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PubMed1989

Hereditary, complete deficiency of complement factor H associated with recurrent meningococcal disease.

Complement factor H (beta-1H globulin) is an important regulatory protein which inhibits the spontaneous complement activation via the alternative pathway. We describe a 15-year-old girl without any detectable factor H in plasma. She has had two episodes of meningococcal disease, but is otherwise completely healthy. Secondary to the factor-H deficiency, the levels of factor B, properdin, C3, and C5-C9 were strongly reduced due to spontaneous in vivo activation of the alternative complement pathway. Plasma C3dg was strongly elevated in spite of the factor-H deficiency; apparently erythrocyte CR1 substitutes for factor H in C3 degradation. Neither C3 nor complement lesions were demonstrable on her erythrocytes which did, however, show increased, spontaneous haemolysis in vitro in citrate plasma, but not in serum. The patient is a single child and her parents, who are unrelated and healthy, had half-normal levels of factor H. This reduction of factor H is sufficient to cause increased, spontaneous activation of the alternative pathway.

Antibodies, Bacterial↗

PubMed1983

Comparison of binding characteristics of factors B and H to C3b on normal and paroxysmal nocturnal hemoglobinuria erythrocytes.

To determine if aberrant interactions of the endogenous control proteins with cell-bound C3b contribute to the greater fixation of C3b to paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes when whole serum complement is activated, we compared the characteristics of binding of factors B and H to normal and PNH red cells bearing C3b (EC3b). Factor B binding is homogeneous, there is 1:1 stoichiometry, and the affinity constant at equilibrium for factor B binding is the same for normal and PNH EC3b. In contrast, analysis by Scatchard's method of factor H binding results in a curvilinear plot, the deviation from linearity being exaggerated for the PNH EC3b. The heterogeneity of binding of factor H appears to be a consequence of the nonrandom distribution of C3b about the alternative pathway convertase site. This nonrandom distribution does not induce negative cooperativity but rather effects a biophysical milieu which enhances factor H binding. The greater heterogeneity of binding of factor H to PNH E bearing nonrandomly distributed C3b appears to be due to the presence of a greater proportion of lower affinity binding sites on the PNH EC3b. However, it appears unlikely that this greater heterogeneity of factor H binding contributes to the enhanced fixation of C3b to PNH erythrocytes.

Binding, Competitive↗

PubMed1991

Cloning of the 1.4-kb mRNA species of human complement factor H reveals a novel member of the short consensus repeat family related to the carboxy terminal of the classical 150-kDa molecule.

Three factor H mRNA species of 4.3 kb, 1.8 kb, and 1.4 kb are constitutively expressed in human liver. Having previously characterized full-length cDNA clones derived from the 4.3-kb and 1.8-kb factor mRNA, we report here the isolation and eucaryotic expression of full-length cDNA clones coding for the 1.4-kb mRNA species. The 1266-bp cDNA codes for a polypeptide of 330 amino acids and contains two polyadenylation signals and a short poly(A)+tail. The protein is composed of a leader peptide followed by five short consensus repeat domains. It shows a hybrid structure with the last three domains being almost identical to the carboxy-terminal of the classical 150-kDa factor H molecule and the two first domains representing unique short consensus repeat structures. Eucaryotic expression in COS7 cells revealed two polypeptides derived from one cDNA clone that are also found in human serum. Differences between the cDNA clones within the last three domains indicate two distinct, possibly allelic sequences that, in addition, differ from the authentic 150-kDa factor H sequence. Southern blot results support the notion that the 4.3-kb factor H and the 1.4-kb factor H-related mRNA are transcribed from two separate but highly homologous genes.

Amino Acid Sequence↗

PubMed1991

Human complement factor H: two factor H proteins are derived from alternatively spliced transcripts.

The human complement factor H is an important component in the control of the alternative pathway of complement activation. We have previously shown that a least three factor H homologous mRNA species of 4.3 kb, 1.8 kb and 1.4 kb in length are constitutively expressed in human liver. In addition, several factor H-related proteins have been detected in human sera using antibodies directed against the classical human factor H glycoprotein of 150 kDa. The structure of the additional polypeptides has not been shown so far. Circumstantial evidence suggests that the 1.8-kb mRNA might encode the 43-kDa factor H-like polypeptide. Here we report the isolation, characterization and eukaryotic expression of the first full-length cDNA representing the major 4.3-kb mRNA and the 1.8-kb mRNA of human factor H. We show that the 4.3-kb transcript encodes the 150-kDa-factor H glycoprotein and the 1.8-kb mRNA the 43-kDa factor H polypeptide. The identity of the two cDNA in a region of 1400 nucleotides suggests that the two factor H-related transcripts are derived from one gene by a process of alternative splicing.

Amino Acid Sequence↗

PubMed1984

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb.

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.

Antibodies, Monoclonal↗

PubMed1987

C3b generation is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from salmonellae.

Salmonellae differing in the O-antigen side chain of their lipopolysaccharide were previously shown to activate the alternative pathway of complement to different extents. We now examine the generation of the major cleavage fragment of the complement component C3 (C3b) on these bacteria in a system that contains the purified components C3, B, D, and P but lacks the regulatory proteins H and I. The deposition of C3b in this system reproduces the same pattern obtained earlier with the use of whole serum, with the expected differences among the strains bearing different O-antigen. However, two distinct mechanisms for these differences in C3b generation became apparent. The intermediate activating strain showed 3 to 4 times less initial deposition of C3b than the other two strains. In contrast, the least activating strain showed adequate initial deposition but poor amplification, as shown by 2 to 3.4 lower amplification indexes as compared with those on the other two strains. Binding studies with factor B showed that decreased C3 convertase formation was responsible for the low amplification on this strain. Only 25% of the C3b bound to its surface was able to bind factor B with a high affinity, in comparison with 90% on the other two strains. No differences were found for the binding of factor H among the strains. These studies identify the molecular mechanisms by which these bacteria avoid complement activation.

Antigens, Bacterial↗

PubMed1985

Expression of complement factor H on the cell surface of the human monocytic cell line U937.

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.

Antibodies, Monoclonal↗

PubMed1984

Factor I deficiency and C3 nephritic factor: immunochemical findings and association with Neisseria meningitidis infection in two patients.

The complement system was examined in two patients with systemic Neisseria meningitidis infections, both of whom had reduced or nondetectable CH50 as analysed by both pathways. C3 measured by conventional technique revealed 19% anti-C3c-reactive protein in the plasma of patient 1 and 3% in patient 2. Patient 1 had circulating C3b but no detectable C3c, C3d, or C4d, whereas patient 2 had normal levels of C3c and C4d and strongly elevated levels of C3d. Factor B analysis revealed no demonstrable native factor B and small amounts of Bb in patient 1 and normal concentration of native factor B plus trace amounts of Bb in patient 2. The depletion of C3 in both patients was due to uncontrolled activation caused by complete factor I deficiency (patient 1) and circulating C3 nephritic factor (patient 2). Both parents of patient 1 had factor I concentrations below (mean-2 SD) that seen in normal healthy individuals (n = 20). Circulating immune complexes (IC) were demonstrated in patient 1 only, whereas serum from both patients had strongly reduced capacity to solubilize preformed IC.

Adolescent↗

PubMed1988

Complement activation in sickle cell disease: a liposome model.

Patients with sickle cell disease (SCD) have poorly defined abnormalities of their alternative complement pathway (ACP). We have previously shown chronic activation of the ACP in these patients. To determine the mechanism of this finding, we studied concentrations of the complement control proteins factors I and H in serum from patients with SCD and found no significant difference when they were compared with a control population. Because certain membrane surfaces promote ACP activation and changes occur in erythrocyte membrane phospholipid organization with sickling, we used a liposome model to determine whether ACP activation could be caused by abnormal phospholipid organization of sickle cells. Liposomes with the composition of the sickle cell outer leaflet, which is enriched in phosphatidylserine and phosphatidylethanolamine, activated the ACP significantly more than liposomes with normal outer leaflet phospholipid content. Similarly, liposomes with the composition of the erythrocyte inner membrane leaflet, containing large amounts of phosphatidylethanolamine and phosphatidylserine, activated the ACP more than liposomes with the phospholipid content of the outer leaflet. These findings suggest that phospholipid composition of membranes may play a role in their ability to promote ACP activation, and that changes in phospholipid organization in sickle cells may contribute to the chronic ACP activation observed in patients with SCD.

Anemia, Sickle Cell↗

PubMed1987

Regulation of the activity of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H.

The alternative pathway of complement is regulated on the surface of homologous blood cells at the C3 amplification step by the membrane protein decay-accelerating factor, as well as by the plasma protein factor H. We have reported elsewhere that platelets from patients with paroxysmal nocturnal hemoglobinuria regulate the activity of the C3 convertase C3bBb, even though they lack decay-accelerating factor. We now report that normal human platelets contain factor H, which was released from the platelet in response to complement deposition or thrombin stimulation. Factor H was localized to the platelet alpha granules by immunocytochemical techniques. As determined by a solid-phase radioimmunoassay, thrombin-stimulated platelets released approximately equal to 54 ng of factor H per 10(8) platelets. The release of factor H in response to complement or thrombin was inhibited by treating the platelets with metabolic inhibitors. Such inhibition resulted in a 3-fold increase in the activity of C3bBb. Platelets that released factor H bound only half as many molecules of radiolabeled factor B to platelet-bound C3b than platelets that could not release factor H. Treatment of platelets with anti-decay-accelerating factor antibody had no effect on the activity of C3bBb unless the release of factor H was blocked. Therefore, so far as we know, human platelets have a unique mechanism for the regulation of the alternative pathway of complement.

Complement Activating Enzymes↗

PubMed1983

Monoclonal antibodies against the complement control protein factor H (beta 1 H).

Two mouse monoclonal antibodies against the human complement control protein, Factor H (beta 1H), are described. The antibodies are both IgG - gamma 1 - subclass and are directed against different epitopes on the human Factor H molecule. One of the antibodies, MRC OX 24, increases the cofactor activity of Factor H in Factor I-mediated cleavage of soluble C3b. The second antibody, MRC OX 23, which has no effect alone, reduces the increase in cofactor activity observed in the presence of the first antibody. However, MRC OX 24 inhibits the binding of 125I-labelled Factor H to surface-bound C3b (EAC3b). Again MRC OX 23 alone does not have an effect but decreases the inhibition in 125I-labelled Factor H binding to EAC3b observed with MRC OX 24. These studies show clearly that the interaction of Factor H with soluble C3b is different to its interaction with surface-bound C3b. In an indirect immunoprecipitation system using these monoclonal antibodies, single-chain molecules of 150 000 mol.wt. are specifically precipitated from human serum and also from the sera of other primates - rhesus monkey, cynomolgus monkey, and African green monkey. There was no precipitation from sera of cow, pig, sheep, chick, or rabbit. Using a radioimmunoassay with radiolabelled monoclonal MRC OX 23, the concentration of Factor H in human plasma was determined.

Animals↗

PubMed1990

Factor H.

While the mouse and human H proteins are structurally and functionally similar, they differ in their genetics. Whereas there is no evidence in humans for more than one gene; in mice the H locus is complex. Based on cDNA sequence and hybridization analysis of genomic cosmid clones, there are at least three distinct genes, all highly related to one another. The consensus repeating unit that comprises this molecule has obviously been duplicated numerous times, since it is present in many other molecules. Thus, it is not surprising to discover that there are several genes related to H in the mouse. A similar case has been described for two other members of this family. In humans, CR1 cDNA hybridizes to two distinct genomic clusters in the CR1 locus (Wong et al. 1989), and in mice, mCRY hybridizes to two regions in the genome, one on chromosome 1 and another on chromosome 8 (Aegerter-Shaw et al. 1987). It will be of interest to see if any other members of this family display as complex a genetic locus as murine H.

Animals↗

PubMed1989

Truncated forms of human complement factor H.

By the use of Western-blot analyses with polyclonal anti-(Factor H) antibodies, two low-Mr protein species of Mr 41,000 and 37,000 under non-reducing conditions and 43,000 and 40,000 under reducing conditions are consistently detected together with the well-known 155,000-Mr Factor H in human plasma and serum. These two additional species are also found in plasma, urine and synovial fluids. The 41,000-Mr species but not the 37,00-Mr species is detected by a monoclonal anti-(Factor H) antibody directed at the N-terminal part of Factor H. The 37,000-Mr species but not the 41,000-Mr species is detected by a monoclonal anti-(Factor H) antibody directed at the C-terminal part of Factor H. The 41,000-Mr and 37,000-Mr species are different from the well-characterized 36,000-Mr N-terminal tryptic fragment of Factor H. They are likely to represent translational products of the short Factor H mRNA species of 1.8 kb and 1.2-1.5 kb occurring in human liver that we have recently described.

Antibodies, Monoclonal↗

PubMed1990

Complement alternative pathway activation and control on membranes of human lymphoid B cell lines.

Membrane regulatory molecules normally prevent complement activation by autologous cells, therefore we compared the membrane control system of human lymphoid cell lines which activate or not human complement through the alternative pathway (AP). Membrane expression of decay-accelerating factor (DAF), membrane cofactor protein (MCP), complement receptors (CR)1, CR2 and H was measured either by radioimmunoassay or enzyme-linked immunosorbent assay on cell lysates. Soluble extracts of isolated membranes were tested functionally for their ability to accelerate the decay of C3bBb C3-convertase and allow the cleavage of C3b by factor I. Both regulatory functions were detected in solubilized membranes of Ramos cells, which do not activate the AP, as well as on the potent AP activator, Raji. Raji cells were found to express CR2, DAF and MCP molecules, while MCP was the only known regulatory protein detected on Ramos cells which expressed neither CR1, nor CR2, H or DAF. The I-cofactor activity of both Raji and Ramos cells was immunoprecipitated by anti-MCP, but the decay-accelerating activity was not adsorbed by anti-DAF nor by any of the available antibodies. Two EBV genome-negative cell lines (BJAB, BL41) were tested before and after in vitro conversion by EBV. As previously shown, EBV-converted cell lines activate the AP more efficiently than EBV- cell lines. At the same time, EBV superinfection induces an increase of both AP regulatory functions of cell membranes and enhances the expression of DAF, MCP and CR2. The results of this study show that complement activation by lymphoid cell lines is not related to an impaired autologous control of these cells, but that the expression of regulatory molecules increases together with the appearance of activating structures on the cell surface. Our results also suggest the occurrence of a new factor involved in the decay-accelerating activity on BL lines.

Antigens, CD↗

PubMed1982

Isolation, characterization, and mechanism of action of rat beta 1H.

beta 1Hrat was purified to homogeneity from fresh rat plasma by precipitation with 28.6% ammonium sulfate followed by sequential chromatography on DEAE-Sephacel, Biorex-70, and gel filtration on Sephacryl-S300. The final material was homogeneous on SDS-PAGE analysis and had an apparent m.w. of 150,000. Reduction with dithiothreitol did not affect its m.w., suggesting that the molecule is composed of one polypeptide chain. The recovery of beta 1H was approximately 10%. A monospecific antibody against beta 1Hrat was obtained from immunized rabbits, which recognized beta 1Hrat as a protein with beta-electrophoretic mobility upon immunoelectrophoresis of fresh rat plasma. The concentration of beta 1H in plasma of normal 4-mo-old Wistar rats was 243.5 +/- 36.3 micrograms/ml (mean +/- S.D.). beta 1Hrat in this study was detected by its capacity to inhibit formation of the P-stabilized cell-bound amplification C3/C5 convertase composed of cell-bound C3bhu and Bbhu. Purified beta 1Hrat produced a dose-related, first-order loss of convertase function and release of 126I-Bbhu from the P-stabilized C3bhuBbhu convertase, indicating a mechanism of action by decay-dissociation of Bbhu from the complex C3bhuBbhuP. beta 1Hrat was at least four times less effective than beta 1Hhu in release of 125I-Bbhu from the homologous convertase composed of C3bhu and Bbhu. On the other hand beta 1H was twice as effective in releasing 125I-Bbrat from the convertase composed of C3bratBbratP when compared to beta 1Hhu. These differences are presumably dependent upon the species-specific affinity of beta 1H from humans or rats for C3bhu or C3brat, respectively.

Animals↗

PubMed1983

The alternative complement pathway control protein H binds to immune complexes and serves their detection.

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125I-H; when fresh serum was chelated with 10 mM EDTA, 125I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.

Antigen-Antibody Complex↗

PubMed1987

Complement activation in patients with amebic liver abscess.

Serum or plasma concentrations of components of the classical (C1q, C4) and alternative (C3, factor B) pathways, regulatory protein factor H, and one of the C3 products of degradation, C3d, were determined in 19 patients with amebic liver abscess (ALA). Patients were divided into two groups. Thirteen patients that recovered under medical treatment who had a significantly shorter clinical course on admission (P less than 0.05) (group 1) exhibited either normal (C1q; C4; factor B; C3d) or increased levels of these components (C3, P less than 0.001; factor B, P less than 0.01). On the other hand, 16 patients that recovered after medical treatment and abscess drainage (group 2) exhibited significantly diminished serum levels of C1q (P less than 0.05), C3 (P less than 0.001), factor B (P less than 0.01) and factor H (P less than 0.05), and normal levels of C4, and C3d as compared to the control group. The relationships among the complement components studied were suggestive of activation of the complement system through the classical pathway in patients within group 1 and through both pathways in group 2. Sera of 3 out of the 5 patients who initially exhibited low plasma levels of C3d showed an increase during convalescence. Plasma levels of C3d were demonstrated to show a direct correlation with serum albumin and SGOT in this group of patients. Possible implications of the complement system in the immunopathogenesis of ALA are discussed.

Complement Activating Enzymes↗

PubMedDate not supplied

Phylogeny of the third component of complement, C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in the C3 from different species.

The third component of complement, C3, binds to several other complement proteins. To study the diverse reactivities of C3, we analyzed the conservation of structural and functional features in the C3 from different species. First, we developed a method to purify swine (Po), rabbit (Rb), mouse (Mo), cobra (Co), Xenopus (Xe), axolotl (Ax), and trout (Tr) C3 from plasma. This involved protein precipitation by polyethylene glycol, followed by anion-exchange, gel filtration, and cation exchange chromatography. All C3's tested were comprised of two chains (alpha/beta-chain) and contain a thiolester bond within the alpha-chain. The two N-linked high-mannose carbohydrates found in human C3 were only conserved (as detected by ConA binding) in Rb C3. In contrast, Xe, Ax, and Tr C3 have this moiety only in the beta-chain and Po and Mo C3 only in the alpha-chain. Co C3, in contrast to cobra venom factor (CVF), lacks ConA binding carbohydrates in both chains. N-terminal amino acid sequence analysis of the alpha-, alpha'-, and beta-chains showed varying degrees of similarity within the different C3's. The N-termini of the Xe and Ax C3 beta-chains were found to be blocked. The conservation of binding sites in the different C3's for human complement receptors type one (CR1) and two (CR2) and for factors H and B was investigated due to the structural and functional similarities of these molecules and to the ability of some of them to bind to the same domain(s) in human C3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗