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First published online 27 June 2006
doi: 10.1242/jcs.02984

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Redox regulation of CD21 shedding involves signaling via PKC and indicates the formation of a juxtamembrane stalk

Annette Aichem¹, Madhan Masilamani^2,* and Harald Illges^1,2,,

¹ Biotechnology Institute Thurgau at the University of Konstanz, Konstanzer Str. 19, 8274 Tägerwilen, Switzerland
² University of Konstanz, Department of Biology, Faculty of Sciences, Universitätsstrasse 10, 78457 Konstanz, Germany

View larger version (32K): [in a new window]   Fig. 1. PMA/CaI-induced CD21 shedding in 293-CD21 cells. (A) 293-CD21cells were stained with the monoclonal anti-CD21 FITC-labeled antibody BL13. Expression and cell surface localization of the exogenous CD21 was verified by confocal microscopy. (B) Western blot analysis of 293-CD21 cells after 4 hours of stimulation with 10 µM PMA, 10 µM PMA + 1 µM CaI or the solvent control DMSO. 30 µg of protein lysate per lane were subjected to western blot analysis using the monoclonal anti-CD21 antibody BU32 (upper panel). The blot was stained with Ponceau Red to control the amount of loaded protein (lower panel). (C) Determination of sCD21 (measured as pg sCD21/ml/median fluorescence intensity of 293-CD21 cells) in cell culture supernatants from 293-CD21 cells by ELISA. Cell culture supernatants were collected after incubation of the cells for 4 hours with 10 µM PMA, 1 µM CaI, 10 µM PMA + 1 µM CaI or the solvent control DMSO. Data are mean of five independent experiments each measured in triplicate.

View larger version (15K): [in a new window]   Fig. 2. Pervanadate induced CD21 shedding in Daudi B cells, PBMC and 293-CD21 cells. 2x106 Daudi B cells/ml (A), 3.4x106 PBMC/ml isolated from four healthy donors (B) and 293-CD21 cells (C) were incubated for 4 hours with increasing concentrations of pervanadate. (A,C) Cell culture supernatants from 293-CD21 and Daudi B cells were collected and the amount of sCD21 was determined by ELISA. Data for 293-CD21 cells are means of three independent experiments, data for Daudi B cells are means of four independent experiments, each measured in triplicate (pg sCD21/ml/median fluorescence intensity of respective cells. (B) PBMC from four healthy donors were collected and cell surface CD21 of primary B cells was measured by flow cytometry after staining with anti-CD21-RPE. n.d.: not determined.

View larger version (17K): [in a new window]   Fig. 3. Induction of CD21 shedding by thiol antioxidants. 293-CD21 cells (A), Daudi B cells (2x106 cells/ml) (B) and PBMC from four healthy donors (5x106 cells/ml) (C) were stimulated for 4 hours with 5 mM reduced glutathione (GSH), 5 mM oxidized glutathione (GSSG), 5 mM NAC, 1 mM or 2.8 mM ß-mercaptoethanol (2-ME). Cell culture supernatants were collected and sCD21 was measured by ELISA; values expressed as pg sCD21/ml/median fluorescence intensity of respective cells. Data are means of three independent experiments each measured in triplicate.

View larger version (24K): [in a new window]   Fig. 4. Oxidants and thiol antioxidants operate at different sites of CD21 shedding induction. (A) Analysis of the tyrosine phosphorylation pattern after incubation of 293 cells stably transfected with construct B (293-CD21-B cells) (left panel) and Daudi B cells (right panel) with the CD21 shedding stimuli pervanadate (200 µM) or NAC (5 mM). Cells were stimulated for 30 seconds or 2 minutes at 37°C, lysed and subjected to western blot analysis (1.6x105 293-CD21-B cells/lane; 1.3x106 Daudi B cells/lane) using an anti-phosphotyrosine antibody. As loading control, both blots were stained with an anti-ß-actin antibody (lower panel). (B) Pervanadate-, but not NAC- and GSH-induced CD21 shedding is inhibited by the PKC inhibitor BIM. 293-CD21-B cells were preincubated for 1 hour with or without 10 µM BIM and subsequently incubated for 4 hours with 1 mM pervanadate, 5 mM NAC or 5 mM GSH in the presence or absence of 10 µM BIM. Cell culture supernatants were collected and sCD21 was measured by ELISA (values expressed as pg sCD21/ml/median fluorescence intensity of 293-CD21-B cells). Data are means of three independent experiments each measured in triplicate.

View larger version (28K): [in a new window]   Fig. 5. CD21 shedding is inhibited by metallo- and serine protease inhibitors. (A) 5x106 Raji cells/ml were cultured for 24 hours in the presence of 200 µM MMPi-I, 50 nM MMPi-II, 200 nM MMPi-III, 1 mg/ml AAT or 2 µM EDTA. Cell culture supernatants were collected and sCD21 was measured by ELISA. (B) 5x106 PBMC/ml from three healthy donors were stimulated for 4 hours with 1 mg/ml AAT, 10 µM PMA or 1 mg/ml AAT and 10 µM PMA. Cell culture supernatants were collected and the amount of sCD21 was measured by ELISA. (C) 293-CD21 cells were preincubated in serum free IMDM for 1 hour at 37°C with TIMP-I (40 nM), TIMP-II (40 nM), TIMP-III (20 nM), MMPi-I (200 µM), MMPi-II (50 nM), MMPi-III (200 nM) or AAT (1 mg/ml) and subsequently stimulated for 4 hours with 10 µM PMA + 1 µM CaI in the presence of the inhibitors. Cell culture supernatants were collected and the amount of sCD21 was measured by ELISA. Data are means of three independent experiments each measured in triplicate. (D) Two 293-CD21-B clones (see Fig. 6 for description) were incubated for 1 hour at 37°C with MMPi-I (200 µM), MMPi-II (50 nM), MMPi-III (200 nM) or 1 mg/ml AAT and subsequently stimulated for 4 hours with 5 mM NAC in presence of the inhibitors. Cell culture supernatants were collected and the amount of sCD21 was measured by ELISA.

View larger version (21K): [in a new window]   Fig. 6. SCR 16 is required for CD21 shedding. (A) Schematic presentation of CD21 constructs. Several CD21 cDNAs containing exon 16 (B-E) and one without (A) were cloned into the eucaryotic expression plasmid pEGFP-C1 as described in Materials and Methods. The resulting CD21 proteins are schematically shown on the right. (B) Verification of the expression and cell surface localization of the different CD21 mutant proteins expressed by 293 cells, stable transfected with the constructs shown in Fig. 6A. Cells were stained with anti-CD21-RPE and subjected to flow cytometry analysis. One clone of each cell line is shown (293-CD21-A, violet; -B, orange; -C, magenta; -D, light blue; -E, green). Untransfected 293 cells (blue) were stained as a control. (C) Determination of the amount of sCD21 in cell culture supernatants after stimulation of the cells for 4 hours at 37°C with 10 µM PMA + 1 µM CaI, 200 µM pervanadate, 5 mM NAC or 5 mM GSH. 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 construct were tested and gave the same results. Data are means of two independent experiments of a single clone of each cell line.

View larger version (32K): [in a new window]   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.

View larger version (27K): [in a new window]   Fig. 8. Schematic representation of proposed mechanisms of CD21 shedding activation. Activation of B cells by BCR and CD40 crosslinking leads to a tyrosine phosphorylation pathway that involves PKC. A similar pathway is activated by pervanadate in B cells, as well as in non-lymphoid cells such as the epithelial cell line 293. Subsequently, a serine protease is activated that is necessary to activate the metalloprotease involved in CD21 shedding. Thiol antioxidants such as NAC directly activate the metalloprotease and open disulfide bridges within the SCRs of CD21. This leads to the formation of a stalk region that permits access of the protease to the cleavage site within SCR16. PMA, phorbol 12-myristate 13-acetate, PTP, protein tyrosine phosphatase; PTK, protein tyrosine kinase; MMP, metalloprotease; TyrP, tyrosine-phosphorylated protein; Bim, bisindolyl maleimid; NAC, N-acetyl cysteine; PKC, protein kinsae C.

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