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PubMed1989

A knowledge-based system for automatic interpretation of an analytical profile of complement factors.

A comprehensive assay to evaluate the complement system includes functional tests of both classical and alternative pathways and immunochemical measurements of C3, C4, B, C1-INA, and C3d. The purpose of this analytical profile is to screen for rare hereditary deficiencies and acquired abnormalities of complement and to define the activation pathway in cases of complement consumptive processes. Based on several years' experience, a routine was established in our laboratory to report the data to the clinician, together with a computer-generated interpretive statement. This routine was formulated into a knowledge base by specifying a series of decision rules for each of the complement disorders. After the rules were tested and updated against some 400 complement profile analyses, reasonable analytical comments were produced by the system. This knowledge-based system for reporting and interpreting complement results offers several advantages: the interpretative work is facilitated and made more reliable; a consistent interpretative comment is generated that is recognized and therefore more meaningful for the clinician; the communication of analytical procedures and policy is enhanced.

Artificial Intelligence↗
PubMed1987

Bactericidal capacity against Neisseria meningitidis of normal human serum and sera with functional deficiencies of the third and eight complement factor.

The bactericidal capacity of serum with C3 deficiency secondary to circulating C3 nephritic factor, serum with C8 deficiency and normal human serum were assessed in vitro against Neisseria meningitidis groups A-C. Normal human serum and C8-deficient serum, originating from a 28-year-old male with two episodes of meningococcal meningitis, showed significant bactericidal capacity against meningococci groups A and B. Against group C meningococci the C8-deficient serum revealed significant bactericidal capacity, whereas normal human serum showed no bactericidal effect. Serum with C3 deficiency, obtained from a 16-year-old female with two episodes of meningococaemia, showed no bactericial effect against meningococci groups A-C. Incubation of normal human serum and C8-deficient serum with isolated nephritic factor resulted in C3 depletion and abolished the bactericidal effect.

Adolescent↗
PubMed1991

Oligomeric domain structure of human complement factor H by X-ray and neutron solution scattering.

Factor H is a regulatory component of the complement system. It has a monomer Mr of 150,000. Primary structure analysis shows that the polypeptide is divided into 20 homologous regions, each 60 amino acid residues long. These are independently folding domains and are termed "short consensus repeats" (SCRs) or "complement control protein" (CCP) repeats. High-flux synchrotron X-ray and neutron scattering studies were performed in order to define its solution structure in conditions close to physiological. The Mr of factor H was determined as 250,000-320,000 to show that factor H is dimeric. This structure is maintained at concentrations between 1 and 11 mg/mL in the pH range 5-9. Zn2+ ions are an inhibitor of C3b cleavage by factor I, a reaction in which factor H acts as a cofactor. Additions of Zn2+ to factor H caused it to form oligomers containing 4-10 monomers. The radius of gyration RG of native factor H by X-rays or by neutrons in 0% or 100% 2H2O buffers is not measurable but is greater than 12.5 nm. Two cross-sectional radii of gyration RXS-1 and RXS-2 were determined as 3.0-3.1 and 1.8 nm, respectively. Analyses of the cross-sectional intensities show that factor H is composed of two distinct subunits. The RXS-1 corresponds to the cross-sectional properties of both subunits and exhibits an unusual radiation dependence on the X-ray flux. Since RXS-2 is close to the corresponding RXS of C4b binding protein (91% of which is formed from SCR/CCP domains), it is inferred that the SCR/CCP domains of factor H and C4b binding protein have similar solution structures. The use of hydrodynamic spheres to reproduce literature sedimentation coefficients of 5.5-5.6 S showed that these were compatible with a V-shaped arrangement of two rods (36 spheres each, length 87 +/- 5 nm) joined at an angle of 5 degrees. The use of a similar arrangement of 244 spheres arranged in two rods (length 77 nm) to fit the experimental X-ray and neutron scattering curves showed that the two rods are joined at an angle of 5 degrees. This model corresponds to an actual RG of 21-23 nm. The separation between each SCR/CCP in factor H is close to 4 nm. In the solution structure of factor H, the SCR/CCP domains are in a highly extended conformation.

Complement C3b↗
PubMed1990

Zinc ions inhibit factor I-mediated release of CR1-bound immune complexes and degradation of cell-bound complement factors C3b and C4b.

ZnCl2 exerted a dose-dependent inhibition of citrate-phosphate-dextrose (CPD) plasma-induced release of 125I-labelled BSA-anti-BSA immune complexes (IC) bound to complement receptor type 1 (CR1, CD35) in human whole blood. Maximal inhibition was observed at 10 mM of ZnCl2. Furthermore, the release of IC bound to erythrocyte (E)-CR1 by purified factor I, factor I-deficient serum plus purified factor I, or normal human serum was reduced by approximately 90%, 64%, and 52%, respectively, in the presence of 10 mM ZnCl2. The effect of ZnCl2 on factor I-mediated degradation of cell-bound C3b/C4b was also investigated employing CPD blood or E from a factor I-deficient donor. These cells expressed covalently bound C3b and C4b as demonstrated by a simple agglutination technique. Upon incubation of CPD whole blood with purified factor I, or of E with purified factor I or normal CPD plasma, the C-fragments were cleaved and the cells were no longer agglutinated by antibodies to C3c and C4c. The presence of ZnCl2 prevented this factor I-mediated degradation of C3b and C4b, as evidenced by the unaffected agglutination of the cells by the antibodies. We conclude that ZnCl2 inhibited factor I activity since: (1) release of complement-preopsonized IC from E-CR1 by purified factor I was markedly inhibited (90%) in the presence of ZnCl2, (2) preincubation of the cells with ZnCl2 caused only a moderate inhibition (32-38%) of the IC release, and (3) degradation by purified factor I of covalently cell-bound C3b and C4b was abrogated in the presence of 10 mM ZnCl2.

Animals↗
PubMed1988

Metabolism of complement factor D in renal failure.

Factor D is an essential enzyme of the alternative pathway of complement. Its plasma concentration increases approximately tenfold in end-stage renal failure (ESRF). To analyze its metabolism in humans, we injected purified radiolabelled factor D into 5 healthy individuals and 12 patients with various renal diseases or renal failure. Fractional metabolic rates (FMR) and extravascular/intravascular distributions (EV/IV) were calculated using a compartmental model. The FMR was very rapid in normal individuals (mean 59.6%/hr; range 74.1 to 50.5), significantly diminished in the five patients with ESRF (5.7%/hr; 7.0 to 2.8; P less than 0.004), and correlated well with the creatinine clearance (r = 0.89; P less than 0.001). The extrarenal catabolic rate was not modified in renal failure. Despite a significant inverse correlation between plasma levels of factor D and creatinine clearance [r = 0.68; P less than 0.002], factor D levels were not a sensitive indicator of renal function because the synthesis rate (SR) varied widely from one individual to another (mean SR: 62.9 micrograms/kg/hr; 14.9 to 136.5). Factor D synthesis was not significantly altered by renal function, and did not correlate with C-reactive protein, suggesting that factor D is not an acute phase protein. The proportion of intact factor D elimination in the urine was increased in patients with tubular dysfunction (up to 15% compared to less than 0.2% in normal individuals) confirming that under normal circumstances factor D is filtered through the glomerulus and catabolized by tubular cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement Activating Enzymes↗
PubMed1989

A new polymorphic variant of human complement factor I.

Sera from 305 individuals were typed for factor I, and a new variant, tentatively designated FI C, was found. All other samples were FI B except for seven samples from Chinese that were of the FI AB phenotype. This suggests that polymorphism of factor I may be rare in Caucasians.

Complement C3b Inactivator Proteins↗
PubMed1988

Sequence polymorphism of human complement factor H.

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2:1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.

Amino Acid Sequence↗
PubMed1986

Human complement factor H: isolation of cDNA clones and partial cDNA sequence of the 38-kDa tryptic fragment containing the binding site for C3b.

We isolated cDNA clones coding for the functionally important tryptic N-terminal 38-kDa fragment of human complement control protein factor H using polyclonal and monoclonal antibodies to screen a human liver cDNA library cloned in a bacterial expression vector, PEX-1. By testing the reactivity of antibodies specific for the recombinant proteins produced by individual clones with proteolytic fragments of serum H the exact position of these cDNA clones within H was mapped. One clone, H-19, coding for the 38-kDa fragment of H was sequenced and found to code for 289 amino acids derived from the 38-kDa N-terminal fragment as well as for the first 108 amino acids belonging to the complementary 142-kDa tryptic fragment. The derived protein sequence could be arranged in 6 highly homologous repeats of about 60 amino acids each, the homology between the repeats being determined by the characteristic position of cysteine, proline, glycine, tyrosine and tryptophane residues. The region coding for the epitope recognized by one of our monoclonal antibodies was localized by subcloning restriction fragments of H-19 into the expression plasmid and testing for the expression of this epitope.

Amino Acid Sequence↗
PubMed1990

Effect of N-methylation on the modulation by synthetic peptides of the activity of the complement-factor-B-derived serine proteinase CVFBb.

Although they share the active-site catalytic triad of less-specific enzymes such as trypsin and chymotrypsin, the serine proteinases of the complement and coagulation cascades each cleave a highly restricted set of substrates. Peptides with sequences similar to that at which C3 is cleaved by the alternative-pathway complement proteinase CVFBb were synthesized by solid-phase methodology and examined for their effects on the activity of this enzyme as measured by three different types of assays. It was found that a peptide methylated at the scissile bond was a far more effective inhibitor of the cleavage of the protein substrate C5 and of the lysis of guinea-pig erythrocytes by the alternative pathway than was the equivalent unmethylated peptide. Whereas the unmethylated peptide inhibited cleavage of the peptide substrate, the methylated peptide actually stimulated cleavage in this assay. This stimulation was found to be due to a 2.8-fold increase in kcat; the dissociation constant for the substrate was not altered significantly. One model consistent with this behaviour is that the binding of the activator peptide in the extended substrate-recognition region stabilizes a catalytically more active conformation of the active site. A small peptide substrate may have access to such an activated active site, whereas the larger substrate, C5, may be excluded from the site. These results demonstrate that the observed effect of a given compound on activity of an enzyme with an extended substrate-recognition region may depend upon the substrate.

Amino Acid Sequence↗
PubMed1989

Membrane-associated complement factor H on lymphoblastoid cell lines Raji expresses a co-factor activity for the factor I-mediated cleavage of C3b.

Evidence of factor H was found on the surface of Raji cells derived from two different lines; a 150,000 MW species identical to 150,000 MW plasma factor H was detected. Rosette experiments performed at physiological ionic strength using EAC3b or EC3b were negative. In contrast, Raji cells expressed a co-factor activity in the factor I-mediated cleavage of fluid-phase C3b. This activity was cell number dependent and was inhibited by anti-factor H but not by anti-CR1. Raji cells did not secrete factor H in the extracellular medium.

B-Lymphocytes↗
PubMed1988

Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents.

The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with [35S]-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression.

Adjuvants, Immunologic↗
PubMed1990

Hereditary subtypic patterns detected in the Ba fragment of complement factor B: occurrence of four common alleles in Japanese.

A procedure which can detect subtype-specific minor bands of factor B (BF) by polyacrylamide gel isoelectric focusing is presented. After zymosan-mediated fragmentation of BF in serum via alternative pathway for complement activation, serum samples are subjected to isoelectric focusing in a narrow pH range (4.2-4.9). The Ba fragments are detected by using immunoblotting. In addition to the previously reported minor bands with subtypic specificities, heterogeneities are observed in other minor band group, where a single minor band corresponds exclusively to a subtype in a regular combination with the previously announced subtypic patterns. A one-to-one correspondence of a single band to each subtype provides an unambiguous determination for three subtypic phenotypes deduced from the two divided BF*F alleles, BF*FA and BF*FB. An autosomal codominant heredity is confirmed through segregation analysis. A population survey reveals that four common alleles, BF*S, BF*FA, BF*FB, BF*Fb1, occur in a Japanese population and the former three alleles, except BF*Fb1, occur in a Cambodian population. The presence or absence of a single anodal minor band was found to be the only difference after neuraminidase treatment of FA and FB, implying that an amino acid substitution responsible for the FA-FB subtypic difference is involved in an additional acquisition in FA of an oligosaccharide unit with a charged sialic acid.

Alleles↗
PubMed1988

Structure of the murine complement factor H gene.

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.

Animals↗
PubMed1992

cis and trans elements differ among mouse strains with high and low extrahepatic complement factor B gene expression.

Factor B (Bf), an enzyme of the alternative pathway of complement activation, is one of four major histocompatibility complex (MHC) class III genes. To ascertain the genetic mechanism for tissue-specific constitutive and regulated expression of Bf, we sequenced the regulatory regions 5' of the gene from mice of different H-2 MHC haplotypes and assessed trans-acting factors, specific DNA binding nucleoproteins, in liver and kidney. Striking tissue-specific differences in constitutive expression of Bf were demonstrated in mice of H-2f or H-2z haplotypes when compared with H-2d or H-2u (kidney and intestinal Bf in H-2d or H-2u much greater than H-2f or H-2z). These differences correlated with a point nucleotide substitution 3 bp downstream of the upstream Bf initiation site that affects interaction with a DNA binding protein. This and additional cis differences localize the sequence substitutions responsible for previously identified restriction fragment length polymorphisms among inbred mouse strains and also reveal two previously unrecognized polymorphisms generated by SmaI and HinfI digestion. Evidence for differences in trans was found in a comparison of DNA binding nucleoproteins from kidney, but not liver, of B10.PL when compared with B10.M. These data, together with the high degree of sequence homology between human and mouse Bf 5' flanking regions, should prompt a search for polymorphic restriction sites and cis binding elements in the Bf promoter that could serve as markers of human MHC-associated renal pathology and variants in local MHC class III gene expression.

Animals↗
PubMed1987

Complement factor H-binding protein of Raji cells and tonsil B lymphocytes.

Several reports have indicated that Factor H has specific effects on certain cell populations, suggesting that Factor H receptors may exist. Lambris & Ross [(1982) J. Exp. Med. 155, 1400-1411] purified a protein from Raji B-lymphoblastoid cell culture supernatants, using Factor H-Sepharose affinity chromatography. This species appeared to consist of two disulphide-linked components each of Mr 50,000, with an additional 50,000-Mr chain attached non-covalently. The existence of cell-surface Factor H-binding proteins has now been re-investigated with 125I surface-labelled Raji and tonsil B cells. Non-ionic-detergent extracts of the cells, in 0.1% Nonidet P40/10 mM-sodium phosphate buffer, pH 7.4, were incubated with Factor H-Sepharose in the presence of proteinase inhibitors. After the beads had been washed, bound components were eluted with 50 mM-NaCl. A single radioactive species was eluted from the resin, which migrates identically with Factor H (apparent Mr 170,000) in SDS/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. Biosynthetic radiolabelling studies confirmed that this species was synthesized by Raji cells. Examination of culture supernatants from biosynthetically radiolabelled Raji cells showed again the presence of a single soluble species that bound to Factor H-Sepharose, but this species was of lower Mr (approx. 105,000) than the membrane-derived protein. The soluble form may be produced by proteolysis of the membrane form, or may be of separate origin. The similarity in size of the cell-surface protein to Factor H was initially confusing, but it is distinct from cell-surface Factor H on the basis of three criteria: (1) it is not recognized by anti-(Factor H) monoclonal antibodies MRC OX23 and MRC OX24, nor by polyclonal F(ab')2 anti-(Factor H); (2) it does not bind to Zn2+-chelate resin, whereas Factor H does; (3) cell-surface Factor H present on U937 cells does not bind to Factor H-Sepharose.

B-Lymphocytes↗
PubMed1985

A quantitative method for assessing the third complement factor (C3) attached to the surface of opsonized Pseudomonas aeruginosa: interrelationship between C3 fixation, phagocytosis and complement consumption.

A direct enzyme-linked immunoassay is described for the determination of C3 that becomes attached to the surface of bacteria upon incubation in serum. The assay uses horseradish peroxidase-conjugated rabbit antiserum specific for human C3. In this study various Pseudomonas aeruginosa strains, Escherichia coli and Staphylococcus aureus were opsonized under different conditions and the amount of C3 fixed to the bacterial surface was measured directly. The extent of C3 fixation was compared with the percentage of phagocytosis by human polymorphonuclear leukocytes and with the complement consumption in the fluid phase. It was demonstrated that C3 fixation closely correlated with the percentage of phagocytosis but not with complement consumption. The method can be performed with relatively simple equipment and could be used in routine laboratories in order to determine the extent of opsonization of Pseudomonas aeruginosa and other bacteria and to detect opsonic defects in the sera of patients.

Complement Activation↗