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PubMed1984

Glomerular deposition and serum levels of complement control proteins in patients with IgA nephropathy.

We examined complement control proteins focusing on the role of modulating the complement activation in IgA nephropathy. Glomerular C4-binding protein (C4-bp) deposits were found in 60% of patients with IgA nephropathy, while C4 deposits were found in 30%. Glomerular deposits of beta 1H globulin (beta 1H) were found in 85% of patients with IgA nephropathy. The frequency of glomerular deposits of C4-bp tended to be higher in the group with deposits of various immunoglobulin types than in the group with deposits of IgA alone, and it increased parallel with the progression of glomerular histologic changes. The serum C4-bp level was higher in IgA nephropathy patients. No significant correlation was found between the serum level of C4-bp or beta 1H and the extent or distribution of tissue deposits of these complement control proteins. These results suggest that glomerular immune deposits in IgA nephropathy may be attributable to the activation not only of the alternative pathway but also of the classical pathway, the latter of which may take place locally in glomeruli with advanced histologic change or various immunoglobulin deposits in IgA nephropathy.

Beta-Globulins↗
PubMed2003

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

Amino Acid Sequence↗
PubMed2002

The yeast Candida albicans binds complement regulators factor H and FHL-1.

The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.

Binding Sites↗
PubMed1999

Complement activation in patients with systemic lupus erythematosus without nephritis.

OBJECTIVE: To study the association between disease activity and complement activation prospectively in patients with systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Twenty-one SLE patients were examined monthly for 1 yr. Disease activity, autoantibodies, conventional complement tests and the following complement activation products were investigated: C1rs-C1inh complexes, C4bc, Bb, C3a, C3bc, C5a and the terminal SC5b-9 complement complex (TCC). RESULTS: Modest variation in disease activity was noted. None of the patients had nephritis. Flare was observed at 27 visits. Four patients had anti-C1q antibodies in conjunction with modestly low C1q concentrations. The complement parameters were rather constant during the observation period. Slightly to moderately decreased C4 (0.05-0.10 g/l) was found in 10 patients and severely decreased C4 (<0.05 g/l) in seven patients. Decreased C4 was not associated with increased complement activation. Complement activation products were either normal or slightly elevated. TCC was the only activation product correlating significantly with score for disease activity at flare. None of the variables tested predicted flares. CONCLUSION: Complement tests are of limited importance in routine examination of SLE without nephritis, although TCC is suggested to be one of the most sensitive markers for disease activity.

Adult↗
PubMed1980

Biosynthesis of the complement components and the regulatory proteins of the alternative complement pathway by human peripheral blood monocytes.

Short-term cultures of human peripheral blood monocytes were shown to synthesize the alternative pathway complement components C3, factors B (B) and D (D), and properdin, the regulatory proteins C3b inactivator (C3bINA) and beta 1H, in addition to C2, C4, and C5. B, D, properdin, C3bINA, and C2 were detected by functional assays, whereas beta 1H, C4, C3, and C5 could only be detected using immunochemical procedures. Immunoperoxidase localization studies showed that all the cells in each culture contained each component, so it is possible that all monocytes synthesize each component. It is concluded that cells of the monocyte-macrophage series form a mobile source of complement components and regulatory proteins which can be concentrated at sites of inflammation.

Cells, Cultured↗
PubMed1991

Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP).

Human C3b bound to the ghost of sheep erythrocytes (E*) via activation of the alternative complement pathway (E*AC3b) consists of four major constituents on SDS-PAGE of 350, 260, 210 and 180 kDa. 350 kDa C3b is a dimeric form of C3b in which the alpha' chain of one C3b binds covalently to that of the other C3b. This complex is presumed to serve as a core for the alternative pathway C5 convertase. The other C3b populations are monomers complexed with membrane proteins or sugars. Using E*AC3b (C3b labeled) as a substrate, we have investigated functional properties of membrane cofactor protein (MCP), which is an integral membrane protein with C3b-binding and factor I-dependent cofactor activities. In conjunction with factor I, MCP was found to degrade the protein-bound C3b preferentially including the 350 kDa dimer. There was a similar but lesser tendency of this selective cleavage of C3b-dimer by CR1 but not by factor H or C4bp. In contrast to CR1 and factor H, detergent solubilization of EAC3b was required for MCP to fully express its cofactor activity for this selective degradation of C3b. We next separated the C3b dimer from the monomers and assessed their ability to assemble the alternative C5 convertase. The C3b dimer but not the monomers expressed C5 convertase activity following the addition of factors B and D, C5 and Ni2+. Kinetic analysis of the degradation of the C3b dimer by MCP and factor I suggested that only one C3b was efficiently converted to C3bi and this occurred concomitant with a decrease in C5 convertase activity. These results suggest that MCP has the ability to more efficiently interact with protein-bound C3b and that this may relate as well to its preferential ability to irreversibly inactivate the C5 convertase.

Antigens, CD↗
PubMed1985

Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator: isolation and characterization of a biologically active fragment, C3d,g.

Limited proteolysis of C3b by C3b inactivator (factor I) consists of a two-step reaction; rapid cleavage of C3b to yield a nicked C3b derivative, iC3b, and followed by slow cleavage of iC3b to yield two antigenically distinct fragments, C3c and C3d,g. Using a fluorescence-labeled C3b as a substrate for I, we have investigated in detail the optimal conditions for the sequential cleavages of C3b by I. The pH optimum for the first cleavage was markedly affected by the ionic strength of buffers. The cleavage was maximum at pH 6.0 under physiological ionic strength but at pH 8.5 under low ionic strength (such as 1.7 mS). The second cleavage was a slow reaction and occurred only under low ionic strength and within a narrow pH range around pH 6.0. One of the products of the second cleavage, C3d,g, was isolated and shown to be a single polypeptide chain of 41,000 daltons with pI 5.0. C3d,g had leucocytosis-inducing activity, like C3d-k, which is a C3d fragment released by the action of plasma kallikrein. Trypsin digestion of C3d,g produced two fragments of 30,000 and 10,000 daltons and the 10,000-dalton fragment retained the leucocytosis inducing activity.

Animals↗
PubMed2000

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi.

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.

Antigens, Bacterial↗
PubMed1985

Human genes for three complement components that regulate the activation of C3 are tightly linked.

A new cluster of complement component genes, including C4BP, C3bR, and FH, is described. Family segregation data indicate that FH is linked to the genes for C4-bp and C4bR, previously reported to be linked and to maintain linkage disequilibrium. This cluster is not linked to the major histocompatibility complex, which contains the genes for the complement components, C4, C2, and factor B, or to the C3 locus. These data further suggest that the organization of genes for functionally related proteins in clusters may be a rule for the complement system.

Carrier Proteins↗
PubMed2005

Identification and functional characterization of complement regulator-acquiring surface protein 1 of the Lyme disease spirochetes Borrelia afzelii and Borrelia garinii.

Complement regulator-acquiring surface protein 1 (CRASP-1) is the dominant factor-H-like protein 1 (FHL-1)- and factor-H-binding protein of Borrelia burgdorferi and is suggested to contribute to persistence of the pathogen. The prototype CRASP-1 of B. burgdorferi sensu stricto (CRASP-1Bb) has been formerly characterized. As shown recently, serum-resistant Borrelia afzelii strains express a unique FHL-1 and factor H-binding protein, designated CRASP-1Ba. Here, we describe for the first time the isolation and functional characterization of the gene encoding the full-length CRASP-1Ba of 28 kDa, which, upon processing, is predicted to be 26.4 kDa. CPASP-1Ba of B. afzelii spirochetes is associated with a genetic locus encoding the orthologous gbb54 gene family that maps to the linear plasmid of approximately 54 kb. Ligand affinity blotting techniques demonstrate that both native and recombinant CRASP-1Ba molecules strongly bind to FHL-1 and much more weakly to factor H. The FHL-1 and factor-H-binding site in CRASP-1Ba is shown to be localized to a 12-amino-acid residue domain at the C terminus of the protein. For comparison, the corresponding cspA-like gene(s) of a serum-sensitive Borrelia garinii strain has also been cloned and characterized. Most notably, two CRASP-1-related B. garinii proteins were identified; however, both molecules bind only weakly to FHL-1 and not at all to factor H. The present identification of the binding site of CRASP-1Ba represents an important step forward in our understanding of the pathogenesis of Lyme disease and may be helpful to design therapeutic regimens to interfere with complement evasion strategies of human pathogenic Borrelia strains.

Amino Acid Sequence↗
PubMed2005

Complement factor H as a marker for detection of bladder cancer.

BACKGROUND: The BTA TRAK and BTA stat tests for bladder cancer use monoclonal antibodies (mAbs) X13.2 and X52.1 to detect factor H (FH)-related material in urine. The exact ligands remain unknown. METHODS: Western blot analyses of purified FH, recombinant factor H-related protein 1 (FHR-1), and serum and urine samples were used to identify the ligands of X13.2 and X52.1. Recombinant FH constructs were used to identify the target sites of X13.2 and X52.1. To analyze whether natural ligands of FH could compete with its recognition by the capture mAb X52.1, we used surface plasmon resonance analysis. The role of the ligands of X52.1 in the BTA TRAK assay was tested with use of purified proteins and FH-depleted samples. RESULTS: X13.2 bound to domain 3 of FH and FH-like protein 1, whereas X52.1 bound to domain 18 of FH and to FHR-1. Using specific FH depletion from a bladder cancer patient's urine and purified FH, we demonstrated that FH is the ligand recognized by the BTA TRAK test. By contrast, FHR-1 in urine reduced the FH-dependent test signal. CONCLUSIONS: FH is a tumor marker for bladder cancer. To reveal the presence of bladder cancer, the BTA TRAK assay detects FH, whereas FHR-1 is able to partly inhibit this detection. This indicates a special mechanism for a diagnostic immunoassay based on the combined effect of simultaneous positive and negative signals in a single sample.

Antibodies, Monoclonal↗
PubMed1984

Inhibition of immune precipitation by complement.

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.

Animals↗
PubMed2001

Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation.

The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.

Animals↗
PubMed1984

Biosynthesis and postsynthetic processing of human C3b/C4b inactivator (factor I) in three hepatoma cell lines.

Human factor I is a two-chain plasma glycoprotein composed of disulfide-linked 50,000- and 38,000-dalton subunits. Analysis of its biosynthesis and postsynthetic processing demonstrated that factor I is synthesized as a single chain precursor (pro-I) that undergoes glycosylation and limited proteolysis to generate the native protein. One of three human hepatoma cell lines, HepG2 , secreted factor I predominantly (70-90%) in a single chain pro-I form. The other cell lines secrete factor I predominantly in its two chain native form. The defect in conversion of pro-I to I in HepG2 was protein specific since other multichain proteins, derived from single chain precursors, the third, fourth, and fifth components of complement were processed normally. Further analysis of the inefficient pro-I to I conversion by HepG2 revealed that Xenopus oocytes injected with HepG2 mRNA secreted factor I in a predominantly two-chain form. In addition, the apparent sizes of native factor I, transferrin, and alpha-1-antitrypsin secreted by the three hepatoma lines differed due to differences in postsynthetic processing.

Animals↗
PubMed1979

Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane.

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.

Binding Sites↗
PubMed1978

Complement C3 convertase: cell surface restriction of beta1H control and generation of restriction on neuraminidase-treated cells.

The alternative or properdin pathway of complement is primarily controlled by the endopeptidase C3b inactivator (C3bINA) and the nonproteolytic glycoprotein beta1H. The molecular mechanisms of control were investigated by performing binding studies of radiolabeled complement proteins to C3b bearing sheep erythrocytes (E(S)C3b). C3b was found to have distinct binding sites for beta1H, C3bINA, Factor B, and properdin. beta1H binding increased C3bINA binding 30-fold, while Factor B binding prevented C3bINA action on C3b and was competitive with beta1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the beta1H and C3bINA sites. Activators such as rabbit erythrocytes (E(R)) have previously been shown to interfere with the effectiveness of the control by C3bINA and beta1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of control does not occur on the surface of E(S), a nonactivator of the alternative pathway. On the basis of comparative binding studies, restriction of control is explained entirely by reduced binding of beta1H to E(R)C3b relative to E(S)C3b. Access of properdin, Factor B, C3bINA, and the Fab fragment of anti-C3 to the two cell types was unrestricted. Restriction of beta1H control could be generated on the surface of E(S) by removal of cell-surface sialic acid with neuraminidase (acylneuraminyl hydrolase; EC 3.2.1.18). This enzymatic treatment converted E(S) from a nonactivator to an activator of the alternative pathway.

Binding Sites↗
PubMed2001

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H.

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B. afzelii and serum-sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H. All serum-resistant B. afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins. The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Adsorption↗