RESUMO
Bacterial DNA contains CpG oligonucleotide (ODN) motifs to trigger innate immune responses through the endosomal receptor Toll-like receptor 9 (TLR9). One of the cell surface receptors to capture and deliver microbial DNA to intracellular TLR9 is the C-type lectin molecule DEC-205 through its N-terminal C-type lectin-like domain (CTLD). CD93 is a cell surface protein and member of the lectin group XIV with a CTLD. We hypothesized that CD93 could interact with CpG motifs, and possibly serve as a novel receptor to deliver bacterial DNA to endosomal TLR9. Using ELISA and tryptophan fluorescence binding studies we observed that the soluble histidine-tagged CD93-CTLD was specifically binding to CpG ODN and bacterial DNA. Moreover, we found that CpG ODN could bind to CD93-expressing IMR32 neuroblastoma cells and induced more robust interleukin-6 secretion when compared with mock-transfected IMR32 control cells. Our data argue for a possible contribution of CD93 to control cell responsiveness to bacterial DNA in a manner reminiscent of DEC-205. We postulate that CD93 may act as a receptor at plasma membrane for DNA or CpG ODN and to grant delivery to endosomal TLR9.
Assuntos
DNA Bacteriano/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Oligodesoxirribonucleotídeos/imunologia , Receptores de Complemento/imunologia , Receptor Toll-Like 9/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Transporte Biológico/genética , Transporte Biológico/imunologia , Linhagem Celular Tumoral , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Modelos Biológicos , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/genéticaRESUMO
CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca2+ was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A).