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PubMed1985

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

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 NaDodSO4/PAGE, and autoradiographs of these gels indicated that C3-binding proteins could be divided into three groups based on Mr: (i) gp200, an approximately 200,000 Mr molecule previously identified as the C3b/C4b receptor or CR1; (ii) gp140, an approximately 140,000 Mr molecule previously identified as the C3d receptor or CR2; and (iii) gp45-70, a heretofore unrecognized group of 45,000-70,000 Mr C3-binding molecules. The cell distribution, Mr, antigenic cross-reactivity, and specificity of gp45-70 were examined. Erythrocytes have no detectable gp45-70, but all leukocyte populations examined possess this group of molecules. On neutrophils and mononuclear phagocytes, CR1 is the predominant C3-binding glycoprotein, but gp45-70 is present on both cell populations and on macrophage and neutrophil cell lines. B plus null cells, chronic lymphocytic leukemia cells, and an Epstein-Barr virus-transformed B-cell line possess CR1, CR2, and gp45-70. On T cells and T-cell lines gp45-70 is the predominant or, in some cases, the only C3-binding protein isolated. gp45-70 is structurally characterized as a broad band or doublet with a mean Mr that is slightly different for each cell population. gp45-70 binds iC3, C3b, and C4b, but not C3d, indicating that the binding region is probably within the C3c portion of C3b. A polyclonal antibody to CR1 and monoclonal antibodies to CR1 and CR2 do not immunoprecipitate gp45-70. While gp45-70 has not been previously characterized on human cells, a C3b-binding glycoprotein of similar Mr is present on rabbit alveolar macrophages. We conclude that gp45-70 is an additional group of membrane proteins present on human leukocytes that possess ligand-binding activity for C3b.

B-Lymphocytes↗

PubMed1992

Macrophage-bound C3 fragments as adhesion molecules modulate presentation of exogenous antigens.

The involvement of complement in the response to T cell dependent antigens is generally accepted, however the mechanism has not been clarified. We compared the T cell response in vitro, using antigen-pulsed macrophages from normal and genetically C3-deficient guinea pigs, and show, that C3-fragments fixed covalently to the surface of the antigen-presenting cells are involved in the triggering of responder T cells. Binding of guinea pig C3-specific mAb to oil-elicited, OVA- and PPD-pulsed macrophages of C3D guinea pigs is reduced compared to normal cells, while the expression of Ia antigens is the same. C3-like peptides can be immunoprecipitated only from the lysate of oil-elicited normal cells. These C3-fragments are fixed to the cell-membrane via ester-bonds, since they are released upon treatment with hydroxylamine. In comparison with normal cells, the antigen-presenting capacity of macrophages derived from C3D animals is strongly impaired in cultures containing 10% normal guinea pig serum. A further impairment is observed in cultures with 10% C3D guinea pig serum. Two of the tested C3-specific mAb inhibited antigen-induced T cell proliferation in a dose-dependent manner. Our data point to the importance of C3, as a bivalent molecule, having the capacity to facilitate the cooperation between the antigen-presenting cell and the responder T lymphocytes.

Adjuvants, Immunologic↗

PubMed1983

Importance of factors H and I for the adherence of C3b-coated erythrocytes to cells.

The role of cell membrane-associated human factor H for the binding of cell-bound C3b to complement receptor-carrying (CR+) cells was investigated. Pretreatment of CR+ cells with antibodies to factor H inhibited the adherence of C3b-coated red cells to human tonsil lymphocytes (TL) and peripheral blood monocytes (M phi). The C3b receptor reactivity of human polymorphonuclear leucocytes (PMN) was not influenced and the one of Raji lymphoblastoid cells only slightly influenced; iC3b and C3d receptor reactivity was in no case affected. When diisopropylfluorophosphate (DFP) in a concentration of 0.1 mM was present during pretreatment of the CR+ cells with anti H, the antibodies gained the capacity to inhibit the adherence of C3b-coated erythrocytes to Raji cells; this effect was dose-dependent with respect to DFP. In contrast, there was no influence of DFP on the inhibition pattern of anti H in the case of TL and M phi. The adherence of C3b-coated erythrocytes to PMN remained unaffected by anti-H antibodies in the presence of DFP. Polyclonal as well as monoclonal antibodies directed against human factor I inhibited the binding of C3b cells to Raji cells but not to TL. Additionally, when anti I and anti H antibodies were both present, C3b receptor reactivity of Raji cells was inhibited to a larger extent than with either antibody alone; again, TL remained unaffected. Results obtained by washing the Raji cells before and after treatment with anti H and anti I suggest that the respective antibodies act on factor H primarily on the level of the cell membrane and on factor I in the fluid phase.

Animals↗

PubMed1988

Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies.

Hydrolysis of the internal thioester bond in native C3 is thought to be a key event in initiating the alternative pathway of C activation, because the resulting C3(H2O) acquires "C3b-like" properties. Therefore, disruption of the internal thioester bond is probably accompanied by conformational changes in the C3 molecule. In this study, we demonstrate that such conformational changes indeed occur; 7 of the 19 mAb raised against C3 or C3 activation products recognized epitopes exposed on C3(H2O) but not on native C3. One of these epitopes is located on the C3a part, three on the C3c part, and another three on the C3d,g part. Because the 7 mAb bound equally well to C3 incubated either with MgCl2 or with methylamine (which primarily disrupts the thioester), the conformational changes detected by the mAb apparently occur after disruption of the thioester. Furthermore, the epitopes were also present on the corresponding C3 activation products. Immunoblotting experiments revealed that the epitopes for the three anti-C3d,g mAb were located on the C3d part, C-terminal to the thioester. The epitopes for 2 of the 3 anti-C3c mAb were located on the C-terminal alpha-chain fragment of C3c. Thus, this study provides immunochemical evidence for the biologic resemblance between C3(H2O) and C3 activation products. Implications of these findings for the activation process of C3 are discussed.

Animals↗

PubMed1989

A morphological and immunohistochemical study of lymphoid germinal centers in synovial and lymph node tissues from rheumatoid arthritis patients with special reference to complement components and their receptors.

Rheumatoid arthritis (RA) is one of the immune complex (IC) diseases in which lymphoid germinal centers (GCs) are found in the synovial tissue. Simultaneously, patients with RA often show swelling of lymph nodes. The morphology and function of the lymph-node GCs in patients with RA is not clear. The aim of this study was to evaluate the differences in morphology and immunoreactions to complement (C) components, their receptors, and IgM-rheumatoid factor (RF) between synovial GCs and lymph-node GCs in RA. Furthermore, the relationship between these immunoreactive substances and follicular dendritic cells (FDCs) in GCs was investigated. The tissues examined were 41 RA synovial specimens, seven RA lymph nodes with massive lymphadenopathy, and 10 non-RA lymph nodes. The number of synovial GCs was relatively decreased in comparison with lymph-node GCs in RA, and the diameter of each synovial GC was smaller than that of each lymph-node GC. The synovial GCs were edematous and less cellular, and moreover, those from RF-seronegative cases were smaller than those from RF-seropositive cases. On the other hand, the lymph-node GCs in RA were larger, more cellular and hyperplastic, but contained more tingible-body macrophages (TBMs) and neutrophils. In the GCs of both synovial tissues and lymph nodes in RA, early C components (C1q, C4, C3c, C3d), IgM-RF, and C3b receptor (C3bR) and C3d receptor (C3dR) were expressed as a lacy network by light microscopy, and were demonstrated on the surfaces of FDCs and lymphocytes, and in the intercellular spaces by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces.

One percent of circulating IgG in humans recognizes galactose alpha 1,3 galactose residues (anti-Gal) and is synthesized in response to stimulation by enteric bacteria. In this study, we found that the prevalence of binding of anti-Gal to blood isolates is significantly higher than its binding to normal stool isolates. When anti-Gal bound onto the lipopolysaccharide of a representative blood isolate, Serratia marcescens #21, it blocked its alternative complement pathway (ACP) lysis and made the organism serum resistant. In contrast, when anti-Gal bound to the capsular polysaccharide of a serum sensitive Serratia, #7, it increased ACP killing of this strain. The mechanism of blockade of ACP lysis by anti-Gal did not involve a decrease in the number of C3 molecules deposited onto Serratia #21 or an inhibition of the binding of C3b to its LPS, nor did it change the iC3b and C3d degradation products of bound C3b or prevent membrane attack complex formation on this organism. Our findings suggest that the effect of anti-Gal on immune lysis is dependent on the bacterial outer membrane structure to which it binds. We postulate that anti-Gal may play a role in the survival of selected Enterobacteriacae in Gram-negative sepsis by blocking ACP-mediated lysis of such bacteria by the nonimmune host, and that this effect depends on where anti-Gal finds its epitope on the bacterial outer membrane.

Complement C3↗

PubMed1986

Ligand-receptor interactions in the phagocytosis of virulent Streptococcus pneumoniae by polymorphonuclear leukocytes.

We used polyclonal and monoclonal antibodies to neutrophil complement receptors CR1 and CR3 to assess the role of these receptors in the phagocytosis of virulent Streptococcus pneumoniae serotypes 3, 6A, and 14, which bear accessible C3 ligands covalently bound to the polysaccharide capsule. When the iC3b receptor (CR3) on normal polymorphonuclear leukocytes (PMNLs) was blocked by the monoclonal antibody OKM10, phagocytosis of pneumococcal serotypes 6A and 14 (which bear exclusively iC3b) was inhibited 50%-80% in pooled human serum and completely in nonimmune serum. Blockade of the PMNL C3b receptor (CR1) failed to inhibit phagocytosis for serotypes 6A and 14. For serotype 3, which bears C3b and C3d (as well as iC3b) on the capsule, CR3-mediated phagocytosis accounted for only 20% of the uptake; again, there was no evidence for CR1-mediated phagocytosis. The iC3b ligand elicited consistently greater release of superoxide, myeloperoxidase, and lactoferrin than did C3b. The iC3b/CR3 interaction is thus the primary trigger for phagocytosis of iC3b-bearing pneumococci and for stimulation of intracellular bactericidal processes.

Complement C3b↗

PubMed1977

Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.

The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/ml (mean +/- 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta1H were removed from these proteins with anti-beta1H Sepharose. However, in the presence of highly purified beta1H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their structure. The action of C3bINA and beta1H on C3b produced two fragments of the alpha1-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta1H on C4b resulted in cleavage of the alpha'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.

Amino Acids↗

PubMed2002

Ceftriaxone induced immune hemolytic anemia: detection of drug-dependent antibody by ex-vivo antigen in urine.

There have been a few reported cases of immune hemolytic anemia induced by ceftriaxone. We encountered a patient with immune hemolytic anemia that seemed to be stimulated by a degradation product of ceftriaxone. The patient's direct antiglobulin test was positive only for C3d, and no ceftriaxone-dependent antibodies were detectable in the patient's serum. To demonstrate the presence of the ceftriaxone-induced antibodies, an ex-vivo antigen in urine was obtained from the patient. In addition, we prepared a 1 mg/mL suspension solution of ceftriaxone, and group AB serum as a complement source. Using several combinations of the above reactants, the indirect antiglobulin test was performed. Only the indirect antiglobulin test using the patient's serum with the ex-vivo urine antigen was found to be positive. Other combinations were not reactive. To our knowledge, this is the first reported case in Korea, in which the causative antibody appeared to be stimulated solely by a degradation product of ceftriaxone.

Anemia, Hemolytic, Autoimmune↗

PubMed2006

The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.

Animals↗

PubMed1994

The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts.

The use of xenografts (Xgs) from distantly related species to relieve the increasing shortage of organs for clinical transplantation is prevented by the occurrence of hyperacute rejection (HAR). This process, in which C activation plays a central role, cannot be inhibited with currently available immunosuppressants. In two clinically relevant xenotransplantation models, this study evaluated the effect of C inhibition using recombinant soluble complement receptor type 1 (sCR1) on HAR. In an ex vivo model in which porcine cardiac Xgs were perfused with human blood, cardiac function ceased within 34 min when the perfusate blood was untreated (n = 3). When the perfusate blood was treated with sCR1 (300 micrograms/ml), cardiac Xg function was maintained for up to 4 hr (n = 3). Immunohistologic examination of these Xgs demonstrated deposition of C3b/iC3b and C3d in Xgs perfused with untreated human blood but only C3d deposition in those Xgs perfused with sCR1-treated human blood. These findings are consistent with the cofactor activity of sCR1 for factor I-mediated degradation of deposited C3b/iC3b to C3d. Treatment with sCR1 also prevented the histopathologic changes of HAR observed when untreated blood was used as the perfusate. In an in vivo pig-to-primate heterotopic cardiac xenotransplantation model, in which porcine Xgs transplanted into untreated cynomolgus monkey recipients underwent HAR in 1 hr or less (n = 3), a single intravenous bolus of sCR1 (15 mg/kg) administered to the recipient immediately before Xg reperfusion markedly inhibited total and alternative pathway serum C activity and prolonged Xg survival to between 48 and 90 hr (n = 5). These studies confirm the important role of C activation in HAR of porcine cardiac Xgs by primates and indicate that sCR1 may be a useful agent for xenotransplantation.

Animals↗

PubMed1997

Local immune response in infertile patients with minimal endometriosis.

OBJECTIVE: To evaluate the role of local immunity in women with minimal endometriosis. METHODS: Uterine endometrium and endometrial implants were obtained simultaneously from 30 infertile women with minimal endometriosis and examined immunohistochemically using antibodies of T cell, B cell, macrophage, Langerhans cell, immunoglobulin (Ig)G, and complement (C) 3d. Serum IgG, IgA, IgM, C3, C4, antinuclear antibody and anti-DNA antibody were also examined in 24 of the women. Data from uterine endometrium and serum were compared with 10 fertile women without endometriosis as a control. RESULTS: Microscopic examination revealed that the endometrial implants were divided into two groups: group 1 (n = 13) showed lymphocytic infiltration in the endometrial implants and group 2 (n = 17) showed no or slight lymphocytic infiltration. The endometrial implants of group 1 showed significantly more dense T-cell infiltration than those of group 2. Other types of infiltrating cells and deposits of IgG and C3d revealed no significant differences between groups 1 and 2. The immunohistochemical examination of the uterine endometrium and the serum data revealed no significant differences among all three groups. Cumulative pregnancy rates showed no significant difference between groups 1 and 2. CONCLUSION: The difference of local immune response in endometrial implants did not affect systemic immunity.

Adult↗

PubMed1976

Observations on the antiglobulin tests. II. C4 components on erythrocytes.

This paper describes anti-C4b(ii) antisera shich agglutinate cells from cases of autoimmune haemolytic anaemia, cold-acquired haemolytic anaemia, and certain test cells, using the antiglobulin test. It is suggested that these particular antisera possess activity against C4d absorbed on to erythrocytes. Methods of preparing test cells coated with C3d and C4d are given, together with methods for producing antisera in rabbits active against these components. The significance of these findings is discussed.

Anemia, Hemolytic↗

PubMed1988

Characterization of a monoclonal antibody MoAb bH6 reacting with a neoepitope of human C3 expressed on C3b, iC3b, and C3c.

Activation products of the complement cascade contain neoepitopes that are not present in the individual native components. Monoclonal antibodies detecting neoepitopes have been used for direct quantification of activation at different steps in the cascade. These methods are suggested to be more sensitive and reliable than conventional complement activation tests, which are hampered by precipitation or fractionation procedures. The present study describes production screening and characterization of a monoclonal antibody (MoAb) bH6. MoAb bH6 exhibited a significantly higher binding capacity to ELISA plates coated with zymosan-activated human serum than to plates coated with EDTA plasma. When fixed to the enzyme-linked immunosorbent assay (ELISA) plates, MoAb bH6 retained material from zymosan-activated serum that only reacted with anti-C3 antibodies. Crossed immunoelectrophoresis performed on zymosan-activated serum demonstrated that MoAb bH6 co-precipitated with anti-C3c antibodies. In experiments using highly purified cell-bound fragments MoAb bH6 showed reactivity with C3b and iC3b, but not with C3d. MoAb bH6 reacted in ELISA with purified C3c, but not with C3dg, both as capture antibody and in tests with the fragments absorbed to the solid phase. Thus, MoAb bH6 is highly specific for a neoepitope of human C3 expressed on the cleavage fragments of C3b, iC3b, and C3c.

Antibodies, Monoclonal↗

PubMed1993

Complement factors H and I synthesized by B cell lines function to generate a growth factor activity from C3.

B lymphocytes and transformed B lymphoblastoid cell lines express CR2 (CD21, C3d/EBV-receptor) that is specific for C3 fragments generated by cleavage of C3b or spontaneously hydrolyzed native C3 (C3i) by the serum enzyme factor I and its cofactor, factor H. It had been shown previously that the Raji B cell line could be cultivated in serum-free medium supplemented with only transferrin and either OKB7 anti-CR2 mAb, C3d, or C3d-derived peptides containing the CR2 binding site. Because these agents appeared to function through ligation of CR2, it was unclear how native C3 could also serve as a growth factor, because C3 does not bind to CR2. It appeared possible that Raji cells might be able to use endogenous factors H and I to generate a CR2 ligand from C3, because previous studies had shown that Raji cells synthesized factor H and probably also synthesized factor I. PCR analysis was used to demonstrate factor I mRNA in Raji cells. Secretion of Raji cell factor I protein was confirmed by a sensitive mAb ELISA. Several B cell lines were examined for C3-dependent growth. Raji cells required both C3 (or OKB7) and transferrin for growth, whereas Wil-2 cells grew with transferrin alone and C3 enhanced the growth-promoting activity of transferrin. Two other B cell lines (Daudi and U698M), the T cell line 8402, and the U937 monocytoid cell line could not be sustained with transferrin plus C3. The C3-dependent growth of Raji cells was inhibited almost completely by either OX-23 anti-factor H or 052.11.3 anti-factor I mAb that also blocked the activity of serum-derived factor H or I, respectively. By contrast, there was no inhibition of growth by either OX-24 anti-factor H or OX-21 anti-factor I mAb that did not block factors H and I activity. After the spontaneous hydrolysis of native C3 to C3i, it is hypothesized that Raji cells convert C3i to iC3i with endogenous factors H and I, and then this iC3i serves as a growth factor by binding to membrane CR2.

Animals↗

PubMed2006

Heart transplantation across antibodies against human leukocyte antigen and ABO-post-transplant follow-up of donor reactive antibodies.

BACKGROUND: We have successfully performed heart transplantation despite the most unfavourable risk factors for graft and patient survival: the presence of a high level of antibodies (Abs) against the donor's human leukocyte antigens (HLA) class I/II and blood group A1 antigens. The present study concerns post-transplant follow-up and characterization of donor reactive antibodies (DRA). METHODS: Pre-transplant treatment consisted of mycophenolate mofetil (MMF), prednisolone, tacrolimus, intravenous immunoglobulin (IVIG), rituximab, protein-A immunoadsorption (PAIA) and per-operative plasma exchange. A standard triple-drug immunosuppressive protocol was used post-operatively. Abs were analyzed by the complement dependent cytotoxicity (CDC) test against donor and panel B/T cells and by flow cytometry (FlowPRA tests detecting isolated HLA class I/II antigens). Abs against the donor's erythrocytes were analyzed using a standard direct agglutination test for immunoglobulin M (IgM) Abs and a Bio-Rad AHG gel card test detecting IgG Abs and C3d. RESULTS: Pre-transplant treatment reduced Ab titers against the donor's lymphocytes from 128 to 16 and against the donor's blood group A1 antigen from 256 to 0. The patient was emergently transplanted with a heart from a blood group incompatible donor (A1 secretor to O). No hyperacute rejection was seen. DRA were present against all mismatched HLA class I and class II antigens at the time of transplantation; two of these DRA Abs disappeared within the first year post-transplant (anti-B62 and anti-DR4), one showed weakened reactivity (anti-A24) and one is still strongly reactive (anti-DQ3). The donor-specific CDC cross-match is still positive (titers 2 to 8). The level of panel reactive antibodies (PRA) remained unchanged from 6 months on post-transplant. Rising anti-A1 blood group Abs preceded the second rejection and were adsorbed by two blood group specific immunoadsorptions (Glycosorb)-ABO) and remained at a low level. IgM anti-A1 blood group Abs disappeared at 1 yr post-transplant and IgG Abs are still reactive with blood group A1 erythrocytes but at low titers (1 to 2). CONCLUSIONS: The patient is clinically well 2 years after heart transplantation despite the constant persistence of donor reactive IgG Abs against blood group A1 and HLA-DQ antigens. The reactivity of DRA against other mismatched HLA antigens disappeared or weakened during the follow-up period.

ABO Blood-Group System↗

PubMed1995

Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b.

Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes.

Binding Sites↗

PubMed1975

Third component of complement (C3): structural properties in relation to functions.

The third component of complement (C3) fulfills a pivotal role in the functions of the complement system. We have investigated the topological relationships among its polypeptide chains, physiologic fragments, enzyme attack regions, and functional sites. C3 consists of two chains (alpha and beta) which are linked by disulfide bonds and noncovalent forces and which have molecular weights of, respectively, 120,000 and 75,000. C3 is activated by action of C3 convertase on the alpha-chain. With hydrolysis of one polypeptide bonds, C3a, the 9000 dalton activation peptide is dislocated from the NH2-terminal portion of the alpha-chain. A previously concealed binding region is thereby transiently revealed in the C3b-fragment (181,000 dalton) which displays affinity for apparently nonspecific acceptors present on biological membranes. Binding of nascent C3b membranes occurs through the C3d portion of the fragment because subsequent action of the C3b-inactivator or trypsin on bound C3b causes release of C3c, but not of C3d. Bound C3b and C3d possess stable sites that are capable of binding to specific receptors present on a limited variety of cells. We propose that all known physiologically occurring fragments of C3 arise by enzymatic cleavage of the alpha-chain: C3a, C3b, C3c, and C3d. Whereas C3a (alpha1) and C3e (alpha2) consist of a single chain and C3b consists of two chains (alpha' and beta), C3c is composed of the entire beta-chain and multiple fragments of the alpha-chain, each of which is linked by disulfide bonds to the beta-chain.

Binding Sites↗