Frontal affinity chromatography analysis of constructs of DC‐SIGN, DC‐SIGNR and LSECtin extend evidence for affinity to agalactosylated N‐glycans

Yabe, Rikio; Tateno, Hiroaki; Hirabayashi, Jun

Dendritic cell‐specific intracellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) is a member of the C‐type lectin family selectively expressed on immune‐related cells. In the present study, we performed a systematic interaction analysis of DC‐SIGN and its related receptors, DC‐SIGN‐related protein (DC‐SIGNR) and liver and lymph node sinusoidal endothelial cell C‐type lectin (LSECtin) using frontal affinity chromatography (FAC). Carbohydrate‐recognition domains of the lectins, expressed as Fc–fusion chimeras, were immobilized to Protein A–Sepharose and subjected to quantitative FAC analysis using 157 pyridylaminated glycans. Both DC‐SIGN–Fc and DC‐SIGNR–Fc showed similar specificities for glycans containing terminal mannose and fucose, but great difference in affinity under the given experimental conditions. By contrast, LSECtin–Fc showed no affinity to these glycans. As a common feature, the DC‐SIGN‐related lectin–Fc chimeras, including LSECtin, exhibited binding affinity to mono‐ and/or bi‐antennary agalactosylated N‐glycans. The detailed FAC analysis further implied that the presence of terminal GlcNAc at the N‐acetylglucosaminyltransferase I position is a key determinant for the binding of these lectins to agalactosylated N‐glycans. By contrast, none of the lectins showed significant affinity to highly branched agalactosylated N‐glycans. All of the lectins expressed on the cells were able to mediate cellular adhesion to agalactosylated cells and endocytosis of a model glycoprotein, agalactosylated α1‐acid glycoprotein. In this context, we also identified three agalactosylated serum glycoproteins recognized by DC‐SIGN‐Fc (i.e. α‐2‐macroglobulin, serotransferrin and IgG heavy chain), by lectin blotting and MS analysis. Hence, we propose that ‘agalactosylated N‐glycans’ are candidate ligands common to these lectins.