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Fig. 7. Replacing the disulfide bridge between cys-2 and cys-4 of SCR16 by a diselenide bridge inhibits CD21 shedding. (A) Schematic presentation of CD21 constructs and the deduced amino acid sequence of SCR16 including the mutated cysteine residues. For the incorporation of selenocysteines instead of cysteines, the codons for cys-2 (Cys941) and cys-4 (Cys968) were exchanged for TGA by site-directed mutagenesis and the SECIS element of the selenium-dependent glutathione peroxidase PHGPx was cloned 3' to exon 19 (construct F). In order to obtain a constitutively open bridge between cys-2 and cys-4, the same codons were replaced by methionine (construct G). (B) Determination of the amount of sCD21 in cell culture supernatants of three 293 clones stable transfected with the construct F (left panel) or G (right panel). Control cells were 293 cells stable transfected with construct B (Fig. 6A). Cells were incubated for 4 hours at 37°C (bars labeled 1, t4 control) or stimulated for 4 hours at 37°C with 10 mM GSH (bars 2), 30 mM GSH (bars 3), 10 mM NAC (bars 4), 30 mM NAC (bars 5), 5 mM GSH (bars 6) or 5 mM NAC (bars 7). Cell culture supernatants were collected and sCD21 was measured by ELISA (values expressed as pg sCD21/ml/median fluorescence intensity of respective clones). Three clones of each cell line were tested in two independent experiments. Data are means of each single clone measured in triplicate.
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