A C-type lectin is involved in the innate immune response of Chinese white shrimp.

C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.

A selenium-dependent glutathione peroxidase (Se-GPx) and two glutathione S-transferases (GSTs) from Chinese shrimp (Fenneropenaeus chinensis).

Glutathione S-transferases (GSTs) and glutathione peroxidases (GPxs) are essential components of cellular detoxification systems that defend cells against reactive oxygen species (ROSs). Two GSTgenes have recently been cloned from Fenneropenaeus chinensis and BLAST P analysis shows that one GST, designated FcMuGST, is similar to members of MuGST while the other has similarities to ThetaGST (FcThetaGST). A selenium-dependent glutathione peroxidase (Se-GPx) has also been cloned from F. chinensis. The alignment of the deduced GST and GPx amino acid sequences with those from other species showed that the residues essential for enzymatic function of these three proteins are highly conserved. Tissue distribution and response to pathogens for the three genes was investigated by RT-PCR analysis, which showed that the transcript of FcMuGST and FcGPx increased in response to Vibrio anguillarum infection, while FcThetaGST showed little change at the transcript level. GPx activity in gill tissues quickly increased at 6 h after V. anguillarum challenge and maintained at a relatively high level from 6 h to 24 h. Total GST activity in hepatopancreas and intestines of the bacterial challenged shrimp was increased at 6 h, and gradually recovered from 12 and 24 h to the normal level. These three genes were all predicted to play an important role in detoxification defense reactions. FcMuGST primarily scavenges excess ROS produced after bacterial infection, while clearance of endogenous hydrophobic electrophile molecules was mainly dependent on activities of FcThetaGST.

A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein.

Lectins are regarded as potential immune recognition proteins. In this study, a novel C-type lectin (Fc-Lec2) was cloned from the hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-Lec2 is 1219 bp with an open reading frame (ORF) of 1002 bp that encodes a protein of 333 amino acids. Fc-Lec2 contains a signal peptide and two different carbohydrate recognition domains (CRDs) arranged in tandem. The first CRD contains a QPD (Gln-Pro-Asp) motif that has a predicted binding specificity for galactose and the second CRD contains a EPN (Glu-Pro-Asn) motif for mannose. Fc-Lec2 was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated in the hepatopancreas of shrimp challenged with bacteria or viruses. Recombinant mature Fc-Lec2 and its two individual CRDs (CRD1 and 2) did not have hemagglutinating activity against animal red blood cells, but agglutinated some gram-positive and gram-negative bacteria in a calcium-dependent manner. The three recombinant proteins also bound to bacteria in the absence of calcium. Fc-Lec2 seems to have broader specificity and higher affinity for bacteria and polysaccharides (peptidoglycan, lipoteichoic acid and lipopolysaccharide) than each of the two individual CRDs. These data suggest that the two CRDs have synergistic effect, and the intact lectin may be more effective in response to bacterial infection, the Fc-Lec2 performs its pattern recognition function by binding to polysaccharides of pathogen cells.

A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity.

Lectins play important roles in animal innate immune responses by serving as pattern recognition receptors, opsonins, or effector molecules. Here, we report a novel hepatopancreas-specific C-type lectin, designated Fc-hsL, from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-hsL is 571 bp long with a 480 bp open reading frame that encodes a 159-residue protein. Fc-hsL contains a signal peptide and a single C-type lectin-like domain (CTLD) or carbohydrate recognition domain (CRD). It has an EPN(Glu-Pro-Asn) motif with a predicted ligand-binding site specific for mannose. Fc-hsL was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated following challenge of shrimp with bacteria or virus. Fc-hsL was not detected in other tissues but was induced in the stomach of immune-challenged shrimp. Fc-hsL protein was detected in both hemolymph and the hepatopancreas of bacteria- and virus-challenged shrimp. Recombinant mature Fc-hsL has no hemagglutinating activity, but calcium-dependent agglutinating activity against some Gram-positive and Gram-negative bacteria was detected. The rFc-hsL also has binding activity to some Gram-positive and Gram-negative bacteria and high antimicrobial activity against some bacteria and fungi. These in vitro functions of recombinant Fc-hsL were calcium-independent. Fc-hsL may act as a pattern recognition receptor in antibacterial defense and as an effector in innate immunity of Chinese shrimp.

Purification and characterisation of an inhibitor of a cathepsin B-like proteinase from sunflower seed.

Cathepsin B is a vitally important enzyme in various physiological processes and in tumor invasion and metastasis. A cathepsin B inhibitor, HCB-SunI, was identified and purified from sunflower seeds, Helianthus annuus, using ammonium sulfate precipitation and two steps of conventional chromatography. The molecular mass of HCB-SunI was estimated to be 12 kDa by SDS-PAGE and 12.32 kDa by MALDI TOF MS. Its N-terminal amino acid sequence was determined to be: PYGGGGTESG. HCB-SunI not only inhibited Helicoverpa cathepsin B (HCB) but also decreased the growth of HeLa and glioma cells by 7-27% and 6-22%, respectively, when the cells were grown in a final concentration of 0.002-0.008 microM inhibitor.

Molecular cloning and characterization of Fc-TSP from the Chinese shrimp Fennerpenaeus chinensis.

Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that modulate cell behavior in homeostasis and during development, wound-healing, immune response and tumor growth of adult tissues in vertebrates. In invertebrates these proteins are a major component of cortical rods in mature oocytes. A fragment of a thrombospondin-like gene was generated by screening a subtractive cDNA library constructed from the hemocytes of Chinese shrimp, Fennerpenaeus chinensis. The full length F. chinensis cDNA of thrombospondin was cloned by 3'- and 5'-rapid amplification of cDNA ends (3'- and 5'-RACE). The complete cDNA sequence, named Fc-TSP, is 2886 bp and the open reading frame of the cDNA encodes a 938-residue protein that contains three ChtBD2 domains, an EGF domain, a TSP-3 domain and a common TSP-C (CTD) domain. The protein shares a high sequence identity with the mj-TSPa (46.3%), mj-TSPb (46.9%) and mj-TSPc (51.9%) of Marsupenaeus japonicus. The expression and distribution of Fc-TSP in both challenged and unchallenged shrimps were studied by Northern blot, RT-PCR and in situ hybridization. Northern blot analysis showed that the Fc-TSP transcripts were detected in the hemocytes, heart, intestine, stomach and ovary of both challenged and unchallenged shrimps, but the signal was much stronger in the challenged tissues. A strong hybridization signal was detected only in challenged hepatopancreas, with no signal in the unchallenged tissue. The RT-PCR showed that the Fc-TSP was detected in both challenged and unchallenged tissues including the hemocytes, heart, hepatopancreas, stomach, gills, intestine, spermary and ovary. Except for the ovary and spermary, the signal of challenged tissues was relatively stronger than that of unchallenged ones, especially in hepatopancreas. These results suggest that the thrombospondin was upregulated in the hemocytes, heart, intestine and stomach of challenged shrimp, and induced in the hepatopancreas of challenged shrimps. Therefore, Fc-TSP may be involved in the defense responses of the shrimp.