Immunologische Untersuchung des humanen Komplementrezeptors Typ II (CR2/CD21). Lösliches CD21 in Gesundheit und Krankheit

In this study, the expression and regulation of membrane associated human complement receptor type-II (CR2/CD21) and its soluble form were investigated.The expression of CD21 was analysed in normal and activated T cells. PCR analyses and immunofluorescence/confocal microscopy of peripheral blood T cells and of activated T cells showed reduction in CD21 mRNA and protein expression upon activation.CD21 is released as a soluble form from the cell surface by proteolytic activity known as Shedding. Soluble CD21 was isolated and purified from human plasma to homogeneity applying affinity chromatography and density gradient centrifugation. Soluble CD21 was found to be a single 126 kDa molecular species. By determining the sedimentation coefficient of sCD21, the hydrodynamic properties such as diffusion coefficient and the frictional coefficient were calculated. These values show that the sCD21 isolated from human plasma is an elongated rod shaped molecule. A specific ELISA was developed using purified sCD21 as a standard for the determination of sCD21 concentration in human sera. Soluble CD21 levels were estimated in serum samples from 235 healthy donors, 209 RA patients, 17 patients with arthritis related disorders, 41 CVID patients, 10 healthy pregnant women and 10 neonates. The normal values of plasma sCD21 in healthy individuals of age between 20-40 years range from 100-500 ng/ml (median 292 ng/ml) with a tendency to decrease with age but did not differ with gender. sCD21 levels were significantly reduced in rheumatoid arthritis (

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

Soluble CD21 (sCD21), released from the plasma membrane by proteolytic cleavage (shedding) of its extracellular domain (ectodomain) blocks B cell/follicular dendritic cell interaction and activates monocytes. We show here that both serine- and metalloproteases are involved in CD21 shedding. Using the oxidant pervanadate to mimic B cell receptor activation and thiol antioxidants such as N-acetylcysteine (NAC) and glutathione (GSH) we show that CD21 shedding is a redox-regulated process inducible by oxidation presumably through activation of a tyrosine kinase-mediated signal pathway involving protein kinase C (PKC), and by reducing agents that either directly activate the metalloprotease and/or modify intramolecular disulfide bridges within CD21 and thereby facilitate access to the cleavage site. Lack of short consensus repeat 16 (SCR16) abolishes CD21 shedding, and opening of the disulfide bridge between cys-2 (Cys941) and cys-4 (Cys968) of SCR16 is a prerequisite for CD21 shedding. Replacing these cysteines with selenocysteines (thereby changing the redox potential from -180 to -381 mV) results in a loss of inducible CD21 shedding, and removing this bridge by exchanging these cysteines with methionines increases CD21 shedding

The Lipid Raft Microdomain-Associated Protein Reggie-1/Flotillin-2 is Expressed in Human B Cells and Localized at the Plasma Membrane and Centrosome in PBMCs

Reggie-1/flotillin-2 is a plasma membrane-associated cytoplasmic protein, which defines non-caveolar raft microdomains. Reggie-1/flotillin-2 is enriched in detergent insoluble (TX100) membrane fractions (DIG), co-localizes with activated GPI-linked proteins and the fyn-kinase in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. In T cells activated by crosslinking the GPI-linked protein Thy-1 or by crosslinking the ganglioside GM1, reggie-1/flotillin-2 co-localizes with the T cell receptor. To determine whether reggie-1/flotillin-2 is also expressed in B cells, primary B cells from human blood and cell lines representing the developmental stages of pro, pre, mature and plasma B cells were analyzed by Western blotting, RT-PCR and immunofluorescence. Here, we show that reggie-1/flotillin-2 is expressed throughout B cell development, as well as in primary B cells, purified by cell sorting. On non-activated mature B cell Raji cell line we found reggie-1/flotillin-2 are exclusively in the detergent (TX100) insoluble membrane fractions that are staining positive for the raft marker GM1. Immunofluorescence microscopy showed that reggie-1/flotillin-2 is localized at the plasma membrane and marks intracellular spots in PBMCs. Confocal co-localization studies showed that reggie-1/flotillin-2 is associated with the plasma membrane, and the centrosomes (microtubule organizing centers) in these PBMCs. Comparison of reggie-1/flotillin-2 cDNA sequences with the genomic sequence database allowed us to determine the exon/intron structures in mouse and human. The gene organizations are highly conserved suggesting an important function of reggie-1/flotillin-2. Since reggie/flotillin proteins co-cluster with the T cell receptor and fyn kinases upon T cell stimulation, our findings of reggie-1/flotillin-2 in B cells suggest a similar role in B cell function