RESUMO
Helicobacter pylori employs unique methods to colonize the stomach, which induces chronic inflammation. It is also able to avoid eradication by macrophages and other immune cells. Leukocyte cell-derived chemotaxin 2 (LECT2), a multi-functional cytokine involved in many pathological conditions, has recently been shown to activate macrophages via the CD209a receptor. Therefore, we aimed to investigate the effects of LECT2 on H. pylori-infected macrophages. Macrophages were treated with recombinant LECT2, and both their ability to kill H. pylori and produce nitric oxide were analyzed. Western blot was performed to determine nuclear translocation and protein phosphorylation of p65, a subunit of nuclear factor (NF)-κB. Transfection experiments were performed to analyze the signaling pathway of LECT2 in macrophages. We found that treatment with LECT2 enhanced H. pylori killing and nitric oxide production in macrophages. In addition, DNA-binding activity and nuclear translocation of p65 were up-regulated by LECT2 treatment. Furthermore, we found that NF-κB activation by LECT2 was mediated by Raf-1 in macrophages, and Raf-1 phosphorylation was specifically altered in response to LECT2. Moreover, LECT2 induced Ser28 phosphorylation in the intracellular domain of CD209a. CD209a Ser28 phosphorylation was required for LECT2-induced Raf-1 and NF-κB activation in RAW264.7 macrophages. Our study showed that the effects of LECT2 on H. pylori killing and nitric oxide production were dependent on CD209a phosphorylation, Raf-1, and NF-κB activation. Together, these results demonstrate for the first time that exposure to LECT2 can modulate specific intracellular mechanisms downstream of CD209a to enhance H. pylori killing and nitric oxide production in macrophages.
Assuntos
Infecções por Helicobacter/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Recombinantes/genética , Fator de Transcrição RelA/biossíntese , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Helicobacter pylori/patogenicidade , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico/genética , Fosforilação , Células RAW 264.7 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/genética , Estômago/imunologia , Estômago/microbiologia , Fator de Transcrição RelA/genética , Ativação Transcricional/genética , TransfecçãoRESUMO
Peritrophic membrane proteins are important components of the insect peritrophic membrane. A novel cDNA gene encoding a chitin-binding protein, named secbp66, was identified by immunization screening of the cDNA library of Spodoptera exigua. The full length of secbp66 is 1806 bp, which encodes 602 amino acids. The predicted weight of the protein is 64.2 kDa. Bioinformatic analysis showed that a signal peptide composed of 17 amino acids located at the N-terminal of SeCBP66 contained seven tandem putative Type-II functional chitin-binding domains and five potential N-glycosylation sites, but no O-linked glycosylation sites. To study the properties of SeCBP66, recombinant SeCBP66 was successfully expressed in the insect cell line BTI-Tn-5B1-4 with a Bac-to-Bac expression system. A chitin binding experiment showed that the recombinant SeCBP66 protein could bind to chitin strongly. This study of the novel chitin-binding protein SeCBP66 provides a basis for developing new control targets for S. exigua.
Assuntos
Quitina/química , Proteínas de Insetos/química , Spodoptera , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Glicosilação , Filogenia , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
This study aims to explore the roles of somatic embryogenesis receptor-like kinase (SERK) in Malus hupehensis (Pingyi Tiancha). The full-length sequences of SERK1 in triploid Pingyi Tiancha (3n) and a tetraploid hybrid strain 33# (4n) were cloned, sequenced, and designated as MhSERK1 and MhdSERK1, respectively. Multiple alignments of amino acid sequences were conducted to identify similarity between MhSERK1 and MhdSERK1 and SERK sequences in other species, and a neighbor-joining phylogenetic tree was constructed to elucidate their phylogenetic relations. Expression levels of MhSERK1 and MhdSERK1 in different tissues and developmental stages were investigated using quantitative real-time PCR. The coding sequence lengths of MhSERK1 and MhdSERK1 were 1899 bp (encoding 632 amino acids) and 1881 bp (encoding 626 amino acids), respectively. Sequence analysis demonstrated that MhSERK1 and MhdSERK1 display high similarity to SERKs in other species, with a conserved intron/exon structure that is unique to members of the SERK family. Additionally, the phylogenetic tree showed that MhSERK1 and MhdSERK1 clustered with orange CitSERK (93%). Furthermore, MhSERK1 and MhdSERK1 were mainly expressed in the reproductive organs, in particular the ovary. Their expression levels were highest in young flowers and they differed among different tissues and organs. Our results suggest that MhSERK1 and MhdSERK1 are related to plant reproduction, and that MhSERK1 is related to apomixis in triploid Pingyi Tiancha.