognition patterns and identification of these penaeid Toll classes remain unknown. Here, we report cDNA cloning of a new type of Toll receptor gene (MjToll) from kuruma shrimp, Marsupenaeus japonicus, and the modulation of expression by immunostimulation. The full length cDNA of MjToll gene has 3095 nucleotides coding for a putative protein of 1009 amino acids. The MjToll gene is constitutively expressed in the gill, gut, lymphoid organ, heart, hematopoietic organ, hemocyte, ventral abdominal nerve cord, eyestalk neural ganglia and brain tissues. The MjToll gene expression was significantly increased (76-fold) as compared to a control in lymphoid organ stimulated with peptidoglycan at 12h, in vitro. lToll gene showed high similarity to PmToll gene with 96.9% identity; however, MjToll gene exhibited a percentage identity of 59% with that of penaeid Toll homologues. Therefore, this suggests that the identified MjToll gene belongs to the other class of Toll receptors in shrimp.     

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.     

A mammalian like interleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediator in intestinal innate immunity of black tiger shrimp (Penaeus monodon)

Interleukin-1 receptor associated kinase-4 (IRAK-4) has been identified as a central signal transduction mediator of the Toll-like receptor (TLR) and Toll/interleukin-1 receptor (TIR) pathways in vertebrate innate immunity. An IRAK-4 homologue was cloned from the black tiger shrimp (Penaeus monodon) (PmIRAK-4) and it shares domains and structures with other IRAK-4s. It was found to be mainly expressed in the hemocytes and midgut but also to a lower extent in several other tissues in shrimp. The PmIRAK-4 responded to bacterial infection in the intestine by an enhancement of its expression level. These results indicate that PmIRAK-4 may play a role at least in the intestinal innate immunity of P. monodon.     

Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage

The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.     

The ribophorin I from Penaeus monodon shrimp: cDNA cloning, expression and phylogenetic analysis

Ribophorin I, a 67 kDa subunit of the oligosaccharyl transferase complex, is involved in facilitating N-linked glycosylation of polypeptides. We have isolated a full length Penaeus monodon cDNA encoding an insect/mammalian ribophorin I homologue by screening a lymphoid cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction of lymphoid RNA. The cDNA clone of shrimp ribophorin I (PmRibI) consists of 2263 nucleotides encoding 601 amino acid residues. Primary structure analysis of PmRibI indicated that it is a type I transmembrane protein, comprising a cleavable signal sequence of 23 residues at the amino terminus, preceding 434 residues of the luminal domain, 17 residues of the transmembrane domain, and 150 residues of the cytoplasmic domain at the carboxy terminus. The protein has a calculated molecular mass of 67.98 kDa with a pI of 6.05. This putative PmRibI cDNA clone was also expressed as PmRibI-6His in Sf9 cells. The recombinant PmRibI has an apparent molecular weight of 70 kDa, similar to the MW calculated from the deduced cDNA sequence. The inferred protein sequence of PmRibI has 52% identity with that of Strongylocentrotus purpuratus, 49% identity with that of Danio rerio, and 47% identity with mammalian ribophorin I. Phylogenetic analysis showed that PmRibI is most closely related to the echinoderm ribophorin I. The expression of the ribophorin I gene is tissue specific, with its mRNA highly abundant in hemocytes, gill, lymphoid organ and hepatopancreas.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon

A novel leucine-rich repeat (LRR) cDNA has been cloned from hemocytes of the black tiger shrimp Penaeus monodon by 5' rapid amplification of cDNA ends. The full-length of P. monodon LRR (PmLRR) consisted of 2604 bp with a 1686-bp open reading frame, encoding 561 amino acids. The deduced protein contained a high proportion of leucine residues (17%) and had significant homology to LRR-containing proteins from bacteria to humans. Sixteen tandem LRR motifs of 23-24 amino acids in length occurred in the primary sequence. The computed 3D structure revealed a horseshoe shape consisting of alternately repeated strand and helical domains. Such structures are generally considered to mediate protein - protein interactions and to our knowledge, this is the first report of an LRR protein from a crustacean. PmLRR expression was tissue-specific (i.e. highest in hemocytes, intestine and lymphoid organ) suggesting that it may play some roles in shrimp defense against pathogens. A preliminary test suggested that PmLRR may be down-regulated after viral injection.     

A Tudor staphylococcal nuclease from Penaeus monodon: cDNA cloning and its involvement in RNA interference

RNA interference (RNAi) plays an important role in an antiviral defense in shrimp. RNAi technology has been extensively used for inhibition of viral replication and studying gene function. However, the mechanism of shrimp RNAi pathway is still poorly understood. In this study, we identified and characterized an additional protein in the RNAi pathway, Tudor staphylococcal nuclease from Penaeus monodon (PmTSN). The full-length cDNA of PmTSN is 2897 bp, with an open reading frame encoding a putative protein of 889 amino acids. Phylogenetic analysis and domain structure comparison revealed that PmTSN is more closely related to vertebrate TSN by sharing the amino acid sequence identity of 57% with TSN of zebrafish. This represents a new type of TSN proteins by exhibiting the four tandem repeat of staphylococcal nuclease-like domain (SN), followed by a Tudor and a partially truncated C-terminal SN domain. Knockdown of PmTSN by dsRNA targeting SN3 domain resulted in the impairment of dsRNA targeting PmRab7 gene to silence PmRab7 expression. In addition, the efficiency of dsRNA targeting YHV-protease gene inhibiting yellow head virus replication was decreased in the PmTSN-knockdown shrimps. Our results imply that PmTSN is involved in dsRNA-mediated gene silencing in shrimp and thus we identified the additional protein involved in shrimp RNAi pathway.