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PubMed2003

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.

Amino Acid Sequence↗
PubMed1994

Analysis of the functional domains of complement receptor type 1 (C3b/C4b receptor; CD35) by substitution mutagenesis.

The complement receptor type 1 (CR1; CD35), carrying 30 short consensus repeats (SCRs), has two sites. Site 1 contains SCR-1 and SCR-2 and binds C4b. Site 2 contains SCR-8 and SCR-9 and was reported to bind mainly C3b (Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A., and Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717). For the functional analysis we used two constructs, each with one site. CR1-4, composed of eight and one-half initial SCRs, carries site 1, binds C4b, and is cofactor for C4b cleavage. CR1-4(8,9), obtained from CR1-4 by converting site 1 to site 2, binds iC3/C3b and, unexpectedly, C4b. It is a cofactor for cleavage of both ligands. Its cofactor activity for C4b cleavage is greater than that of site 1. Analysis of the mutants constructed by interchanging homologous peptides between the two sites identified no sequences necessary for cofactor activity other than those required for binding. In site 2, peptides important for both ligands were found. Some modifications of either site led to higher activity for both ligands. Thus the activity of complement regulators can be increased by changing a few amino acids within SCRs, an important step toward the generation of more effective inhibitors of complement activation. Knowledge of the active sites of CR1 should be applicable to other SCR-containing proteins and should provide insights into the evolution of these proteins.

Amino Acid Sequence↗
PubMed1988

Molecular heterogeneity of complement component C4-null and 21-hydroxylase genes in systemic lupus erythematosus.

C4A-null alleles (C4A*Q0) and hereditary complete C4 deficiency (homozygous C4A*Q0,C4B*Q0) are associated with systemic lupus erythematosus (SLE). Using Southern blot analysis with C4 and 21-hydroxylase (21-OH) DNA probes, we studied SLE patients and normal control subjects with or without C4A*Q0, and 2 C4-deficient SLE patients. A previously reported large C4A,21-OHA gene deletion associated in normal subjects with the HLA-A1;B8;DR3;C4AQ0 haplotype was detected by the appearance of a new C4 Hind III 8.5-kb fragment and disappearance of a 3.2-kb 21-OH Taq I fragment. In 3 SLE patients with homozygous C4A*Q0 and 15 with heterozygous C4A*Q0, this deletion pattern occurred almost exclusively in association with the HLA-B8;DR3;C4A*Q0 phenotype; the one exception was a black SLE patient. Other C4A*Q0-bearing HLA phenotypes in white patients and black patients with SLE, and the 2 completely C4-deficient SLE patients, had normal DNA hybridization to both C4 and 21-OH probes. The genetic basis for C4-null alleles in SLE is heterogeneous. A large C4A,21-OHA deletion occurs mainly on the HLA-B8;DR3;C4AQ0 haplotype in SLE and controls. Other HLA haplotypes bearing C4A*Q0 have normal C4 and 21-OH genes, as demonstrated by Southern blot analysis.

Alleles↗
PubMed1995

Length polymorphism of the human complement component C4 gene is due to an ancient retroviral integration.

The fourth component of the complement system, C4, is encoded by two highly homologous MHC-linked genes expressing the two isotypes C4A and C4B. A gene size polymorphism (either 22.5 or 16 kb) has been described which depends on the presence or absence of a 6.5-kb insertion in intron 9 of the C4 gene. By sequencing a C4A-specific lambda clone from a human genomic library containing the long intron 9 as well as PCR-amplified DNA containing the short intron, the DNA sequences of both introns were determined. The long and short introns have lengths of 6,787 bp and 415 bp, respectively. The sequence of the short intron is almost identical (96%) to the corresponding parts of the long intron. At position 282 of the short intron, a 6,372-bp insertion is present in the long intron which has all characteristics of a full-length endogenous retrovirus. The proviral DNA is flanked by two 6-bp target site repeats. The orientation of the proviral sequence is opposite to that of the C4 coding strand. Long terminal repeats (LTRs) of 548 bp were found at both ends of the provirus. A TATA box and an SV40 enhancer core as well as a polyadenylation signal are present in the LTR. A 5' primer binding site for lysine tRNA was identified. The strongest sequence homologies were found in comparison to human endogenous retrovirus (HERV-K): between 65-88% for gag, pol and env genes. However, a search for open reading frames in these regions indicated the presence of multiple stop codons in all three reading frames.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1982

Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

Antigen-Antibody Complex↗
PubMedDate not supplied

Assembly and regulation of the complement amplification loop in blood: the role of C3b-C3b-IgG complexes.

Amplification of complement activation in blood and serum starts on multi-protein complexes that act as precursors of an alternative C3 convertase. Among these covalently linked C4b-, C3b-, and IgG-containing complexes C3b-C3b-IgG complexes represent the major species containing C3b and IgG. Recent work on their purification and characterization is discussed. Special emphasis is placed on the arrangement of ester bonds in these complexes and their dual type of partial protection from inactivation. Partial protection from inactivation is mediated by properdin which binds to these complexes in the complete absence of any other complement protein. High dose IgG, known to stimulate inactivation of these complexes, appears to lower properdin binding in a process that also involves factor H. Properdin stimulates factor B binding to these complexes and renders them far better precursors of a C3 convertase than C3b. The available information allows a suggestion for a new scheme on how the amplification loop is assembled and regulated in blood and serum.

Antibody Formation↗
PubMed1984

MHC Class III products: an electron microscopic study of the C3 convertases of human complement.

We have reported a transmission electron microscopic study of the two C3 convertases of human complement and their precursors. The corresponding proteins and complexes of the classical and alternative pathway appear very similar. Cofactors C3b and C4b are nearly indistinguishable and display a characteristic but highly irregular substructure. C2 and Factor B are globular with diameters of 85 +/- 8 A and 80 +/- 8 A and both consist of three discrete globular domains each approximately 40 A in diameter. Bb and C2a each contain two domains connected by a short linker segment. Both domains of Bb and one domain of C2a are 42 A in diameter (28 kd), while the second domain of C2 is 47 A in diameter (39 kd). Attachment of the enzymatic subunits to cofactors occurs through one domain only.

Complement Activating Enzymes↗
PubMed2007

Molecular interactions between MASP-2, C4, and C2 and their activation fragments leading to complement activation via the lectin pathway.

Activation of component C3 is central to the pathways of complement and leads directly to neutralization of pathogens and stimulation of adaptive immune responses. The convertases that catalyze this reaction assemble from fragments of complement components via multistep reactions. In the lectin pathway, mannose-binding lectin (MBL) and ficolins bind to pathogens and activate MBL-associated serine protease-2 (MASP-2). MASP-2 cleaves C4 releasing C4a and generating C4b, which attaches covalently to the pathogen surface upon exposure of its reactive thioester. C2 binds to C4b and is also cleaved by MASP-2 to form the C3 convertase (C4b2a). To understand how this complex process is coordinated, we have analyzed the interactions between MASP-2, C4, C2, and their activation fragments and have compared MASP-2-catalyzed cleavage of C4b2 and C2. The data show that C2 binds tightly to C4b but not to C4, implying that C4 and C2 do not circulate as preformed complexes but that C2 is recruited only after prior activation of C4. Following cleavage of C4, C4b still binds to MASP-2 (KD approximately 0.6 microM) and dissociates relatively slowly (koff approximately 0.06 s-1) compared with the half-life of the thioester (<or=0.7 s) (Sepp, A., Dodds, A. W., Anderson, M. J., Campbell, R. D., Willis, A. C., and Law, S. K. (1993) Protein Sci. 2, 706-716). We propose that the C4b.MASP-2 interaction favors attachment of C4b near to the activating MBL.MASP complex on the bacterial surface so that, following recruitment of C2, the proximity of enzyme and substrate (C4b2) combined with more favorable reaction kinetics drive the formation of the C3 convertase, promoting complement activation.

Animals↗
PubMedDate not supplied

High variability of CYP21 gene rearrangements in Spanish patients with classic form of congenital adrenal hyperplasia.

Mutations that cause deficiency of the 21-hydroxylase (21-OH) appear as a result of recombinations between the CYP21B coding gene and the highly homologous CYP21A pseudogene, which are tandemly arranged with the C4A and C4B genes. We report a detailed analysis of a major chromosomic rearrangement by Southern blot using 21-OH and complement C4 cDNA probes, in a wide sample of classic Spanish congenital adrenal hyperplasia (CAH) patients. This study made it possible to observe that 50% of the patients carried at least one allele with gross abnormalities and that the frequencies of alleles with large deletions (16.66%) and gene conversions (14.16%) in the CYP21B gene were very similar. Moreover, our analysis revealed the existence of sixteen different restriction patterns of C4/CYP21 genes. Besides the detection of a new haplotype, which does not seem to appear from unequal crossing-over mechanisms, we observed the highest frequency on CYP21A duplications reported, as well as no duplications of the CYP21B gene. We also observed that although gross abnormalities of the CYP21A pseudogene did not affect 21-OH activity, alleles carrying deleterious point mutations had more rearrangements of the CYP21A gene than normal alleles. Even though the 21-OH deficiency is an autosomic trait, boys in our sample carried 2.6 times more deletions than girls. In contrast, conversion alleles were found equally frequently.

Adrenal Hyperplasia, Congenital↗
PubMed1988

Heterogeneity in the gene locus for steroid 21-hydroxylase deficiency.

DNA was analysed from 33 patients with congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. In each case Southern blots were prepared from a number of restriction enzyme digests and hybridised with probes for both the 21-hydroxylase and the adjacent fourth component of complement (C4). Evidence for deletion of the active 21-hydroxylase gene (CYP21B) was found in 13 cases and in 10 of these the deletion included the adjacent C4B gene, leading to a hybrid CYP21A/CYP21B gene. Deletion of CYP21B alone was found in one patient, the remaining two cases appearing to have the active gene replaced by the inactive pseudogene. Duplications of the CYP21A-C4B region and deletion of the pseudogene are also described. In a further 12 cases no gross abnormality could be found.

Adrenal Hyperplasia, Congenital↗
PubMed1990

The complement component C4 of mammals.

Human complement component C4 is coded by tandem genes located in the HLA class III region. The products of the two genes, C4A and C4B, are different in their activity. This difference is due to a degree of 'substrate' specificity in the covalent binding reactions of the two isotypes. Mouse also has a duplicated locus, but only one gene produces active C4, while the other codes for the closely related sex-limited protein (Slp). In order to gain some insight into the evolutionary history of the duplicated C4 locus, we have purified C4 from a number of other mammalian species, and tested their binding specificities. Like man, chimpanzee and rhesus monkey appear to produce two C4 types with reactivities similar to C4A and C4B. Rat, guinea pig, whale, rabbit, dog and pig each expresses C4 with a single binding specificity, which is C4B-like. Sheep and cattle express two C4 types, one C4B-like, the other C4A-like, in their binding properties. These results suggest that more than one locus may be present in these species. If this is so, then the duplication of the C4 locus is either very ancient, having occurred before the divergence of the modern mammals, or there have been three separate duplication events in the lines leading to the primates, rodents and ungulates.

Animals↗
PubMed1988

Influence of C4 null genes on infection with human immunodeficiency virus.

The hypothesis that complement is important in the host response to human immunodeficiency virus (HIV) was tested. Complement C4 and Bf allotypes were determined in 26 patients who fulfilled the diagnostic criteria for persistent generalised lymphadenopathy due to HIV, 72 homosexuals who were negative for antibody to HIV, and 185 control subjects drawn from the local population. HLA-A, B, and DR were also typed and the phenotypes examined for the presence of supratypes and C4BQ0. Eleven patients (42%) had C4B null alleles compared with only 13 (18%) homosexuals who were negative for antibody and 28 (15%) controls. From estimates of gene frequencies the difference between the patients with lymphadenopathy and the controls was significant after conservative correction. In the patients only a minority (six) of the C4B null alleles were contained within ancestral haplotypes. Together with the fact that C4 null alleles result in partial deficiency of C4, this finding suggests that products of complement genes are important in infection with HIV or its consequences, or both. A role is proposed for complement and Fc receptors.

Acquired Immunodeficiency Syndrome↗
PubMed1983

Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S.

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.

Amino Acids↗
PubMedDate not supplied

Glycemic control and coagulation inhibitors in diabetic patients.

OBJECTIVE: To investigate the plasma antigenic levels and functional activities of coagulation inhibitors in poorly controlled diabetic patients and the possible effect of good glycemic control on these parameters. RESEARCH DESIGN AND METHODS: Both functional activities and plasma antigenic levels of coagulation inhibitors (antithrombin III, heparin cofactor II, protein C, and protein S) and plasma levels of C4b-binding protein were measured in 28 diabetic patients (13 males, 15 females; 2 IDDM, 26 NIDDM; median age 56.5 years; median duration of diabetes 5.5 years) with poor glycemic control (median HbA(1c) 11.8%). Twenty-three healthy subjects were enrolled as controls. Following a 3-month intensification of antihyperglycemic therapy, good glycemic control (HbA(1c) <8%) was achieved in 17 patients, and the plasma levels of the same parameters during this period were compared with baseline values. RESULTS: Functional activities and plasma antigenic levels of coagulation inhibitors were comparable in poorly controlled diabetic patients and healthy subjects. In patients achieving good control after 3 months, there was a significant reduction in plasma antigenic levels of protein S (p = 0.005) and C4b-binding protein (p = 0.03); however, no difference could be observed in other parameters. HbA(1c) did not show any correlation with plasma antigenic levels or functional activities of coagulation inhibitors either at baseline or at 3 months of good glycemic control. CONCLUSIONS: Our findings suggest that in poorly controlled diabetic patients, coagulation inhibitors are not different from healthy controls. Short-term good glycemic control may not exert a profound effect on coagulation inhibitors except protein S and its binding protein, C4b-binding protein.

Antithrombin III↗
PubMed1991

Clinical manifestations in humans of combined C7 and C4 deficiency associated with low levels of C2, C8, and C9.

64 members of a large kindred with inherited deficiency of the seventh component of complement, C7, were studied for plasma levels of antigenetic and functional components of complement as well as for clinical manifestations of infections and autoimmune diseases. Thirty-six individuals showed a low level of C2, C7, C8, and/or C9, including null alleles for C4A and C4B. Two subjects had a complete C7 deficiency. One of them concomitantly presented a low C2 level and a C4BQ0 allele. HLA allotyping strongly suggested C2 depression associated with a C4BQ0 allele. The 2 individuals with total absence of C7 suffered from fulminant disseminated meningococcal infections. The partial depression of one or more complement components associated with apparent good health. These results may indicate that simultaneous partial depressions of up to four complement components do not lead to clinical manifestation of infectious and autoimmune disease.

Adult↗
PubMed2003

Effect of hybrid complement regulatory proteins on xenogeneic cells.

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.

Animals↗
PubMedDate not supplied

[Membrane proteins as regulators of the complement system].

The data on structure, biochemical properties and functions of membrane proteins, performing cell defence against complement lysis, are summarized. Proteins DAE (decay accelerating factor) and CR1 (complement receptor of type 1) reduce the stability of complement convertases and cause their dissociation. MCP (membrane cofactor protein) and CR1 act as cofactors. In factor I mediated proteolysis of the convertase fragments C3b and C4b. The proteins C8bp-(C8-binding protein) and protectin affect membrane attack complex assembly. In contrast to MCP and CR1, which are integrative proteins, DAF, C8bp and protectin are bound to membranes with their glycophospholypid anchors. Tissue distribution of the proteins and the ways of their solubilization into biological fluids are reviewed.

Cell Membrane↗