Qilin Yi

An LRR-only protein representing a new type of pattern recognition receptor in Chlamys farreri

Accumulating evidence has demonstrated that leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and were involved in the immune response. In the present study, an LRR-only protein (designed as CfLRRop-1) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfLRRop-1 contained an open reading frame (ORF) of 1377 bp, which encoded a protein of 458 amino acids. An LRRNT motif, an LRR_7 motif and seven LRR motifs were found in the deduced amino acid sequence of CfLRRop-1. And these seven LRR motifs contained a conserved signature sequence LxxLxLxxNxL. The mRNA transcripts of CfLRRop-1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hepatopancreas. After the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), the mRNA transcripts of CfLRRop-1 in haemocytes all increased firstly within the first 6 h and secondly during 12-24 h post stimulation. The mRNA expression level of CfLRRop-1 was continuously up-regulated, after the expression of CfTLR (previously identified Toll-like receptor in C. farreri) was suppressed via RNA interference (RNAi). The recombinant CfLRRop-1 protein could directly bind LPS, PGN, GLU and poly I:C, and induce the release of TNF-α in mixed primary cultured scallop haemocytes. These results collectively indicated that CfLRRop-1 would function as a powerful pattern recognition receptor (PRR) and play a pivotal role in the immune response of scallops.

A galectin from Eriocheir sinensis functions as pattern recognition receptor enhancing microbe agglutination and haemocytes encapsulation.

Galectins are a family of β-galactoside binding lectins that function as pattern recognition receptors (PRRs) in innate immune system of both vertebrates and invertebrates. The cDNA of Chinese mitten crab Eriocheir sinensis galectin (designated as EsGal) was cloned via rapid amplification of cDNA ends (RACE) technique based on expressed sequence tags (ESTs) analysis. The full-length cDNA of EsGal was 999xa0bp. Its open reading frame encoded a polypeptide of 218 amino acids containing a GLECT/Gal-bind_lectin domain and a proline/glycine rich low complexity region. The deduced amino acid sequence and domain organization of EsGal were highly similar to those of crustacean galectins. The mRNA transcripts of EsGal were found to be constitutively expressed in a wide range of tissues and mainly in hepatopancreas, gill and haemocytes. The mRNA expression level of EsGal increased rapidly and significantly after crabs were stimulated by different microbes. The recombinant EsGal (rEsGal) could bind various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (GLU), and exhibited strong activity to agglutinate Escherichia coli, Vibrio anguillarum, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and Pichia pastoris, and such agglutinating activity could be inhibited by both d-galactose and α-lactose. The inxa0vitro encapsulation assay revealed that rEsGal could enhance the encapsulation of haemocytes towards agarose beads. These results collectively suggested that EsGal played crucial roles in the immune recognition and elimination of pathogens and contributed to the innate immune response against various microbes in crabs.

The neuroendocrine immunomodulatory axis-like pathway mediated by circulating haemocytes in pacific oyster Crassostrea gigas

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of host. In this study, a neuroendocrine immunomodulatory axis (NIA)-like pathway mediated by the nervous system and haemocytes was characterized in the oyster Crassostrea gigas. Once invaded pathogen was recognized by the host, the nervous system would temporally release neurotransmitters to modulate the immune response. Instead of acting passively, oyster haemocytes were able to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities, thus regulating intracellular concentration of Ca2+. This neural immunomodulation mediated by the nervous system and haemocytes could influence cellular immunity in oyster by affecting mRNA expression level of TNF genes, and humoral immunity by affecting the activities of key immune-related enzymes. In summary, though simple in structure, the ‘nervous-haemocyte’ NIA-like pathway regulates both cellular and humoral immunity in oyster, meaning a world to the effective immune regulation of the NEI network.

Molecular cloning and characterization of a cytoplasmic manganese superoxide dismutase and a mitochondrial manganese superoxide dismutase from Chinese mitten crab Eriocheir sinensis.

Superoxide dismutase (SOD) functions as the first and essential enzyme in the antioxidant system and is ubiquitously existed in both prokaryotes and eukaryotes. In the present study, both cytoplasmic and mitochondrial manganese SOD were identified from Chinese mitten crab Eriocheir sinensis (designed as EscytMnSOD and EsmtMnSOD). The complete nucleotide sequence of EscytMnSOD comprised 1349 bp and consisted of a 5 untranslated regions (UTR) of 43 bp, a 3 UTR of 445 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acid residues. The full-length cDNA sequence of EsmtMnSOD comprised 990 bp, containing a 5 UTR of 55 bp, a 3 UTR of 278 bp and an ORF of 657 bp encoding a polypeptide of 218 amino acid residues. The deduced amino acid sequences of EscytMnSOD and EsmtMnSOD contained highly conserved MnSOD signature and typical functional domain, and exhibited high similarity with their reported homologues. In the phylogenetic tree, EscytMnSOD and EsmtMnSOD were clustered with their homologues from the land crab Cardisoma armatum. The EscytMnSOD and EsmtMnSOD transcripts were constitutively expressed in haemocytes, muscle, heart, gill, haepatopancreas and gonad, with the highest expression level in gills and haepatopancreas, respectively. The mRNA expression levels of them were all up-regulated in haemocytes with similar profiles after the stimulation of Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The EsmtMnSOD with low basal expression level responded to invading microbes intensely, while the EscytMnSOD with high basal expression level exhibited mild responses against stimulating microbes. The purified rEscytMnSOD and rEsmtMnSOD proteins exhibited specific Mn(2+)-dependent enzymatic activities, while rEscytMnSOD with lower basic activity displayed higher stability than rEsmtMnSOD. All these results indicated that EscytMnSOD and EsmtMnSOD were efficiently antioxidant enzymes and potentially involved in the innate immune responses of E. sinensis with different roles, the former might play a routine role in the innate immune system in crabs, while the later might be involved in the immune response against invading microbes specifically.

The versatile functions of LRR-only proteins in mollusk Chlamys farreri

Leucine-rich repeat (LRR)-only proteins are involved in the innate immune responses as they mediate protein-ligand interactions. In the present study, three novel LRR-only proteins, CfLRRop-4, CfLRRop-5 and CfLRRop-6, were identified and characterized from Zhikong scallop Chlamys farreri. They all contained LRR motifs with consensus signature sequences of LxxLxLxxNxL or LxxLxLxxCxxL. All the mRNA transcripts of three CfLRRops were high abundant in hepatopancreas, gills and gonads, and their mRNA transcripts in hemocytes could respond to the stimulations of different microbes, including Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. These three CfLRRops exhibited similar ligand binding and recognition characteristics as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The immune effectors, including tumor necrosis factor α, superoxide dismutase, catalase and lysozyme, varied significantly after the scallops were stimulated by recombinant LRR-only proteins. All these results indicated that LRR-only proteins are functionally differentiated and exhibit different immunomodulation activities on various downstream immune effectors.

Two short peptidoglycan recognition proteins from Crassostrea gigas with similar structure exhibited different PAMP binding activity.

ABSTRACT Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in specifically recognizing bacterial peptidoglycan (PGN) and eliminating the invading bacteria. In the present study, the full length cDNA of two PGRP genes, CgPGRPS2 and CgPGRPS4, were cloned from oyster Crassostrea gigas. Their amino acid sequences both contained one signal peptide, one typical PGRP/amidase domain with conserved catalytic residues responsible for amidase activity (55H, 90Y, 164H, 172C in CgPGRPS2, and 98H, 133Y, 207H, 215C in CgPGRPS4), and specific PGN recognition (84R, 85W, 104R, 109V in CgPGRPS2, and 127G, 128W, 147R, 152V in CgPGRPS4), and they shared 55.9% sequence similarity. The mRNA transcripts of CgPGRPS2 and CgPGRPS4 were constitutively expressed in all the examined tissues, including haemocytes, hepatopancreas, mantle, gonad, heart, adductor muscle and gill, with the highest expression level in adductor muscle and hepatopancreas, respectively. Both CgPGRPS2 and CgPGRPS4 proteins were mainly localized in the cytoplasma. The recombinant protein of CgPGRPS2 (rCgPGRPS2) could bind lipopolysaccharide (LPS), PGN and mannan (Man), as well as various microorganisms including Gram‐negative bacteria Escherichia coli, Vibrio anguillarum, Gram‐positive bacteria Staphylococcus aureus and fungi Yarrowia lipolytica. The recombinant protein of CgPGRPS4 (rCgPGRPS4) exhibited higher binding affinity to PGN, lower binding affinity to LPS, while no binding activity to Man and Y. lipolytica. The results indicated that CgPGRPS2 and CgPGRPS4 could function as pattern recognition receptors (PRR) in the innate immune response of oyster, and they exhibited a certain degree of functional differentiation in recognition of Man. HIGHLIGHTSCgPGRPS2 and CgPGRPS4 were identified as short type PGRP genes from Pacific oyster.CgPGRPS2 and CgPGRPS4 showed highest mRNA expression in adductor muscle and hepatopancreas, respectively.CgPGRPS2 and CgPGRPS4 proteins were mainly localized in cytoplasma of oyster haemocytes.rCgPGRPS2 could bind LPS, PGN, Man, E. coli, V. anguillarum, S. aureus and Y. lipolytica.rCgPGRPS4 could bind PGN, LPS, but not Man and Y. lipolytica.

Functional characterization of hemocytes from Chinese mitten crab Eriocheir sinensis by flow cytometry

Hemocytes comprise a diversity of cell types with functional and structural heterogeneity, and they play key roles in the host defense of invertebrates. In the present study, the hemocytes from Chinese mitten crab Eriocheir sinensis were directly separated into two groups by flow cytometry. The hemocytes in P1 group were full of round and abundant granules with deeply staining cytoplasm, while P2 hemocytes were more diverse with a wide range of sizes and less granularity. Both P1 and P2 hemocytes exhibited phagocytic ability, but the phagocytic rate of P1 hemocytes increased which was significantly higher than that of P2 hemocytes after LPS stimulations. The levels of ROS production and intracellular Calcium as well as lysosome content were higher in P1 hemocytes than that in P2 hemocytes under both normal and immune-activated situations. The genes involved in phagocytosis, antimicrobial and antioxidant activities were mainly expressed in P1 hemocytes, while the genes involved in proPO activation system were highly expressed in P2 hemocytes. These results collectively suggested that P1 hemocytes were the main immunocompetent hemocytes in Chinese mitten crab and P2 hemocytes mainly participated in proPO activation system.

The first CUB-domain containing serine protease from Chlamys farreri which might be involved in larval development and immune response

Abstract Serine proteases (SPs) are one of the most well understood enzyme families, which play an important role in regulating many physiological events. In the present study, one CUB‐domain containing serine protease was identified from Chlamys farreri (designated as CfCUBSP). The full‐length cDNA of CfCUBSP was of 3181 bp with an open reading frame of 2688 bp encoding a polypeptide of 896 amino acids. CfCUBSP shared closer phylogenetic relationship with those multi‐domain SPs which consisted of one SP domain, and different numbers of CUB domain and LDLa domain than other SPs. The mRNA transcripts of CfCUBSP were detected in all developmental stages with the highest expression level in fertilized eggs and the lowest in trochophore larvae. In adult scallop, the CfCUBSP mRNA could be detected in all examined tissues with the highest level in hepatopancreas, and CfCUBSP protein was dominantly located in the gills, hepatopancreas, gonad and kidney. The mRNA expression of CfCUBSP in hemocytes was significantly up‐regulated after the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and &bgr;‐glucan (GLU) (P < 0.05). All the results collectively indicated that CfCUBSP was a primitive member of the invertebrate SPs which might be involved in larval development and immune response against Gram‐negative (G−) and Gram‐positive (G+) bacteria and fungus in scallop. HighlightsA CUBSP protein was identified from Zhikong scallop.CfCUBSP mRNA was ubiquitous with the highest level in fertilized eggs and hepatopancreas of adults.CfCUBSP protein was dominantly located in the gills, hepatopancreas, gonad and kidney.The mRNA expression of CfCUBSP was significantly induced by LPS, PGN and GLU.

Glycogen synthase kinase-3 (GSK3) regulates TNF production and haemocyte phagocytosis in the immune response of Chinese mitten crab Eriocheir sinensis

&NA; Glycogen synthase kinase‐3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3‐&bgr; from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide‐induced tumor necrosis factor (TNF)‐&agr; factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF‐&agr; production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF‐&agr; production and inhibiting haemocyte phagocytosis. HighlightsA homologue of glycogen synthase kinase‐3 (EsGSK3) gene was identified from Chinese mitten carb, E. sinensis.The EsGSK3 transcripts in haemocytes could be induced significantly by immune challenges.The mRNA expression of EsLITAF and TNF‐&agr; production were suppressed after EsGSK3 was blocked in vivo.The activity of EsGSK3 could regulate the phagocytosis of crab haemocytes.

The various components implied the diversified Toll-like receptor (TLR) signaling pathway in mollusk Chlamys farreri

ABSTRACT Toll‐like receptor (TLR) signaling pathway, composed of various components, plays pivotal roles in host innate immune defense mechanism. In the present study, twenty‐nine TLR signaling pathway components, including receptors, adaptors, transduction molecules and immune effectors, were identified in Zhikong scallop Chlamys farreri via assembling and screening public available transcriptomic data and expression sequence tags (ESTs). These identified TLR signaling pathway components were constitutively expressed and detectable in various tissues, and almost all of them were highly expressed in gill and hepatopancreas. These results indicated the presence of TLR signaling pathways in both MyD88‐dependent and MyD88‐independent forms in scallop, and implied the diversified TLR signaling pathway in mollusk C. farreri. HIGHLIGHTSTwenty‐nine TLR signaling pathway components were identified in Chlamys farreri.Forty‐nine LRR‐only proteins were identified in C. farreri.An outline was sketched based on these identified elements.