TACI-Fc binding requires the heparan sulfate side chains of syndecan-2. (A) Parental Jurkat cells and stable Synd2/Jurkat cells were assayed for their ability to bind TACI-Fc in the presence or absence of heparin (as indicated). Addition of heparin completely blocked binding to syndecan-2-expressing Jurkat cells. (B) Treatment of Synd2/Jurkat cells with the heparan sulfate-cleaving enzymes heparinase or heparitinase reduced the ability of cells to bind TACI-Fc. Curves shown in panels A and B were analyzed by flow cytometry in the same experiment but are shown in separate panels for clarity. Negative and positive control curves (Jurkat and Synd2/Jurkat) in panels A and B are the same data. (C) Syndecan-2 does not bind a TACI ligand noncovalently. Synd2/Jurkats were incubated with heparin and then washed extensively to remove heparin and any putative ligands that might have been bound to cell surface syndecan-2. Washed Synd2/Jurkat cells recover their ability to bind TACI-Fc. (D) TACI-Fc binds directly to immobilized heparin or heparan sulfate. 0-Fc (lanes 3 and 4) or TACI-Fc (lanes 6 and 7) were mixed with carbozone resin conjugated to heparin (Hi) or heparan sulfate (Ha). Unbound fusion proteins were removed by extensive washing. Immobilized recombinant protein was subsequently eluted with SDS and analyzed by SDS-PAGE followed by Coomassie blue staining. Lanes 2 and 5 (labeled “S”) indicate the input amount of 0-Fc or TACI-Fc.