C-type Lectin-like Receptor Dectin-1 And Clec-2 Study

Partâ… Study on the subcellular localization of hDectin-1E and its interaction with RanBPMDectin-1 is a typeâ…¡transmembrane receptor with high homology to typeâ…¡lectin receptors expressed by natural killer(NK) cells and was identified as a pattern recognition receptor forβ-1,3/β-1,6-linked glucans.Dectin-1 is predominantly expressed on the surface of dendritic cells,monocyte/macrophages and neutrophil lineages.It is also detected on the surface of non-professional antigen-processing cells, such as HEK293T cells.It possesses a single extracellular C-type lectin-like domain (CTLD) connected to transmembrane region by a stalk and an immunoreceptor tyrosine activation-like motif(ITAM) within the cytoplasmic tail.Pre-mRNA of human Dectin-1 is alternatively spliced,resulting in two major(A and B) and six minor(C-H) isoforms.The two major isoforms are the major receptors forβ-glucans. The function of the six minor isoforms,however,remains poorly understood.Among these six isoforms,isoform E(hDectin-1E) is structurally unique,containing a complete C-type lectin-like domain as well as an ITAM-like sequence.Compared with the isoform A(hDectin-1A),hDectin-1E lacks part of putative cytoplasmic domain,transmembane region and the stalk.However,little is known about its function.It is possible that the lack of transmemebrane region results in a different subcellular localization from its full-length form.In the present study,we demonstrated that hDectin-1E was not secreted and it mainly resided in the cytoplasm. Using yeast two-hybrid screening,we identified a Ran-binding protein,RanBPM,as an interacting partner of hDectin-1E.GST pull-down assays showed that RanBPM interacted directly with hDectin-1E and the region containing SPRY domain was sufficient for the interaction.The binding of hDectin-1E and RanBPM was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis.RanBMP functions as a scaffold with protein-interaction motifs and multiple canonical docking sites for signaling intermediates.RanBPM might provide a platform for the activation of hDectin-1E. Partâ…¡Study on Hsp60 as a potential ligand for Dectin-1 and the mechanism of Hsp60-induced maturation of macrophagesDectin-1,a receptor forβ-glucans,can internalizeβ-glucans and induce an intracellular signaling cascade that results in various cell-specific responses.The cell walls of fungi are predominantly composed ofβ-glucans and thus Dectin-1 has a key role in anti-fungal immunity.On ligand binding,the cytoplasmic ITAM motif of Dectin-1 becomes tyrosine phosphorylated by SRC kinases and recruits Syk to induce intracellular signaling cascade that results in the production of ROS and respiratory burst to kill microbes as well as induction of inflammatory cytokines such as TNF-αto regulate the immune system.In addition to recognizingβ-glucans,Dectin-1 also recognizes an endogenous ligand on T cells,acting as a co-stimulatory molecule in inducing the proliferation of T cells in vitro.This binding to T-lymphocytes can not be inhibited byβ-glucans,which shows that Dectin-1 possesses a distinct binding site for T-lymphocytes from theβ-glucan binding site.Additionally,Dectin-1 has a low binding capacity to HEK293 cells and an increased binding after induction of apoptosis,showing that the ligand has an increased expression level after induction of apoptosis.However,the ligands,Dectin-1 recognizes,remain to be identified.Using its complete C-type lectin-like domain as bait,we performed a yeast two-hybrid screening of a human thymus cDNA library and identified a serum soluble protein Hsp60.Hsp60 mainly presents in the mitochondria and serves as a chaperone. Hsp60 has been detected on the cell surface or in the circulation on stress and inflammation and has a regulatory role in the immune system.Hsp60 can be internalized and activates antigen-presenting cells,followed by the release of inflammatory cytokines.However,the receptors for Hsp60 remain to be identified.In this study,we reported that Dectin-1 could bind and internalize Hsp60.The BIAcore analysis revealed the equilibrium dissociation constant(K_D) of 1.04e-6M, Additionally,we demonstrated that Hsp60 could rapidly activate Spleen tyrosine kinase(Syk) from resident peritoneal macrophages.Further,piceatannol,an inhibitor for Syk,diminished the phosphorylation of ERK1/2 and p38,suggesting that Syk resides upstream of MAP kinases in this signal transduction pathway.Moreover the inhibition of Syk impaired the maturation and reduced cytokine release of peritoneal macrophages treated with Hsp60.Taken together,our data demonstrate that Hsp60 is an endogenous ligand of Dectin-1 and employs Syk to propagate its signals and thus regulate macrophage functions,which will present a novel strategy for Hsp60 as a therapeutic agent. Partâ…¢Molecular characterization of two novel isoforms and a soluble form of mouse CLEC-2CLEC-2 was first identified as one member of non-classical C-type lectins by sequence similarity to C-type lectin-like molecules with immune functions.Human CLEC-2 is a typeâ…¡transmembrane receptor displaying a single extracellular CTLD connected to transmembrane region by a stalk and one D-x-Y-x-x-L motif in its cytoplasm.Recently,CLEC-2 has been demonstrated as a novel activating receptor that is likely to underlie platelet activation by the snake toxin rhodocytin through Syk signaling pathway.Additionally,it has been reported to facilitate the capture of HIV-1 by platelets.More recently,several studies have identified podoplanin on tumor cells as an endogenous ligand for CLEC-2.Mouse CLEC-2(mCLEC-2) shares high homology with human counterpart with a conservation of key features including the lack of a Ca²⁺-dependent sugar-binding site and the presence of the D-x-Y-x-x-L cytoplasmic motif.To clone previously reported mCLEC-2,we performed RT-PCR reaction and identified two new alternatively spliced transcripts(GenBank accession nos. EF070191 and EF070192):one,named mCLEC-2B,possesses a 594bp open reading frame(ORF) via lacking exon 2 which encodes the transmembrane region,and the other,named mCLEC-2C,omits exons 2 and 4,which causes a frameshift and generates an ORF of only 252bp.Both isoforms were further confirmed by northern blot analysis.These two variants had different expression profiles from full-length mCLEC-2.Moreover,mCLEC-2Bå’ŒmCLEC-2C could not be secreted into culture media and mainly were localized in the cytoplasm.We also observed that full-length mCLEC-2 could be cleaved probably by proteases sensitive to aprotinin and PMSF into a soluble form.Additionally,we observed that mCLEC-2 partially existed as a disulfide-linked homodimer and Cys80 in the neck region play an important role in dimerization.The results presented here represent a further advancement toward the understanding of mCLEC-2.