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1.
Fish Shellfish Immunol ; 43(1): 264-74, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25559445

RESUMO

Intracellular fatty acid-binding proteins (FABPs) are members of the lipid-binding protein superfamily. Aside from the main functions of FABPs in the uptake and transport of fatty acids, they are also critical in innate immunology. In this work, the full-length cDNA for a Chinese mitten crab Eriocheir sinensis FABP (Es-FABP3) was cloned with an open reading frame of 402 bp encoding a 133 amino acid polypeptide. Analysis using quantitative real-time PCR (qPCR) revealed that Es-FABP3 transcripts were widely distributed in gills, muscle, intestine, hepatopancreas, eyestalk, heart, stomach, brain, thoracic ganglia and hemocytes. After challenge with pathogen associated molecular pattern molecules (PAMPs), the relative mRNA expression levels of Es-FABP3 increased in hepatopancreas, gills and hemocytes. Moreover, the mature recombinant Es-FABP3 protein exhibited different binding activities to bacteria and fungus and inhibited the growth of different microbes. These collective results demonstrated the role of Es-FABP3 in the immunoreactions of E. sinensis to PAMPs.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Artrópodes/genética , Braquiúros/genética , Braquiúros/imunologia , Proteínas de Ligação a Ácido Graxo/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Braquiúros/metabolismo , Braquiúros/microbiologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Pichia/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência
2.
PLoS One ; 8(10): e76132, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146827

RESUMO

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate.


Assuntos
Antibacterianos/farmacologia , Proteínas de Artrópodes/farmacologia , Braquiúros/imunologia , Hepatopâncreas/imunologia , Lectinas Tipo C/imunologia , Sequência de Aminoácidos , Animais , Antibacterianos/imunologia , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Braquiúros/efeitos dos fármacos , Braquiúros/genética , Braquiúros/microbiologia , Cálcio/metabolismo , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Etiquetas de Sequências Expressas , Biblioteca Gênica , Hepatopâncreas/microbiologia , Lectinas Tipo C/química , Lectinas Tipo C/genética , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
3.
Fish Shellfish Immunol ; 35(5): 1554-65, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24012749

RESUMO

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play significant roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (EsLecD) was identified from the crustacean Eriocheir sinensis. The cloning of full-length EsLecD cDNA was based on the initial expressed sequence tags (ESTs) isolated from a hepatopancreatic cDNA library. The full-length EsLecD cDNA of 686 bp with an open reading frame of 468 bp encodes a putative protein of 155 aa residues, including an N-terminal signal peptide and a single carbohydrate-recognition domain (CRD). By quantitative RT-PCR analysis, the EsLecD transcript was mainly detected in the hepatopancreas but rarely in other tissues, and it was significantly upregulated in the hepatopancreas after immune challenge with lipopolysaccharides. The recombinant EsLecD protein (rEsLecD) exhibited the ability to bind to all tested microorganisms, including bacteria and yeast. Meanwhile, calcium significantly increased the binding affinity of rEsLecD toward microorganisms, but it was not essential. The binding of rEsLecD induced the aggregation of microbial pathogens. Moreover, rEsLecD was capable of inhibiting the growth of microorganisms and even directly killing bacteria. Interestingly, rEsLecD could stimulate cellular encapsulation in vitro. In conclusion, results of this study suggest that EsLecD acts as an antibacterial PRR participating in the innate immunity of invertebrates.


Assuntos
Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/imunologia , Lectinas Tipo C/imunologia , Análise de Variância , Animais , Sequência de Bases , Western Blotting , Cálcio/metabolismo , China , Análise por Conglomerados , Biologia Computacional , Primers do DNA/genética , Biblioteca Gênica , Hepatopâncreas/metabolismo , Lectinas Tipo C/metabolismo , Lipopolissacarídeos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA
4.
PLoS One ; 8(8): e73563, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23967346

RESUMO

Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab (Eriocheirsinensis). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1 (rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichiapastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.


Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Inibidores de Proteases/farmacologia , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Bases , Braquiúros/química , Braquiúros/genética , Clonagem Molecular , Expressão Gênica , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia
5.
Dev Comp Immunol ; 41(4): 544-52, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23911906

RESUMO

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate.


Assuntos
Antibacterianos/imunologia , Crustáceos/genética , Crustáceos/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Receptores de Reconhecimento de Padrão/genética , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Crustáceos/metabolismo , Imunidade Inata/imunologia , Lectinas Tipo C/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Alinhamento de Sequência
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