Single Site <i>N</i>‑Glycosylation of B Cell Maturation Antigen (BCMA) Inhibits γ‑Secretase-Mediated Shedding and Improves Surface Retention and Cell Survival

B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor (TNFR) family, on the cell surface plays a key role in maintaining the survival of plasma cells and malignant as well as inflammatory accessory cells. Therefore, targeting BCMA or disrupting its interaction with ligands has been a potential approach to cancer therapy. BCMA contains a single N-glycosylation site, but the function of N-glycan on BCMA is not understood. Here, we found that the N-glycosylation of BCMA promoted its cell-surface retention while removing the N-glycan increased BCMA secretion through γ-secretase-mediated shedding. Addition of γ-secretase inhibitor prevented nonglycosylated BCMA from shedding and protected cells from dexamethasone and TRAIL-induced apoptosis

Investigation of SSEA‑4 Binding Protein in Breast Cancer Cells

SSEA-4, a sialyl-glycolipid, has been commonly used as a pluripotent human embryonic stem cell marker, and its expression is correlated with the metastasis of some malignant tumors. However, there is no in-depth functional study related to the receptor and the role of this glycolipid. Here, we report the identification of an SSEA-4-binding protein in a breast cancer cell line, MCF-7. By using affinity capture and glycan microarray techniques, the intracellular FK-506 binding protein 4 (FKBP4) was identified to bind directly to SSEA-4. The biological significance of SSEA-4/FKBP4 interaction was investigated

Dengue virus infection is through a cooperative interaction between a mannose receptor and CLEC5A on macrophage as a multivalent hetero-complex.

<p><b>(a)</b> Binding model of dengue virus with MR/DC-SIGN and CLEC5A on GM-MDM cell membrane. <b>(b)</b> The strong avidity receptor captures dengue virus to facilitate the interaction with a weak-binding signal receptor in proximity to induce innate immune response.</p

Global kinetic analysis of dengue virus binding to different Fc-lectins that immobilized on Octet AHC biosensor.

<p>Global kinetic analysis of dengue virus binding to different Fc-lectins that immobilized on Octet AHC biosensor.</p

Immunogold assisted TEM revealed close proximity of CLEC5A/MR/DC-SIGN.

<p>GM-MDM cells were cultured on grids for eight days, with or without IL-4 treatment. After standard TEM procedure, the image was further analyzed as described. <b>(a)</b> Immunogold TEM image of GM-MDM cell membrane with MR (15 nm gold particles), and CLEC5A (5nm gold particles) stained. <b>(b)</b> Immunogold TEM image of GM-MDM cell stimulated by IL-4; the membrane was stained with anti-MR (15 nm gold particles), anti-DC-SIGN (10 nm gold particles) and anti-CLEC5A (5 nm gold particles) antibodies. <b>(c)</b> Statistical result of the number of CLEC5A-immunogold dots counted within various radius ranges using MR/DC-SIGN as the centre of a circle.</p

Dengue virus-induced DAP12 Tyr-phosphorylation is mannose binding receptor and DC-SIGN dependent.

<p><b>(a)</b> Human GM-MDM cells challenged with 0, 0.1, 1 or 10 MOI dengue virus were treated with anti-mannose receptor (hMR) antibody as a blocker, then immunoprecipitated with phosphor-tyrosine (pY) antibody for quantification of the endogenous DAP12 with Western blot. <b>(b)</b> GM-MDM cells were stimulated by IL-4 for 48 hr and then challenged with 1 M.O.I. dengue virus in the presence of the indicated blocker, then the endogenous DAP12 was analyzed with Western blot.</p

The Pearson Coefficient Correlation (PCC) analysis of co-localization revealed that CLEC5A/MR and CLEC5A/DC-SIGN colocalization is significantly increased in the dengue virus presence area of virus-infected human GM-MDM cells.

<p>GM-MDM cells were cultured on coverslips for eight days, with or without IL-4 treatment as described, and then infected with 0.1 M.O.I. C6/36-derived dengue virus. After the cell was fixed and stained, and analyzed by confocal fluorescence microscopy, the confocal fluorescence micrographs were further analyzed for localization using the PCC analysis by Volocity (PerkinElmer). Then the data collections go further followed by Student's <i>t</i>-test or ANOVA test to see if the means of two or several groups are equal. Every data point in the figures accounts for a single cell. More than 45 cells are counted in each donor. *p < 0.05, **p < 0.01, ***p < 0.001, ns = no significant. <b>(a)</b> PCC analysis of colocalization of CLEC5A with MR in GM-MDM cells in various donors. Comparison of results with or without dengue virus challenge (Left). Comparison of dengue virus gated positive and negative results in dengue virus tested GM-MDM cells (Right). <b>(b)</b> PCC analysis of colocalization of DC-SIGN with MR, and CLEC5A with MR, and CLEC5A with DC-SIGN in GM-MDM cells stimulated with IL-4. Various donors are compared with or without dengue virus challenge. <b>(c)</b> CC analysis of colocalization of DC-SIGN with MR, and CLEC5A with MR, and CLEC5A with DC-SIGN in GM-MDM + IL-4 stimulated cells challenged with dengue virus. Various donors are compared with dengue virus gated positive and negative cells.</p

Induction of CXCL8 and IL-1β is decreased in CLEC9A-silenced THP-1 cells treated with H37Ra.

<p>Control and CLEC9A-silenced THP-1 cells were stimulated with heat-killed H37Ra for the indicated times. (A) Total RNA was extracted using TRIzol. After reverse transcription, the expression levels of the indicated mRNAs were measured by Q-PCR and normalized against GAPDH mRNA. The relative mRNA level in knock-down control THP-1 cells (shluc) at 0 hour was set as 1.0. (B) The medium was collected and analyzed for TNF, CXCL8 and IL-1β level by ELISA. The results are mean ± SD of three separate experiments. Two-tailed multiple t-tests were performed (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01).</p

Activation of SYK and MAPK is decreased in CLEC9A-knocked down cells that had been H37Ra stimulated.

<p>CLEC9A in THP-1 cells was knocked down using lentiviral-based shRNA. After treating with heat-killed H37Ra for the indicated times, the cells were lysed and subjected to immunoblot analysis. (A) Syk activation was analyzed by measuring protein phosphorylation using the indicated specific antibodies. (B) MAPK signal activation was analyzed by measuring protein phosphorylation using the indicated specific antibodies. Representative data from three independent experiments are showed. The western blots were quantified with densitometry, the relative amount normalized to β-actin was calculated. The densitometry data was presented as mean values from three experiments.</p

Leukocyte infiltration <i>in vivo</i> is decreased by CLEC9A inhibition with H37Ra.

<p>C57BL/6 mice were infected intratracheally with heat-killed H37Ra with or without pre-incubation with human CLEC9A-Fc fusion protein. The mice were sacrificed 24 hours post-H37Ra challenge. <b>(A)</b> Formalin-fixed, paraffin embedded lung sections were stained for acid-fast bacteria by the Ziehl-Neelsen method. 1000× magnification, and <b>(B)</b> hematoxylin and eosin (H&E)-stained lung histology was examined. 100× magnification. Representative data of three independent experiments are shown. <b>(C)</b> The quantification of lung infiltration using H&E staining. <b>(D)</b> The amount of apoptosis present was determined by TUNEL stain using sections of lung. 100× magnification. Representative data from three independent experiments are shown. <b>(E)</b> The quantification of cell death by TUNEL staining. <b>(F)</b> The amounts of TNF and CXCL8 in bronchoalveolar lavage (BAL) fluid were analyzed by ELISA. <b>(G)</b> The total cell number was counted in the cytocentrifuged BAL fluid samples. The data points represent values from n  =  3–6 mice per group. Two-tailed multiple t-tests were performed (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01).</p