Functional Characterization Of A New Human Dectin-1Isoform

Candida albicans is a major opportunistic fungal pathogen that can cause a widerange of diseases ranging from superficial infections to disseminated disease, whichmanifests primarily in immuno-compromised individuals. Despite the currentlyapplied anti-fungal therapies, both mortality and morbidity caused by this humanpathogenic fungus are still unacceptably high. In order to define new targets forcombating fungal disease, there is a need to understand the immune evasionstrategies of C. albicans in detail. C. albicans is a dimorphic pathogen that colonizesthe mucosal surfaces. During C. albicans infection,DC and macrophage canrecognize the PAMP (Pathogen Associated Molecular Pattern) from fungal cell wallthrough their cell surface PRR(pattern recognition receptors),which triggers a seriesof signaling cascades leading to activation of various transcription factors. As aconsequence, chemokines and pro-inflammatory cytokines are released,which inturn attract leukocytes such as neutrophils to the site of pathogen invasion.PRRs on the surface of professional phagocytes recognize both mannans andmannoproteins that cover the fungus, as well as β-glucans and chitin at the site of thebudding scar. These PAMPs are recognized by receptors grouped into severalfamilies: the Toll-like receptors (TLRs), the C-type lectin receptors(CLRs), and thenucleotide-binding domain leucine-rich repeat-containing receptors (NLRs). TheCLRs are believed that it is important for the immune responses to fungal. CLR arepart of a heterogeneous superfamily of soluble and transmembrane proteins definedby a characteristic C-type lectin domain,they bind to most, if not all,fungal speciesthat cause disease in humans. These receptors recognize the major carbohydratestructures that are found in fungal cell walls, including β-glucan and mannan.Dectin-1(also known as CLEC7A) is the best characted CLR.It is expressed bymyeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular antimicrobialactivity,including phagocytosis and production of reactive oxygen species(ROS). Dectin-1is a type II transmembrane receptor with a single extracellularcarbohydrate recognition domain and an immunoreceptor tyrosine activation motif inits cytoplasmic tail.Dectin-1triggers intracellular signalling via the cytoplasmicITAM-like motif. Downstream signalling pathways induce a number of innate immune responses including recruitment of Syk, activation of NF-κB via CARD9, aswell as the activation of MAPKs and NFAT．The human Dectin-1mRNA is alternatively spliced, resulting in two major (Aand B) and six minor isoforms. Of these isoforms, there are three isoforms,whichcontaining a complete C-type lectin-like domain as well as an ITAM-like sequence.Here in the first part,We found that RAW264.7cells, which infected withlentiviral expression vectors encoding Dectin-1isoform E,can phosphorylate IκB aand induced nuclear translocation of NF-κB (p65) when these cells were stimulatedwith C.albican. In the second part,we maked the anti-Dectin-1antibody. Theconstruct pET28a-Dectin-1(203-744) was transformed into the competent E.coliBL21to induce the expression of fusion protein His-Dectin-1(203-744) under theIPTG stimulation. The fusion proteins, which are purified with His affinitychromatography, subjected to immunized Mouse to make the monoclonal antibodyagansit Dectin-1.We got4antibodies,which can dectect Dectin-1E.