Xiao-Fan Zhao

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 novel C-type lectin (FcLec4) facilitates the clearance of Vibrio anguillarum in vivo in Chinese white shrimp.

C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps.

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.

Molecular cloning and expression analysis of chymotrypsin-like serine protease from the Chinese shrimp, Fenneropenaeus chinensis

A new member of the serine protease (SP) chymotrypsin (designated Fc-chy) was isolated from the hepatopancreas of Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA of Fc-chy contained 951 nucleotides with a polyadenylation sequence and a polyA tail. It encoded a peptide of 271 amino acids with a signal peptide of 17 amino acids and an activation peptide of 28 amino acids. The mature peptide concludes 226 amino acids. It contained the conserved catalytic triad (H, D, and S). Similarity analysis showed that Fc-chy shared high identity with chymotrypsins from the Pacific white shrimp, Litopenaeus vannamei. Northern blot, quantitative real-time PCR, in situ hybridization, and western blot analysis were carried out to analyze the expression pattern and distribution profiles of Fc-chy after bacteria and virus challenges. The results showed that Fc-chy transcription and Fc-chy protein levels were upregulated in the hepatopancreas after bacterial and viral infection. Fc-chy from the hepatopancreas was purified by affinity chromatography. It showed high hydrolytic activity toward the substrate N-succinyl-ala-ala-pro-phe p-nitroanilide (AAPF), and its activity was inhibited by Kazal-type SP inhibitor from Chinese shrimp. All of these may indicate that Fc-chy is involved in the innate immune reactions in Chinese shrimp.

A Shrimp C-type Lectin Inhibits Proliferation of the Hemolymph Microbiota by Maintaining the Expression of Antimicrobial Peptides

Background: The hemolymph of healthy shrimp contains low albeit stable numbers of bacteria. Results: Knockdown of C-type lectin MjHeCL led to suppressed expression of antimicrobial peptides, bacterial proliferation, and shrimp death. Conclusion: MjHeCL protects shrimp by inhibiting the proliferation of hemolymph microbiota. Significance: This study demonstrated a novel role for soluble C-type lectins in antibacterial response. Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species.

Establishment of a New Cell Line from Lepidopteran Epidermis and Hormonal Regulation on the Genes

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.

C-type lectin binds to β-integrin to promote hemocytic phagocytosis in an invertebrate

Background: Our previous study found shrimp C-type lectin (FcLec4) facilitated bacterial clearance in vivo. Results: Native FcLec4 promoted hemocytic phagocytosis. The FcLec4 receptor was identified as β-integrin. Conclusion: FcLec4 functions as an opsonin, and β-integrin determines its opsonic activity. Significance: This is the first study to show the structural basis of an opsonin in crustaceans. Phagocytosis is a conserved cellular response among metazoans. Opsonins are some molecules that label targets to increase their susceptibility to phagocytosis. Opsonins are usually captured by receptors on the surface of phagocytes. Our previous study found the C-type lectin FcLec4 from Chinese white shrimp Fenneropenaeus chinensis might function as an opsonin to facilitate bacterial clearance. In the present study we purified the native FcLec4 protein and confirmed its opsonic activity in the near relation, kuruma shrimp Marsupenaeus japonicus. The possible receptor of FcLec4 was identified as β-integrin by panning a T7 phage display library of shrimp hemocytes and then confirmed by co-immunoprecipitation assay. We further proved that the interaction between FcLec4 and β-integrin did not rely on the carbohydrate recognition domain but on the N terminus of FcLec4. In addition, inhibition of FcLec4 expression using RNAi delayed bacterial clearance, and β-integrin knockdown suppressed the opsonic activity of FcLec4. This study is the first to show the direct interaction between an opsonin and its receptor in crustaceans. Our study provides new insights into invertebrate phagocytosis and the functions of C-type lectins.

Identification and molecular characterization of a Spätzle-like protein from Chinese shrimp (Fenneropenaeus chinensis)

In invertebrates, the Toll signaling pathway is important for activation of antimicrobial peptides in the innate immune system. Activation of the Toll pathway requires binding of Toll with its ligand Spätzle. Here we described a Spätzle-like protein, designated as Fc-Spz, from hemocytes of Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequence of Fc-Spz shares 54% identity with Spätzle-like protein of salmon louse (Lepeophtheirus salmonis). Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time PCR, and Western blot analyses were carried out to analyze the expression pattern and distribution profile of Fc-Spz in shrimp after challenged with bacteria and virus. The results showed that Fc-Spz mRNA was up-regulated in all the tissues tested in shrimp injected with Vibrio anguillarum and white spot syndrome virus (WSSV). The C-terminal active Fc-Spz domain (114 residues) was expressed in Escherichia coli and purified by affinity chromatography. The recombinant Fc-Spz C-114 was injected into crayfish (Procambarus clarkii) to determine the expression levels of several antimicrobial peptide genes. The results showed that recombinant Fc-Spz C-114 could up-regulate crustin 2 expression in crayfish. These results suggest that Fc-Spz may play a role in the innate immune defence of Chinese shrimp and crayfish.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to white spot syndrome virus.

White spot syndrome virus (WSSV) infects all shrimp species and is the greatest detriment to shrimp culture. To better understand the mechanism of molecular responses to WSSV infection in Chinese white shrimp Fenneropenaeus chinensis, two dimensional electrophoresis (2-DE) was used. Differentially expressed proteins in the hepatopancreas of control and WSSV-injected Chinese white shrimp between (6, 12 and 24 h post-injection) were screened. Quantitative intensity analysis and mass spectrometry revealed that 54 spots of 47 proteins were significantly up-regulated, including some immune-related proteins, such as Toll receptor precursor, Leu-rich repeat protein, peroxinectin and serine proteinase-like protein. Fourteen spots of 13 proteins, such as heat shock protein, ATP synthase sub-unit beta and thrombospondin, were down-regulated in WSSV-infected shrimps. Protein expression patterns of the infected shrimp were drastically altered by WSSV infection. Some of the altered proteins were further investigated at the mRNA level using semi-quantitative reverse transcript PCR. These data may provide some information about shrimp proteins that participate in the WSSV infection process.

An anti-lipopolysaccharide factor from red swamp crayfish, Procambarus clarkii, exhibited antimicrobial activities in vitro and in vivo

The anti-lipopolysaccharide factors (ALFs) are a group of effector molecules of innate immunity in arthropods, exhibiting binding and neutralizing activities to lipopolysaccharides. In this study, an ALF cDNA sequence (PcALF1) was identified from red swamp crayfish, Procambarus clarkii. The deduced peptide of PcALF1 was conserved; it manifested the signal peptide and lipopolysaccharide (LPS)-binding domain, especially the two conserved cysteine residues at both ends of the domain. Transcripts of PcALF1 were detected in multiple tissues. Results of quantitative real-time PCR exhibited that the expression level of PcALF1 was induced by virus and Gram-positive and Gram-negative bacteria. Purified recombinant protein of PcALF1 revealed multiple biological activities: it gave all the tested bacteria and fungi a tight binding; it could bind microbial polysaccharides (LPS, LTA, and β-glucan) as well. In vitro, the antimicrobial activity assay was demonstrated as a broad spectrum against Gram-positive and Gram-negative bacteria and a fungus. The rPcALF1 also exhibited a clearance activity on Vibrio anguillarum in a dose-dependent manner in vivo.