Complement component factor B has thrombin-like activity.

Serine proteases are fundamental components of biology, including innate immunity, which is systematically orchestrated in an orderly, balanced fashion in the healthy host. Such serine proteases are found in two well-recognized pathways of an innate immune network, coagulation and complement. Both pathways, if uncontrolled due to a variety of causes, are pathogenic in numerous diseases, including coagulation disorders and infectious diseases. Previous studies have reported sequence homologies, functional similarities and interplay between these two pathways with some implications in health and disease. The current study newly reveals that complement component factor B (Bf), the second component of the alternative complement pathway, has thrombin-like activity, which is supported by a characteristic homology of the trypsin-like domain of Bf to that of thrombin. Moreover, we newly report that the trypsin-like domain of Bf is closely related to Limulus clotting factor C, the LPS sensitive clotting factor of the innate immune system. We will also discuss potential implications of our findings in diseases.

Therapeutic inhibition of the alternative complement pathway attenuates chronic EAE.

Previous studies from our laboratory using complement-mutant mice demonstrated that the alternative pathway is the dominant activation pathway responsible for complement-mediated pathology in demyelinating disease. Using a well-characterized inhibitory monoclonal antibody (mAb 1379) directed against mouse factor B, we assessed the therapeutic value of inhibiting the alternative complement pathway in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Administration of anti-factor B antibody to mice prior to the onset of clinical signs of active EAE had no affect on the onset or acute phase of disease, but significantly attenuated the chronic phase of disease resulting in reduced cellular infiltration, inflammation and demyelination in antibody-treated mice. Attenuation of the chronic phase of disease was long lasting even though antibody administration was terminated shortly after disease onset. Chronic disease was also attenuated in transferred EAE when anti-factor B antibody was administered before or after disease onset. Similar levels of disease attenuation were observed in transferred EAE using MOG-specific encephalitogenic T cells. These studies demonstrate the therapeutic potential for inhibition of factor B in the chronic phase of demyelinating disease, where treatment options are limited.

Reference typing report for complement factor B.

Factor B (BF) reference typing was carried out on the occasion of the VIIth Complement Genetics Workshop in Mainz, May 1998. Two different sets of samples were analysed by agarose electrophoresis and/or isoelectric focusing at the protein level, and by PCR-RFLP analysis at the DNA level. These results confirmed the reliability of the standard agarose electrophoresis technique for the identification of the major BF alleles as well as for the identification of cathodic and anodic variants. However, the exact alphanumeric designation of individual variants relative to the reference distance between alleles S and F1 turned out to be more difficult. Using PCR-RFLP analysis, the common alleles F and S as well as the FA and FB subtypes in 6 samples containing an F allele were all assigned correctly. However, the variants F1 and S07 were not detected by this method, as they could not be distinguished from the accompanying S allele. Therefore, a combined application of all three typing methods is recommended for a reliable identification of factor B alleles, variants and FA/FB subtypes.

Genomic analysis of the F subtypes of human complement factor B.

Factor B of human complement is encoded within the Major Histocompatibility Complex (MHC) and is polymorphic, with up to 30 alleles defined by electrophoretic mobility. One of the most common alleles, BF*F, is subdivided into the FA and FB subtypes, which differ at the gene level by non-synonymous base substitutions in the seventh codon. We have found at this position a new restriction site polymorphism, as a Bsl I site absent from the FB allele. Using this restriction polymorphism, we have developed a method for BF F subtype determination, based on amplification by polymerase chain reaction of the 5' end of the BF gene, and digestion with Bsl I. This new method has been applied to a panel of 29 selected BF F individuals. A single strand DNA conformation analysis of the same region of the gene allowed us to confirm the above DNA-based BF F subtyping. During this study, two BF*F1 alleles showed discrepancies between protein and DNA typing, which were confirmed by our sequencing data. These were identical, in the 5' region, to BF*S and BF*FB genes, respectively. In a comparison with two protein subtyping methods, identical results were found for only one third of the selected samples. The conflicting results may arise, in part, from previously undescribed molecular heterogeneity within BF F subtypes, or from the presence of a null allele. Our new method allows BF*F subtyping to be used with confidence in the definition of disease-associated MHC haplotypes.

Nomenclature for human complement factor B. WHO-IUIS Nomenclature Sub-Committee.

In this note is recommended a unified nomenclature for allotypes and variants of human complement factor B, which was approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).

Factor B (BF) nomenclature statement.

A common nomenclature for factor B (BF) allotypes is recommended as a result of the BF Reference Typing of the VIth Complement Genetics Workshop and Conference, Mainz, FRG, 1989. It has generally been agreed that the alphanumeric BF nomenclature according to Mauff et al. should also be used in the future for all major BF allotypes distinguishable by standard agarose gel electrophoresis (AGE). The common BF F subtypes and further described rarer subtype variants are not detectable by standard AGE. Therefore, the nomenclature had to be extended. For the subtypes of BF F an alphabetical designation with capital letters will now be used: FA and FB. The designation of the five rarer subtype variants was modified after the reference typing to FB1, FB2, SB1, SB2, and SB3. Hyposynthetic variants detected in samples with previously assumed non-expressed (BF*Q0) alleles are now designated as SQL, M1QL, and M2QL, HQL' characterizing their lower concentration.

Two forms of guinea pig factor B of the alternative complement pathway with different molecular weights.

Two forms of guinea pig factor B (B) of the alternative complement pathway with different mol. wts (Mr) have been isolated from plasma and characterized. The Mr of the two B species, tentatively termed B1 and B2, were estimated to be about 100,000 and 96,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Incubation of B with guinea pig C3 and human factor D (D) in the presence of Mg2+ generated two cleavage fragments of B, namely Ba and Bb. Although the Bb fragments showed the same migration corresponding to an Mr of 62,000, Ba fragments showed different mobilities corresponding to an Mr of 38,000 from B1 and 34,000 from B2. Digestion of B1-Ba, the Ba fragment derived from B1, and B2-Ba, the Ba fragment derived from B2, with endoglycosidase F resulted in a band at Mr 30,000 on an SDS-PAGE in both cases, indicating a difference in structure of the asparagine-linked oligosaccharide moiety in B1-Ba and B2-Ba. No difference in antigenicity was noted between B1 and B2 on immunodiffusion with anti-B sera. Immunoblotting analysis showed that all individual Hartley guinea pigs examined in this study possessed both B1 and B2 at similar levels, as determined by the intensity of staining of their sera. Furthermore, treatment of their serum with zymosan led to the generation of two Ba species corresponding to the Ba fragments from B1 and B2. The capacity to form C3/C5 convertase, as determined by hemolytic assay, was found to be similar between B1 and B2. Furthermore, kinetics of the decay of C3 convertase showed the same half-life of 3.0 min at 30 degrees C. The NH2-terminal amino acid sequences of B1 and B2 and their Bb fragments were determined and found to be identical.

Detection of a new BF F subtype variant by isoelectric focusing.

The paper reports a new BF F variant which was observed in a family, i.e. in the father and in 2 of the 4 children. The variant can only be seen by means of isoelectric focusing and appears as an additional cathodic band of the BF subtype FB. We suggest FB1 as a preliminary name for this variant. The family studied suggested an autosomal-codominant inheritance.

Two BF F subtypes, but no BF S, BF F1 or BF S07 subdivision, are found by isoelectric focusing.

When studied by immunoelectrophoresis the factor B (BF)*F allele is a monomorphic protein, but by using isoelectric focusing (IEF), it turns out to be polymorphic. Two BF*F allelic subdivisions, BF*FA and BF*FB, were detected in samples from unrelated Spanish donors; *FA and *FB are in Hardy-Weinberg equilibrium, segregate as autosomal codominant alleles in families, and BF*FA (but not BF*FB) is found to be in linkage disequilibrium with B44 and DR7 and is within the A29, B44, Bw4, Cx, CFA31, DR7, DRw53, DQ2 haplotype. Furthermore, BF FA individuals have a higher BF serum concentration than the BF FB individuals. The subdivision of BF S observed with IEF was found to be due to nonspecific BF S cleavage by serum proteases in inadequately collected or aged samples. Thus, the subdivision of BF S is spurious and was not found in our sera. BF F1 and BF S07 could not be further subdivided by IEF in our subjects. The BF F1 banding pattern was characterised by the presence of a cathodal band which corresponds to the Bb activation fragment. Finally, IEF combined with immunoblotting and monoclonal Ba and Bb antibodies may be used for accurately distinguishing BF phenotypes and doubtful bands.