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Induction of CD21 shedding

CVID is a disease where diagnosis used to be mainly based on antibody deficiencies and recurrent infections. The cause of CVID was poorly understood up to now and this consortium contributed in several ways towards characterization and diagnosis of CVID clearly improving both. The following is a summary of the results obtained/contributed by partner 8 (BITg).

We have developed a new purification method to isolate pure sCD21 from human sera and used this to establish an ELISA. This was necessary because sCD21 was biochemically purely characterized and a high quality ELISA was and is not available on the market.

Determining the sedimentation coefficient, the partial specific volume, the diffusion coefficient and the frictional coefficient of the sCD21 protein we were able to conclude that sCD21 is an elongated rod-shaped molecule of 126 kDa. Using our ELISA we have analysed sCD21 content in > 400 healthy donors and from > 150 CVID patient´s sera. We found that CD21 in healthy donors is on average about 300ng/ml and declines with age (statistically significant). The sCD21 content varies significantly in the CVID subgroups. In rheumatoid arthritis, Soegren´s syndrome, scleroderma, systemic lupus erythomatosus and others we found a severe reduction of sCD21 and concluded that autoimmune processes act on the regulation of CD21 shedding. Mass spectrometrical analysis of sCD21 showed only fragments derived from the extracellular portion of the molecule and representing the short version of CD21. During our analysis of CD21 in activation processes, we found for the first time that two lipid microdomain resident proteins, flotillin-1 and -2 form pre-assembled platforms (PAPs) in haematopoietic cells. These platforms recruit signalling molecules such as CD21 upon activation through lipid rafts. We have shown that activation of lymphocytes via PMA and Ca2+ Ionophores or via crosslinking using anti-IgM/anti-CD40 antibodies induced the proteolytic cleavage of CD21.

Analyzing signalling pathways leading to CD21 shedding we found that CD21 is susceptible to BzATP induced shedding via purinergic receptors. Shedding could be inhibited with P2X7 receptor inhibitors oxATP and KN62. CD21-shedding is redox regulated. Oxidants and thiol-antioxidants operate at different sites of CD21-shedding induction and serine- and metalloproteases are involved in CD21-shedding. CVID patients differ in sCD21 comparing Bryant´s classification A and B groups.

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Harald ILLGES, (Head of Unit)
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