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At least 19 recordsLinked to original sources
PubMed1989

Common binding region of complement factors B, H and CR1 on C3b revealed by monoclonal anti-C3d.

Fab fragments of a murine C3d-specific monoclonal antibody, clone 4C2, strongly inhibited the binding of H, and up to about 80% that of B, to C3b on sheep erythrocytes, as shown by using radiolabelled purified proteins. The inhibition was also detected in the fluid phase. Conversion of C3 by preformed solid phase alternative pathway C3 convertase was not affected by 4C2. Also, it did not affect the decay rate of the convertase, but reduced the decay-accelerating effect of H on it. The results suggest that the binding sites of B and H on C3b are close to each other, and their competition for binding to C3b is focused around the C3d region. In addition, 4C2 partially blocked noncovalent binding of C3b to human erythrocytes, which suggests that CR1 also binds close to the H- and B-binding region in C3b.

Antibodies, Monoclonal↗
PubMed2001

Characterization of IFN-gamma regulation of the complement factor B gene in macrophages.

Complement factor B (Bf) is involved in the activation of the alternative complement cascade. Bf is induced by IFN-gamma; however, the mechanisms of Bf gene regulation have not been well characterized in general, and not in macrophages specifically. Northern analysis reveals that IFN-gamma induces a dose- and time-dependent increase in Bf mRNA expression in primary macrophages and macrophage cell lines. MH-S cells transfected with reporter constructs containing truncated regions of the Bf promoter reveal that IFN-gamma responsiveness lies between -154 and -53 bp on the Bf promoter. This region of the Bf promoter contains both an IFN-gamma-activation site (GAS) and an interferon-stimulated response element (ISRE). Site-directed mutagenesis of the GAS binding site or the ISRE binding site in this region of the Bf promoter partially inhibits IFN-gamma responsiveness. Mutagenesis of both the GAS and ISRE cis elements totally abrogates IFN-gamma responsiveness of the Bf promoter. Electrophoretic mobility shift assays reveal nuclear binding complexes involving both Bf-GAS and Bf-ISRE oligonucleotide sequences upon IFN-gamma stimulation. In competition assays, both Bf-GAS and Bf-ISRE oligonucleotides, but not mutant Bf-GAS nor mutant Bf-ISRE oligonucleotides, compete for the DNA binding. Supershift analysis reveals that Stat1-GAS and IRF-1-ISRE nuclear binding complexes contribute to induction of Bf by IFN-gamma. Western analysis confirms an IFN-gamma-stimulated increase in tyrosine phosphorylation of Stat1. These findings suggest that both GAS and ISRE cis binding sites have an additive effect on IFN-gamma-stimulated Bf gene expression and that both are required for full expression of Bf by IFN-gamma. Stat1 and IRF-1 take part in IFN-gamma-stimulated Bf gene induction in macrophages through their respective cis binding elements.

Animals↗
PubMed1989

Regulation of human and murine complement: comparison of 5' structural and functional elements regulating human and murine complement factor B gene expression.

The serine protease complement factor B (Bf), an acute phase plasma protein, is a component of the alternative pathway of complement activation. Previous studies revealed that several cytokines including IFN-gamma and IL-1 are involved in mediating acute phase Bf expression. To determine the molecular details of Bf expression we isolated, sequenced and characterized the 5' flanking regions of the human and murine Bf genes. In both species the Bf transcriptional start site in liver was located less than 400 bp 3' to the polyadenylation site of the upstream C2 gene. This upstream intergenic region contained greater than 65% nucleotide homology between species. Within this region, an IRS and three heat shock consensus elements were found in the murine sequence in an identical position to that of the human. To examine the functional details of Bf expression, a series of mouse and human Bf promoter-chloramphenicol acetyltransferase (CAT) chimeric gene constructs were transfected into mouse L or human HepG2 cells. Analysis of expression of these fusion gene constructs revealed that 1) cis-acting DNA sequences identified, at least in part, in the 3' untranslated region of the C2 gene (within the 400 bp upstream of the Bf cap site) mediate responsiveness to IL-1 and IFN-gamma, 2) the responsiveness to each mediator appears to be conferred by separate upstream regions similar in position and homologous in man and mouse, and 3) the IL-1 responsive region in both species appears to have the characteristics of an enhancer element. The results of this analysis suggest a selective pressure to conserve the intergenic sequence between C2 and Bf genes and that further studies of these sequences will be useful in elucidating mechanisms controlling the acute phase response.

Animals↗
PubMed2000

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding.

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.

Amino Acid Sequence↗
PubMed2004

Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation.

Complement factor B is a 90 kDa protein consisting of three domains: a three-module complement control protein, a von Willebrand factor A domain, and a C-terminal serine protease (SP) domain that adopts a default inactive (zymogen) conformation. The interaction between factor B and pathogen-bound C3b is mediated by its A domain, triggering a conformational change in factor B that ultimately creates the "C3 convertase" of the alternative complement pathway. We report the crystal structure of the A domain from factor B and show that it contains an integrin-like MIDAS motif that adopts the "open" conformation typical of integrin-ligand complexes, with an acidic residue (provided by a fortuitous crystal contact) completing the coordination of the metal ion. Modeling studies indicate that the factor B A domain can also adopt the closed conformation, supporting the hypothesis that an "integrin-like switch" is conserved in complement proteins and perhaps in 60 other A domains found within the human proteome.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of the porcine complement factor B.

A genomic clone, SSBf1, containing the complement factor B (BF), a major histocompatibility class III antigen, has been isolated from a porcine genomic library. Partial sequencing and comparison with a human BF gene has identified seven exons coding for amino acids of Ba and Bb, the two subunits of BF. The protein sequence similarity with the human BF is on the average 87%. Southern blot analysis confirmed the existence of only one BF gene per haploid genome. Restriction fragment length polymorphism typing with Taq I showed that there are at least three different porcine BF-haplotypes.

Amino Acid Sequence↗
PubMed1989

Rapid determination of the human complement factor B phenotypes.

A rapid phenotyping method for human complement factor B (Bf) was worked out using high voltage acetate electrophoresis. The results were compared with those obtained by conventional agarose gel electrophoresis. Separation of the bands was performed in the same way, but only 2.5 hours were required for the evaluation of 24 samples. Furthermore, at least ten times less specific antibody was needed than for the conventional technique.

Complement Factor B↗
PubMed1984

Complete primary structure for the zymogen of human complement factor B.

The entire amino acid sequence of complement factor B has been established combining both protein and DNA sequencing strategies. The zymogen consists of 739 amino acids, has four asparagine-linked carbohydrate sites, and has independently disulfide-bonded NH2- and COOH-terminal regions. The catalytic subunit, Bb, is a unique serine protease containing 259 amino acids that are not integral to any of the classical serine proteases. It is proposed that this region of the Bb fragment functions as a cofactor-binding domain for C3b. The Ba fragment was found to contain three regions of internal sequence homology which were unrelated to the "kringle" regions of prothrombin and plasminogen and which suggest an independent evolution for the B genome. Sequence alignment of the active site of B to the serine proteases was made using the three-dimensional structures of chymotrypsin and trypsin as molecular models. Three stretches within the hypothetical model for B contrast markedly with all known serine proteases in both amino acid sequences and predicted configuration. It is suggested that these "altered" regions contribute at least in part to the formation of the catalytic region of the C3 convertase.

Amino Acid Sequence↗
PubMed1985

A DNA restriction fragment length polymorphism in the complement region of the human MHC shows an absolute correlation with polymorphism of complement factor B(Bf) defined by isoelectric focusing.

The gene coding for properdin factor Bf is located in the human major histocompatibility complex and is closely linked to the genes coding for the complement components C2 and C4. Recently, by Southern blotting techniques, a restriction fragment length polymorphism was identified using the endonuclease Taq I, which subdivides haplotypes carrying the F allele of factor Bf. The F allotype has also been subdivided at the protein level by isoelectric focusing into two subtypes Fa and Fb. We have investigated the DNA of 41 healthy unrelated individuals with known BfF subtypes using the 2.3 kb factor Bf cDNA probe to determine if there is any correlation between the Taq I polymorphism and F subtype. We have found that in 23 individuals who carried the Fb subtype a 6.6 kb Taq I fragment was present. The remaining 18 individuals carried the Fa subtype and showed only the 4.5 kb Taq I fragment on Southern blotting (P = 10(-12). This striking correlation (r = 1) between the Fb protein and DNA polymorphism is surprising especially as the 4.5 kb and 6.6 kb Taq I fragments overlap the Bf and C2 genes and the polymorphic Taq I site is located within the C2 gene.

Alleles↗
PubMed1990

A new complement factor B variant (BF S075) in Japanese.

A new slow-moving variant of the complement factor B, named BF S075, was found in a Japanese patient with cerebral thrombosis and urticaria. The variant was inherited in a codominant manner. The protein concentration and functional hemolytic activity of the complement factor B in the patient's serum were within normal limits. The BF S075 is the fourth rare BF variant found in the Japanese population.

Complement Factor B↗
PubMed1992

Properdin factor B and complement factor C4 allotypes in rheumatoid arthritis: results of a follow-up study.

The association between allotypes of properdin factor B (Bf), the fourth component of complement (C4A and C4B), and rheumatoid arthritis (RA), was investigated in a well-characterized cohort of RA patients who were followed from an early phase of the disease for a mean duration of 6 years. The frequencies of probable heterozygous C4AQ0 and of C4A3 were lower in RA patients compared to controls, irrespective of the presence of DR4 [relative risk (RR): 0.52 and 0.49, respectively, 95% confidence intervals (95% CI): 0.34-0.80 and 0.29-0.82]. The frequency of C4A4 was higher in RA patients compared to controls (RR: 1.86, 95% CI: 1.03-3.35), especially in DR4 positive RA patients compared to DR4 positive controls (RR: 2.58, 95% CI: 1.07-6.25), indicating a positive association of this allotype with RA additional to DR4. Bf and C4B allotypes were comparable in RA patients and controls. We did not find significant differences in Bf and C4 allotype frequencies in RA patients subdivided according to severity of the disease into a mild group and a progressive group. Because of inconsistent results in all studies on Bf and C4 allotypes, we conclude that C4 and Bf allotypes do not seem to have an important independent effect on determining disease susceptibility.

Adult↗
PubMed1993

Human complement factor B: functional properties of a recombinant zymogen of the alternative activation pathway convertase.

The human complement factor B is a centrally important component of the alternative pathway activation of the complement system. Here we report the isolation, characterization and eukaryotic expression of the first full length cDNA transcript for human factor B. In a factor B dependent haemolysis assay, the recombinant human factor B generated by transient COS cell transfection was shown to reconstitute haemolytic activity of factor B depleted human serum. To study the biological activities assigned to factor B, the availability of recombinant polypeptides representing definite portions of the human factor B molecule is desirable.

Animals↗
PubMed2026

A First-in-Japanese Phase 1, Double-Blind, Placebo-Controlled, Parallel-Cohort Study of Sefaxersen, an Antisense Oligonucleotide Targeting Complement Factor B, in Healthy Participants.

Increased activity in the complement alternative pathway (AP) plays a key role in diseases such as IgA nephropathy (IgAN). This first-in-Japanese double-blind Phase 1 study investigated the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of sefaxersen (RO7434656), an antisense oligonucleotide targeting complement factor B messenger RNA. Healthy participants were randomized equally into four cohorts: placebo or sefaxersen 20, 40, or 70 mg. The PK, PD, and safety endpoints were monitored throughout the study and during the 90-day follow-up period. All 24 participants completed the study, with no new safety signals or clinically meaningful changes in blood chemistry, electrocardiogram, or vital signs observed. Plasma sefaxersen concentration demonstrated a biphasic PK profile, characterized by an initial rapid decline followed by a slow elimination. Sefaxersen decreased PD markers related to the complement AP selectively, without affecting the classical pathway, in a dose-dependent manner, and the PD effects persisted over 2 to 3 months. Sefaxersen was well tolerated by healthy Japanese participants, with a manageable safety profile. These findings support the inclusion of Japanese patients with IgAN in the global Phase 3 study (IMAGINATION, NCT05797610).

Humans↗
PubMed2002

Complement factor B and the alternative pathway of complement activation in bovine milk.

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2.06 microg/ml (+/- 0.18, SEM), 0.57% of the mean value found in serum (360 microg/ml). This proportion was comparable to that of serum albumin (0.63% of serum value) but less than the proportion of C3 in milk (2.71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 degrees C for 3 min or 50 degrees C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 degrees C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.

Animals↗
PubMed1988

Molecular basis for the microheterogeneity of human complement factor B.

The involvement of sialic acids in the microheterogeneity of human complement factor B was investigated. Desialylation kinetics revealed all the charge intermediates from a complex native to a homogeneous form. The relation between this heterogeneity and posttranslational events was explored in cultured hepatoma cells. Intracellular factor B exhibited the same isoelectric focusing pattern as the desialylated purified protein, whereas a highly heterogeneous form was secreted. In contrast, when N-glycosylation was prevented by tunicamycin, both intracellular and secreted forms focused like intracellular factor B from control cultures. These data lead to the conclusion that the microheterogeneity of human factor B results from different degrees of sialylation of its N-glycans.

Carcinoma, Hepatocellular↗
PubMed1990

Fragment Bb of bovine complement factor B: stimulatory effect on the microbicidal activity of bovine monocytes.

We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.

Animals↗
PubMed1994

Molecular cloning of a lamprey homologue of the mammalian MHC class III gene, complement factor B.

To elucidate the origin and evolution of the complement system and the MHC, we isolated cDNA clones for the MHC class III complement factor B (Bf) gene from lamprey, one of the most primitive extant vertebrates. A part of the serine protease domain of the lamprey Bf was amplified by reverse transcriptase-PCR using the degenerated primers corresponding to the conserved amino acid stretches between the mouse Bf and C2 sequences. A full-length lamprey Bf cDNA clone was isolated from the lamprey liver cDNA library using a PCR-amplified DNA clone as a probe. The deduced amino acid sequence of 763 residues showed essentially the same domain structure as mammalian Bf or C2, consisting of three short consensus repeat domains, a von Willebrand domain, and a serine protease domain. Lamprey Bf showed 33 and 29% overall amino acid similarity to mouse Bf and mouse C2, respectively, whereas amino acid similarity between mouse Bf and mouse C2 was 36%, suggesting that the gene duplication of Bf/C2 occurred in the main line of vertebrate evolution after the divergence of cyclostomes. This is the first report of the molecular cloning from cyclostomes of a component of the mammalian MHC that offers the possibility of genetic analysis of the presumably primitive MHC of cyclostomes.

Amino Acid Sequence↗
PubMed2002

Complement factor B gene regulation: synergistic effects of TNF-alpha and IFN-gamma in macrophages.

Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-alpha and IFN-gamma, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-alpha and IFN-gamma in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-alpha and IFN-gamma in MH-S cells. Truncations of the 5' Bf promoter identified a region between -556 and -282 bp that mediated TNF-alpha responsiveness as well as the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. Site-directed mutagenesis of a NF-kappaB-binding element in this region (-433 to -423 bp) abrogated TNF-alpha responsiveness and decreased the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. EMSAs revealed nuclear protein binding to this NF-kappaB cis-binding element on TNF-alpha stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-alpha. An I-kappaB dominant negative mutant blocked Bf induction by TNF-alpha and reduced the synergistic induction by TNF-alpha and IFN-gamma. In addition, the proteasome inhibitor MG132, which blocks NF-kappaB induction, blocked TNF-alpha-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-alpha and IFN-gamma. LPS was found to induce Bf promoter activity through the same NF-kappaB cis-binding site. These findings suggest that a NF-kappaB cis-binding site between -433 and -423 bp is required for TNF-alpha responsiveness and for TNF-alpha- and IFN-gamma-stimulated synergistic responsiveness of the Bf gene.

5' Untranslated Regions↗