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PubMed1992

Comparison of antithrombin III, protein C and protein S levels in capillary and venous blood of newborn infants.

The plasma concentrations of antithrombin III, protein C and protein S in capillary and venous blood samples obtained simultaneously from 30 neonates were compared in order to determine the suitability of using capillary blood for estimation of these proteins with anticoagulant action. Our findings showed that while capillary and venous blood did not differ significantly in antithrombin III functional activity and protein C antigen levels, the capillary samples had significantly lower protein C functional activity and higher antithrombin III antigen level. Protein S antigen level was also significantly higher in the capillary samples although the difference was relatively small. The capillary and venous concentrations of the binding protein of protein S, C4b binding protein, were almost identical.

Antigens↗
PubMed1985

Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3.

Human C3d (try-C3d), prepared from trypsin-digested C3, was fragmented by cleavage with CNBr. Eight peptides were defined and separated by h.p.l.c. on reversed-phase columns. By automatic Edman degradation the complete sequences of five peptides and partial sequences of three peptides were determined. To obtain overlapping peptides the latter three fragments were digested with trypsin, chymotrypsin or Staphylococcus aureus V8 proteinase, after which the fragments were separated on reversed-phase columns. Two of the CNBr-cleavage peptides were completely sequenced, and 70% of the sequence of the remaining CNBr-cleavage peptide was determined. The non-sequenced part represents a very hydrophobic segment of try-C3d. The sequence data obtained represent 90% of the primary structure of try-C3d. Alignment of the CNBr-cleavage fragments was made easier by comparison with the cDNA sequence of mouse pro-C3 [Wetsel, Lundwall, Davidson, Gibson, Tack & Fey (1984) J. Biol. Chem. 259, 13857-13862]. Comparison of try-C3d with the equivalent part of human C4B revealed an extensive sequence homology in the N-terminal half of the molecules.

Amino Acid Sequence↗
PubMed1996

Visual demonstration of the organization of the human complement C4 and 21-hydroxylase genes by high-resolution fluorescence in situ hybridization.

We analyzed the gene organization in the complement component C4 and 21-hydroxylase (21OH) gene region of the human major histocompatibility complex using visual mapping of stretched DNA by multicolor fluorescence in situ hybridization (FISH). Normally, this region contains a duplicated 21OH-C4 gene cluster (21OHB-C4B-21OHA-C4A). Duplication and deletion of one or more copies of the 21OH-C4 gene unit are known to occur frequently. Biotin-labeled cDNA of the C4 gene and digoxigenin-labeled cDNA of the 21OH gene were hybridized to decondensed nuclei of peripheral blood lymphocytes obtained from individuals with various 21OH-C4 haplotypes. Hybridization signals of the C4 and 21OH probes were detected with fluorescein isothiocyanate (green) and rhodamine (red), respectively. Two linear green and red signal clusters were observed in each nucleus showing the normal haplotype. Gene duplication and deletion were visualized as addition and deletion of the signal cluster, respectively. The DNA types of the 21OH-C4 region determined by FISH were concordant with the results previously obtained by conventional molecular studies. Our high-resolution FISH technique is found to be useful for screening gene duplications and deletions.

Complement C4↗
PubMed1991

Major histocompatibility complex (MHC) complement deficiency, ancestral haplotypes and systemic lupus erythematosus (SLE): C4 deficiency explains some but not all of the influence of the MHC.

In 1982 we reported that among Caucasians with systemic lupus erythematosus (SLE) there is an increased frequency of C4A null. As this allele occurs on the HLA-A1,B8,BfS, C4AQO,B1,DR3 (8.1) supratype, we suggested this accounted for the reported association of B8 and DR3. Since then we have shown that many supratypes including 8.1 identify unique segments of DNA conserved from a common but remote ancestor. Many of these ancestral haplotypes (AH), including 8.1, carry disease genes and some bear C4 null. We have therefore tested the hypothesis that in SLE C4 null alleles are directly involved by examining (1) whether all or only some AH bearing C4 null alleles are increased, (2) whether C4 null is increased in all racial groups examined, and (3) whether C4 null is associated with the presence of antinuclear antibodies (ANA) in the absence of SLE. We performed HLA and complement allotyping on 62 Australian Caucasians and 9 Australian aborigines with SLE and on the 10 out of 133 healthy individuals with 7 or more international units of ANA. Our data confirm an association of C4A null in Australian Caucasians (gene frequency 0.30 versus 0.15 in controls) and show an increased frequency of C4B null in Australian aborigines (gene frequency 0.33 versus 0.22). A review of an extensive literature shows C4A and/or C4B null are increased in all racial groups examined. On the other hand, the HLA-A3,B7,BfS,C4A3,B1,DR2 (7.1) AH rather than C4 null is associated with ANA in health. Our data indicate that while C4 nulls contribute to MHC susceptibility, other genes are likely to be involved.

Alleles↗
PubMed1990

Organization of the chimpanzee C4-CYP21 region: implications for the evolution of human genes.

We prepared a cosmid library from chimpanzee DNA and screened it with a mouse probe specific for the complement component 4 (C4)-encoding gene. We isolated 29 clones and constructed restriction maps for 20 of these. The clones could be arranged into two overlapping clusters covering the entire C4 region of both chromosomes in this particular heterozygous chimpanzee. The region is about 100 kilobases (kb) long and contains two C4 and two CYP21 genes, the latter coding for the enzyme 21-hydroxylase. Using oligonucleotide probes we identified the genes as corresponding to human C4A, C4B, CYP21 and CYP21P genes. The last gene apparently contains an 8-base pair (bp) deletion (as does the corresponding human gene), which renders it a pseudogene. The genes are arranged in the order C4A...CYP21P...C4B...CYP21. Each of the two C4 genes is 16 kb long and thus corresponds to the short version of the human C4 genes. We suggest that the duplication of the basic C4-CYP21 unit that generated the standard arrangement of the human C4-CYP21 region occurred before the separation of the evolutionary lineages leading to humans and chimpanzees (i.e., more than five million years ago). We suggest further that the original form of the C4 gene was of the long variety and was generated by the insertion of a 6.8-kb element into one of the C4 introns. The element was subsequently excised in the ancestors of the chimpanzees and in at least one lineage of the human C4B gene. We speculate that the presence of the 6.8-kb insert in the human C4A and some C4B genes might largely be responsible for the great instability of this chromosomal region which leads to frequent duplications and deletions, some of which cause 21-hydroxylase deficiency.

Animals↗
PubMed1988

Serum protein markers in systemic lupus erythematosus.

Serum protein markers (alpha 1-AT, Bf, C3, C4A, C4B, Hp and Tf) were studied in a series of 36 patients with systemic lupus erythematosus (SLE) and compared to normal blood donors. In agreement with the results of previous investigations a significant increase of complement C4 deficiency was found among the SLE patients. The relative risks for AQ0 and BQ0 homozygosity were 7.2 and 4.1, respectively. Simultaneous occurrence of AQ0 and BQ0 was found in three patients with a calculated relative risk of about 65. A significant increase of the haptoglobin type 2-2 (p less than 0.05) was found among SLE patients. The remaining serum protein systems showed no statistically significant associations with SLE.

Blood Proteins↗
PubMed1985

Ultrastructure of human C4-binding protein: proposition for a new model.

The structure of human C4-binding protein (C4bp), a regulatory factor of the classical C3 convertase of complement, has been under investigation for several years, but remains poorly understood. For example, the number of subunits in the C4bp molecule has not been established. In this report, we use two different techniques (partial reduction and electron microscopy) to clarify the structure of the C4bp. Our results lead us to propose a structural model which is quite different to that suggested before, i.e. the C4bp molecule appears to be a decamer. In addition to the disulfide bonds which link each subunit to another, a second disulfide interaction leads to the association of the subunits in pairs. Each pair of subunits appears as a filament ending in a globular head at the N-terminal extremity. The pairs of subunits join to form a conical central domain (at the C-terminal extremity) linked by disulfide bonds. The proposed pentameric shape of the C4bp is consistent with the stoichiometry of the C4b-C4bp interactions. The proposed model indicates an overall structural homology between C4bp and other binding proteins.

Carrier Proteins↗
PubMed1998

Molecular genetics of the human MHC complement gene cluster.

The human major histocompatibility complex (MHC) complement gene cluster (MCGC) is a highly variable region that is characterized by polymorphisms, variations in gene size and gene number, and associations with diseases. Deficiencies in complement C2 are either due to abolition of C2 protein synthesis by mini-deletions that caused frameshift mutations, or blocked secretion of the C2 protein by single amino acid substitutions. One, two or three C4 genes may be present in a human MCGC haplotype and these genes may code for C4A, C4B, or both. Deficiencies of C4A or C4B proteins are attributed to the expression of identical C4 isotypes or allotypes from the C4 loci, the absence or deletion of a C4 gene, 2-bp insertion at exon 29 or 1-bp deletion at exon 20 that caused frameshift mutations. The C4 genes are either 21 or 14.6 kb in size due to the presence of endogenous retrovirus HERV-K(C4) in the intron 9 of long C4 genes. A deletion or duplication of a C4 gene is always accompanied by its neighboring genes, RP at the 5' region, and CYP21 and TNX at the 3' region. These four genes form a genetic unit termed the RCCX module. In an RCCX bimodular structure, the pseudogene CYP21A, and partially duplicated gene segments TNXA and RP2 are present between the two C4 loci. The RCCX modular variations in gene number and gene size contributed to unequal crossovers and exchanges of polymorphic sequences/mutations, resulting in the homogenization of C4 polymorphisms and acquisitions of deleterious mutations in RP1, C4A, C4B, CYP21B and TNXB genes. RD, SKI2W, DOM3Z and RP1 are the four novel genes found between Bf and C4. RD and Ski2w proteins may be related to RNA splicing, RNA turnover and regulation of translation. The functions of Dom3z and RP1 are being investigated. The complete genomic DNA sequence between C2 and TNX is now available. This should facilitate a complete documentation of polymorphisms, mutations and disease associations for the MCGC.

Chromosome Mapping↗
PubMed1988

Levels and plasma distribution of free and C4b-BP-bound protein S in human fetuses and full-term newborns.

Levels and plasma distribution of protein S were measured on umbilical cord plasmas from 25 normal full-term newborns and 25 normal fetuses which were between 20 and 31 weeks of gestation. Fetal blood samples were obtained by direct puncture of the umbilical vein under high resolution real-time ultrasound. Total and Free protein S levels were found to be lower in all fetuses and newborns as compared to normal adults. The calculated ratio Total protein S/Free protein S and crossed immunoelectrophoresis patterns indicate that protein S circulates essentially in the free form in fetuses and newborns. These data may be explained by the low levels of C4b-binding protein observed at these stages of development.

Adult↗
PubMed1996

Characterization of platelet-reactive antibodies in children with varicella-associated acute immune thrombocytopenic purpura (ITP).

Biochemical analyses were performed on blood samples obtained from two children (P1, P2) who presented with acute immune thrombocytopenic purpura (ITP) following a recent varicella zoster virus (VZV) infection. Patient sera had antibodies that were reactive with normal blood-group O platelets as measured by flow-cytometric assay. Western blot analysis of electrophoretically separated normal blood-group O platelets under reducing and non-reducing conditions demonstrated that these sera were reactive with platelet antigens of approximately 50 and approximately 110 kD, respectively. These 50/110 kD antigens were not reactive with seven sera from acute ITP patients whose illness was not preceded by VZV infection, with serum from a patient with a prior history of VZV and no thrombocytopenia, nor with normal healthy control sera. VZV antibodies (IgG and IgM), isolated from patient sera by affinity chromatography using immobilized purified VZV glycoproteins, were found to bind to gel-filtered autologous platelets and with normal blood-group O platelets, as analysed by flow cytometry. No binding was observed using antibodies similarly prepared from healthy volunteer sera. To investigate their ability to sensitize platelets to complement activation, affinity-purified VZV antibodies were incubated with platelets and then with purified complement components C1 and 125 I-labelled C4. Platelets reacted with VZV-specific antibodies from the two patients and showed increases of 2.3-2.4-fold of platelet-surface deposition of 125 I-C4b, compared to controls. These data provide evidence that virus-specific antibodies occurring in children with varicella-associated acute ITP cross-react with normal platelet antigens, and may contribute to platelet clearance.

Antibodies, Viral↗
PubMed1979

Complement components detected on normal red blood cells taken into EDTA and CPD.

Normal red blood cells (RBC) from fresh EDTA and CPD blood and from stored CPD blood were examined for the presence of bound subcomponents of C3 and C4. By serologic agglutination tests, only C3d was detectable on the cells. Incubation in compatible fresh normal serum (FNS) at 37 degrees C appeared to increase the amount of 3Cd on the RBC. C3b was serologically detectable only on stored CPD cells and only after incubation in compatible FNS. No. C4 components were detected on the cell surfaces in agglutination tests. Using an indirect labeling technique, small, but significant, amounts of C3d and C4d were found on all three types of untreated cells. C3b was present on stored CPD cells only. The indirect labeling technique showed a significant increase in C3d and C4d on all cells following incubation i- compatible FNS, whereas bound C3b was significantly increased only with stored CPD cells. There was no increase in bound C4b following serum incubation. The average number of C3d molecules per cell on normal EDTA cells was 557 and average Ko was 3.6 x 10(7) l/mol.

Animals↗
PubMed1989

Major-histocompatibility-complex gene markers and restriction-fragment analysis of steroid 21-hydroxylase (CYP21) and complement C4 genes in classical congenital adrenal hyperplasia patients in a single population.

The gene CYP21B, encoding the steroid 21-hydroxylase enzyme of adrenal steroid biosynthesis, has been mapped to the human major histocompatibility complex (MHC). Deficiency of this enzyme leads to congenital adrenal hyperplasia (CAH). We report the phenotypes of the HLA and complement C4 and Bf genes, which are closely linked to the CYP21B gene, together with a detailed analysis of the CYP21 and C4 RFLP, in 17 Finnish families with CAH. The RFLP analysis with six restriction enzymes suggested that, altogether, 35% of the affected chromosomes had a CYP21B + C4B gene deletion, 9% an obvious gene conversion of the CYP21B gene to a CYP21A-like gene, and 3% a CYP21A + C4B duplication. The remaining 53% gave the RFLP patterns also found in nonaffected chromosomes. We also found that a 14.0-kb EcoRI RFLP marker of the CYP21 genes was strongly associated with the presence of a short C4B gene, suggesting that some of the RFLP markers found with the CYP21 probe may actually derive from C4B gene polymorphism. Three particular MHC haplotypes, each with a characteristic RFLP pattern, were found in many unrelated families. These three haplotypes accounted for 59% of the affected chromosomes in our study group, the rest (41%) of the affected chromosomes being distributed among various subtypes. The results suggest that, within a single, well-defined population such as in Finland, only a few CYP21B gene defects may constitute a substantial part of the affected chromosomes. This finding will help in genetic studies of CAH in such populations.

Adrenal Hyperplasia, Congenital↗
PubMed1988

Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma.

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.

Animals↗
PubMed1983

Binding of human C4 to C-reactive protein-pneumococcal C-polysaccharide complexes during activation of the classical complement pathway.

Sequential interaction of CRP-PnC aggregates, made at slight CRP excess, with purified human C1, C4 and C2oxy resulted in formation of an effective C3-convertase, indicating the binding of C1, C4 and C2 on the aggregates. Immunoprecipitation experiments demonstrated that, following cleavage of 125I-C4 by CRP-PnC-C1 complexes, approximately 3% of the 125I-C4 was bound to CRP while a lower percentage was bound to PnC, CRP-C4 complexes could also be demonstrated by substituting 125I-CRP for 125I-C4. The nature of the CRP-C4 bond was examined by electrophoretic analysis. Complexes of 125I-C4-CRP prepared as earlier were incubated at 100 degrees C for 2 min in buffer containing 2% SDS and 5% beta-mercaptoethanol and subjected to electrophoresis in SDS-containing polyacrylamide gradient slab gels. Autoradiography of the dried gels revealed the presence of high mol. wt bands containing the alpha'-chain of C4b. CRP could also be demonstrated in these high mol. wt bands which apparently represented covalent complexes between the alpha'-chain of C4b and CRP monomers. Since CRP contains no detectable carbohydrate, it seems likely that an amide bond is formed between the two proteins.

Amides↗
PubMed2002

X-ray crystal structure of the C4d fragment of human complement component C4.

C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.

Amino Acid Sequence↗
PubMed2006

Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio).

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.

Animals↗
PubMed1991

Determination of total, free and complexed protein S in plasma by ELISA, and comparison with a standard electroimmunoassay.

An enzyme-linked immunosorbent assay (ELISA) for measuring total, free and complexed protein S in plasma was developed. To assay free protein S, C4b-binding protein-bound protein S (C4b-BP-PS) was extracted by addition of polyethyleneglycol (PEG) 6000 (5%, final concentration) to plasma samples. Microtiter plates were coated with rabbit anti-human protein S, and bound protein S was detected with labelled anti-protein S antibody. Diluted plasma samples were incubated in the plates overnight at 22 degrees C to permit C4b-BP-PS complexes to dissociate. Mean variation coefficients of 2.1 and 3.2% (intra-assay) and 4.3 and 7.9% (inter-assay) were found for total and free protein S assays, respectively. The ELISA measures free and complexed protein S with equal efficiency as is demonstrated by the fact that the sum of free protein S and C4b-BP-PS complex levels in normal individuals, women in their third trimester of gestation and patients with acute deep vein thrombosis (DVT), equaled the level of total protein S present in the corresponding plasma. Total protein S values obtained with the ELISA, in all groups studied, correlated well with those obtained with a standard electroimmunoassay (EIA) (r = 0.93; n = 40). However, total protein S levels measured by EIA were lower than those assayed by ELISA in pregnant women and in DVT patients. Furthermore, addition of several amounts of purified C4b-BP to NHP, which reduced the recovery of free protein S, did not influence the total protein S values measured by ELISA but slightly decreased the recovery of total protein S measured by EIA. These results indicate the necessity of using assays which accurately and reliably measure the total amount of protein S antigen. After addition of C4b-BP to NHP, the residual functional protein S level was lower than the residual level of free protein S antigen; this lends support to the idea that C4b-BP-PS complex inhibits the activated protein C cofactor activity of protein S.

Carrier Proteins↗