Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines.

Abstract

Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NlttodSO4/PAGE, and autoradiographs of these gels indicpe. that C3-binding [1][2][3][4]. This molecule has a number of interesting structural and regulatory features (1, 2, 5-11), including an unusual polymorphism in which there is an -60,000 difference in Mr among the three recognized alleles (5-7). CR2, the C3d receptor of human peripheral blood cells, is an -140,000 Mr integral membrane glycoprotein found on human B lymphocytes (12, 13). It is probably identical to a B-cell antigen defined by the monoclonal antibody anti-B2 (12, 14) and a C3b-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.binding protein purified from Raji cells (15). CR2 apparently binds to the C3d fragment within intact C3b (15).Our laboratory has employed affinity chromatography to isolate CR1 (5,7,8) and CR2 (16), as well as a C3b-specific integral membrane glycoprotein from rabbit alveolar macrophages (17, 18). In our studies of CR1 and CR2 on human leukocytes, we routinely observed a third group of molecules with Mrs of 45,000 to 70,000 (gp45-70) with binding specificity for C3. This report represents our initial evaluation of these C3-binding proteins of human leukocytes, including their isolation, cell distribution, electrophoretic mobility, and specificity for C3 and its proteolytic fragments. MATERIALS AND METHODSIsolation of Human Peripheral Blood Cells. Normal individuals donated "30 ml of blood for preparation of erythrocytes (E) or up to 500 ml for preparation of leukocytes. E were washed in phosphate-buffered saline (Pi/NaCl; 10 mM potassium phosphate/150 mM NaCl, pH 7.40) and resuspended in a 10% suspension. Mononuclear and polymorphonuclear (PMN) cell populations were separated by dextran sedimentation followed by Ficoll/Hypaque centrifugation (5, 8). Cell populations enriched for T lymphocytes, B plus null lymphocytes, or monocytes were prepared and analyzed as previously described (19). Greater than 80% of the monocyte-enriched population was esterase positive. Analysis of cell surface characteristics for the enriched lymphocyte populations showed that >75% of the T-cell-enriched population formed E rosettes with only 1% possessing surface immunoglobulin, and >60% of the B plus null cells had detectable surface immunoglobulin with <5% forming E rosettes.Cell Lines. The Raji cell line (an Epstein-Barr-virus-negative B-lymphoid line), the GM3104, GM3161, and GM3164 cell lines (all Epstein-Barr-virus-transformed B-cell lines), and the Jurkat T-cell line were grown in T-150 flasks at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% fetal bovine serum (Sterile Systems, Logan, UT), 4 mM glutamine, and an antibioti...

Elevated serum levels of soluble membrane cofactor protein (CD46, MCP) in patients with systemic lupus erythematosus (SLE)

“…CD46, CD55 and CD59 are important cell surface molecules which protect autologous cells from injury by complement [1,2,13,14]. Among these molecules, up-regulation of CD59 was ®rst shown on the glomerular cells of lupus nephritis patients [15].…”

“…Membrane cofactor protein (MCP, CD46) is a cell surface glycoprotein which acts as a cofactor for the factor I-mediated cleavage of the activated complement components C3b/C4b [1,2]. This molecule is thought to be crucial in protecting autologous cells against attack by the complement system.…”

“…CD46 is diffusely distributed in the human body and is found on endothelial and epithelial cells, ®broblasts and almost all human blood cells except for erythrocytes [1,20,21]. With regard to variants of sCD46, two groups of sCD46 of different molecular weights whose origins are assumed to be different have been detected [5].…”

Defective B cell ontogeny and immune response in human complement receptor 2 (CR2, CD21) transgenic mice is partially recovered in the absence of C3

“…Recently, it was shown that after antigen binding to BCR, the BCR itself could form a platform for activation of the classical complement pathway which resulted in the deposition of C3 fragments on the antigen which would presumably allow direct binding with CR1/2 and provide co-stimulatory signals into the B cell in a cell bound amplification loop (Manderson et al, 2006;Rossbacher and Shlomchik, 2003). It is also worth remembering that CR2 was shown to bind to sepharose bound inactive C3 (Cole et al, 1985). Thus, the ability of fB/C2 deficient sera to cause down regulation of hCR2 expression (Figure 6) demonstrates that the natural generation of inactive C3 could provide ligand to interact with CR2 as well as suggesting that the generation of C3d is not necessarily required for this outcome.…”

“…CR2, in both mouse and man, binds with high affinity to the C3 breakdown fragment C3d (as well as iC3b and C3dg, (Cole et al, 1985;Farries et al, 1990;Iida et al, 1983;Kalli et al, 1991;Molina et al, 1994;Weis et al, 1984) which remains covalently bound to complement activating surfaces or antigen (Law and Dodds, 1997). Consistent with a key role for C3 fragments in amplifying the immune response via the CR2/CD19, C3 −/− mice have weak humoral immune responses and defects in germinal center formation, Wessels et al, 1995).…”