CONICET | Buscador de Institutos y Recursos Humanos

Clusterin (CLU) is a glycoprotein found in different fluids and tissues. It interacts with misfolded proteins preventing their aggregation. We have shown that semen CLU (sCLU), but not serum CLU, bears a set of complex glycans with high affinity for DC-SIGN. Here, we show that sCLU interacts with misfolded proteins targeting them to DC-SIGN, promoting antigen endocytosis and presentation. Clusterin was purified from seminal plasma by affinity chromatography. The ability of sCLU to interact with misfolded proteins was analyzed by using BSA. sCLU (100 μg/ml) inhibited the aggregation of BSA (200 μg/ml) induced by heat treatment (60°C, 30 min) followed by measuring the turbidity associated with the formation of precipitated protein at 360nm: BSA=0.54±0.11 vs BSA+sCLU=0.13±0.04, absorbance, mean ± ES, n= 6, p<0.01. The ability of sCLU to target stressed-BSA to DC-SIGN was study using Raji cells. Biotin-labeled BSA (200 μg/ml) was stressed in the absence or presence of sCLU (100 μg/ml) and the binding of BSA to Raji cells was analyzed by flow cytomtery. sCLU increased the binding of stressed-BSA to DC-SIGN transfected Raji cells (430±24 vs 1360±36, mean fluorescence intensity ± ES, n= 5, p<0.01) but not to control Raji cells (not shown). Similar results were obtained using dendritic cells (DCs) instead of Raji cells (not shown). Analysis by confocal microscopy showed that BSA colocalized with sCLU in DC-SIGN and EEA1+ compartments inside DCs, suggesting that sCLU drives targeted proteins into a classical endocytic pathway. Additional experiments showed that DCs from PPD+ patients cultured with proteins extracted from M tuberculosis (PPD) in the presence of sCLU stimulated the production of IFN- by autologous lymphocytes evaluated by ELISPOT assays (PPD=6±2 vs PPD+sCLU=43±14, spots x106 células, n=3, p<0.01). Our observations demonstrate that sCLU promotes the presentation of stressed antigens.