Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes

After separating the *F and *S alleles by electrophoresis the allele-specific hemolytic activity was detected by agarose overlay method using the programmable densitometer for scanning. The hemolytic activity of BF allotypes was analyzed from 81 individuals. In thirteen FS heterozygous serum samples BF F had lower hemolysis than BF S. Four FF homozygous samples also exhibited lower hemolysis than a homozygous control sample. The low hemolytic activity of F in FS heterozygotes was not due to decreased protein concentrations relative to S. On the contrary, BF F was associated with higher protein concentration than BF S. The relative quantitation of the allele specific BF protein was done by crossed immunoelectrophoresis. BF F with low hemolytic activity but with high protein concentration associated strongly with HLA B35 phenotype and the family material confirmed the association with the haplotypes A3, Cw4, B35, DR1, BFFB, C4A3BQO (or A2BQO, A3,2BQO). The results suggest that particular MHC haplotypes contain a factor B allele with encoding for poor hemolytic activity or that MHC haplotype specific regulatory elements affect pre- or post-translational activity levels.     

There are two C4 genetic loci and a null allele in the chimpanzee

Genetic polymorphism in C4 in the chimpanzee was studied by agarose gel electrophoresis of desialated plasma and development of patterns by immunofixation with antiserum to human C4 and by a C4-sensitive hemolytic overlay. In general, immunofixation patterns showed multiple partially overlapping bands of which only the most cathodal had strong hemolytic activity. In analogy to human C4, the latter were designated C4B, whereas those detected by immunofixation which had little hemolytic activity were designated C4A. Chimp C4A and C4B reacted with human and mouse (monoclonal) anti-C4B and human anti-Ch1 but neither reacted with monoclonal anti-C4A or human anti-Ch2, Ch3, Rg1, or Rg2. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the alpha chain of C4B showed a slightly lower apparent relative mass than that of C4A at around M _r 93 000. There were three C4A variants and two C4B variants inherited in families as autosomal codominant traits, as C4A-C4B cosegregating pairs with no detectable crossing-over. These pairs were inherited with chimpanzee leukocyte antigen types C2 and BF variants without detectable crossing-over. Half-null C4 haplotypes with C4B ^*Q0 were observed in family studies. Nine BF, C2, C4A, C4B allelic haplotypic combinations (complotypes) were identified among presumably unrelated chimpanzees.Abbreviations used in this paper: ChLA chimpanzee leukocyte antigen - HLA human leukocyte antigen - EDTA ethylenediaminetetraacetate     

Apparently non-expressed alleles of factor B (BF) code for hypomorphic proteins

In three families with an apparent non-expressed factor B (BF) allele (BF ^* Q0), advanced methods of isoelectric focusing for the determination of BF F subtypes revealed different hypomorphic BF products (BF QL) with functional hemolytic activity expressed by the assumed BF ^* Q0 allele. A Taq I and a Msp I restriction fragment length polymorphism as well as the Ba fragment of the expression products showed banding patters for the BF ^* QL alleles corresponding to BF S types, whereas an altered Bb fragment was seen in two BF QL products. In one family an intragenic recombination site within the Bb part of the BF gene was assumed. Investigations of factor B and its conversion fragments, as demonstrated by the used methods, allow to complement molecular genetic investigations of BF ^* Q0 alleles in heterozygous genotypes on a protein level. We conclude that apparently non-expressed alleles of factor B code for hypomorphic but functionally active proteins. Correspondence to : I. Siemens.     

Human C4 polymorphism: Pedigree analysis of qualitative,quantiative, and functional parameters as a basis for phenotype interpretations

Summary Ten families with 82 members were investigated for C4A- and B polymorphism in a blind trial. Phenotyping was done on neuraminidase treated sera by immunofixation and simulataneously by hemolytic overlay electrophoresis. In addition Rg, Ch, BF, C2, HLA-A, B, C, DR, and GLO were determined. After decoding the samples the reliability of blind typing was found to be 84.4% according to segregation patters. Inconsistencies occurred mostly when A 4, A 2, or A 92 were present. The detection of silent A*Q0 and B*Q0 alleles was more critical than that of difficult allotypes. The quantitation of the C4A/B ratio by densitometry of stained gels or by conventional immunochemical measurements of serum C4 level could not substantially improve the identification of A*Q0 or B*Q0. C4 dependent activity in radial diffusion hemolysis showed satisfactory correspondence with the number of expressed C4B alleles. At least three haplotypes with two C4A genes (duplicated A genes) were observed as ascertained from offspring analysis in accordance with the MHC segregation pattern. Individuals with the duplicated C4A gene (C4A*3. A*2. in the absence of any other expressed A allele or together with C4A*92) showed only partial inhibition of Rodgers antisera. Partial inhibition of Chido antisera was seen in individuals with C4B 2 (in the absence of other B allotypes). The findings support the hypothesis of at least two structural C4 loci. The also demonstrate the inconsistency of quantitative data in the recognition of silent alleles.     

Immunofixation for C2 typing: C2 allotypes in Spaniards in relation to HLA,Bf and C4

Summary C2 typing is performed by immunofixation with anti-C2 antiserum instead of by a hemolytic overlay. This method gives sharp band definition, is less cumbersome than the hemolytic overlay, gel files are easily made, and it also enables one to describe putative new nonhemolytic variants. C2 allele frequencies were studied in a sample of the normal Spanish population and were found to be similar to other Caucasoids. HLA-Bw62,-Cw3, and-DR4 were significantly associated with C2 B. Concordantly, the only C2^*B extended HLA haplotype found in family material was Bw62-Cw3-Bw6-(DR4)-Bf^*S-C2^*B-C4A^*3 B^*2-(GLO^*1). C4A^*4 B^*2 and C4A^*4 B^*4 are not found within the same haplotype together with C2^*B and Bw62 or Bw22 respectively, nor do other C2^*B haplotypes occur with common HLA-B alleles. These results may favour the hypothesis that the Bw62-C2^*B haplotype is produced by one mutation arising in the Bw62-C2^*C haplotype and that subsequent crossovers can explain other C2^*B haplotypes (including Bw22-C2^*B).     

Structural and functional studies in C1q deficiency

The sera of two brothers were found totally lacking hemolytic C activity. One of them, a 16-yr-old male, presented a severe lupus-like syndrome, whereas the other was apparently healthy. Immunochemical quantitation of C components in both sera showed depressed levels of C1q, whereas the levels of C1r, C1s, and C1 inhibitor were elevated. C4, C3, C5, factor B, and beta 1H levels were in the normal range. Hemolytic C1 activity was totally lacking. C4 titers were elevated (150% of normal). C2 hemolytic activity was about one-third of normal, and the titers of the terminal components C3-C9 were also reduced in the two siblings. Double immunodiffusion against anti-C1q antiserum showed a partial loss of C1q antigenic determinants in the two siblings. Furthermore, the C1q of both siblings was unable to interact with immunoglobulins or to associate with C1r and C1s. Addition of purified human C1q to the sera restored their total C and C1 hemolytic activity. The dose response to the C1q addition was linear, indicating that the functional deficiency was not due to the presence of a serum inhibitor. Although antigenically deficient in comparison with normal C1q, the abnormal C1q appeared to have a larger m.w., as determined by gel chromatography. Investigation of other members of this family suggests a genetically linked disorder, because four out of six siblings had the same dysfunctional C1q in their serum.     

An unusual "morphologic" variant of BF S

In the course of family studies of haplotypes of the alleles of the sixth chromosome loci HLA-A, C, B, D/DR, BF, C2, C4A, C4B, and glyoxalase I, we encountered an unusual BF variant. Its mobility was similar to BF F but it appeared to have a lesser intensity after straining with Coomassie Blue, and it was demonstrated by crossed immunoelectrophoresis to be present in lower concentration. It was therefore designated BF FQL. This variant was found on the haplotype HLA-A1, B17, DR7, BF*FQL, C2*C, C4A*6, C4B*1, GLO2. All other haplotypes of this type so far identified carry the BF variant BF S. Following activation of serum samples with zymosan, BF was analyzed by both agarose electrophoresis and isoelectric focusing and immunofixation. On both treatments, serum with BF SFQL produced a Ba pattern identical to that of a sample which was BF S. The Bb pattern for F and S are similar but differ from those of the rare variants BF F1 and BF S1. The Bb pattern of BF FQL was, thus, as expected, the same as BF F or BF S. Hence, we conclude that the variant is a mutant from BF S with mobility similar to BF F. The mutation seemed also to have resulted in a lower concentration of product than normal.     

Further study on a BF silent allele

Summary A family was found which indicated the existence of a silent allele (BF ^* QO) at the locus for complement factor B. Three generations with eight members were studied. Four individuals were considered to be heterozygous for B deficiency because of unusual segregation patters of the BF electrophoretic variants and low levels of B. Haplotype study on the other HLA-linked markers supported the presumption. No unusual products were detected by immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

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.     

A silent gene (C3-) producing partial deficiency of the third component of human complement

A family is described with 3 members in 3 generations being heterozygous for the silent gene C3-: one girl, her mother and grandfather had half normal C3 levels and were apparently incompatible homozygous. No significant deviation in the total hemolytic complement activity, serum concentration of Bf and C4 component was found in the affected individuals.     

Human BF*F-subtypes: segregation analysis with inclusion of MHC haplotypes

Summary The segregation of factor B(BF)F subtypes was analyzed in conjunction with other MHC markers in 15 families with 89 offspring. Informative data for BF F subtypes were obtained from 11 families, 6 of them with known recombinant individuals for the HLA-B/DR/GLO region. The subtypes did not contribute further to the localization of the cross-overs, but followed the known segregation of conventional BF allotypes. In 2 families of one kinship, the recognition of heterozygous BF^*FAFB individuals could be established following the inclusion of three generations. The rarer of the two BF F subtype alleles, BF^*FA, is positively associated with the HLA haplotypes BW62, CW3, C4A^*3 and A29, CWX, B44, C4A^*3, B^*1, DR7. BF F subtypes are regarded as a very useful additional tool for studies of MHC organization and disease association.     

Characterization of pneumolysin from Streptococcus pneumoniae,interacting with carbohydrate moiety and cholesterol as a component of cell membrane

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MβCD, which is in a 3β configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1–3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins.     

BF types and the mode of inheritance of insulin-dependent diabetes mellitus (IDDM)

Insulin-dependent diabetes mellitus (IDDM) has been found to be highly associated with a rare allele of the complement protein, properdin factor B (BF). Assuming that there is a susceptibility gene for IDDM tightly linked to the genetic locus forBF and the major histocompatibility complex (MHC), the distribution of BF types in more than 1100 North American IDDM patients strongly argues for the rejection of dominant, epistatic, and overdominant modes of inheritance. Other evidence suggesting complex modes of inheritance for IDDM is reviewed and it is concluded that our observations and published data are consistent with the idea of susceptibility to IDDM being inherited as a simple autosomal recessive trait. — C4 and C2 types, also linked toBF and theMHC, were investigated too. C4 Fs⁰ was found to be increased in association with BF F1, while C4 f⁰S and C2 B were each found to occur twice as frequently as in a control population and will be of value in defining haplotypes associated with susceptibility to IDDM.     

A single arginine to tryptophan interchange at beta-chain residue 458 of human complement component C4 accounts for the defect in classical pathway C5 convertase activity of allotype C4A6. Implications for the location of a C5 binding site in C4.

In general, C4A allotypes of human C4 show one-fourth to one-third the hemolytic activity of C4B allotypes. An exception to this rule is C4A6 which is almost totally deficient in hemolytic activity. Previous studies have localized the defect in C4A6 to the C5 convertase stage. Of the two critical events required for C5 cleavage, namely formation of a covalent adduct between C3b and the C4b subunit of the C3 convertase (C4b2a), and binding of C5 to this C4b-C3b complex, it is a defect in the latter step that accounts for the aberrant activity of C4A6. DNA sequencing studies described in a companion paper have suggested that the sole C4A6-specific difference was a Trp for Arg replacement at beta-chain residue 458. To directly ascertain whether this single substitution was responsible for the hemolytic defect in C4A6, we have used site-directed mutagenesis to introduce this change into both C4A and C4B cDNA expression plasmids. We found that the R to W replacement totally abrogated hemolytic activity. However, irrespective of the amino acid at residue 458, the mutant proteins behaved like their wild-type counterparts with respect to covalent binding to C1-bearing targets, i.e., the C4B recombinants displayed higher binding to sheep and human red cells than did the C4A counterparts. Furthermore, the mutants were able to form covalent C4b-C3b adducts. There was, however, substantially less C5 cleavage produced by cell-bound C4boxy23b complexes made with R458W mutant C4B than with wild-type C4B. These results are consistent with the sole defect in the mutants being at the C5 binding stage and strongly suggest that Arg 458 of the C4 beta-chain contributes to the C5 binding site of the molecule.     

Chironex fleckeri (Box Jellyfish) Venom Proteins: EXPANSION OF A CNIDARIAN TOXIN FAMILY THAT ELICITS VARIABLE CYTOLYTIC AND CARDIOVASCULAR EFFECTS*

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg⁻¹) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU₅₀ = 5 ng ml⁻¹) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.     

Nonlinkage between C6 and chromosome 6 markers

Summary C6 typing was performed in a family material by two different techniques: serum or plasma samples were subjected either to high-voltage agarose gel electrophoresis or to isoelectric focusing in polyacrylamide gel slabs. Proteins with C6 activity were then visualized by a specific, hemolytic assay.In 81 unrelated adults within the family material the following allele frequencies were found: C6 ^A:0.61 and C6 ^B:0.39.Linkage studies exclude linkage between C6 and HLA region marker loci, and also between C6 and another chromosome 6 marker locus PGM3.     

Structural and functional changes in complement protein C4 under UV irradiation

The influence of ultraviolet (UV) light on the structural and functional states of the complement factor C4 was investigated using hemolytic and acid-base titration, PAG electrophoresis, and IR and UV spectrophotometry. UV doses of 75.5 and 755 J/m² initiated C4 activation through changes in the globule structure (increased number of aromatic amino acids and ionogenic groups at the surface). The maximal dose of 2265 J/m² has a destructive effect and decreases its C4 activity in the cascade of hemolytic reactions of the complement system.     

Genetic polymorphism of the second component of human complement (C2)

Summary Proteins were separated by prolonged isoelectric focusing in polyacrylamide gels, whereupon C2 bands were detected by a specific hemolytic assay. This was performed by treating the gel with iodine to increase C2 activity, and then developing C2 bands with an agarose gel overlay containing sensitized sheep cells and diluted human serum as a complement source deficient in functional C2. The gene frequencies observed in a material of 122 unrelated adults were: C2¹:0.97 and C2²:0.03.C2 linkage relations and C2 haplotype associations have been examined a family material. It is concluded that C2 is very closely linked to HLA loci.     

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.