A physical map of the C6 and C7 complement component gene region on chromosome 5p13.
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Base Sequence↗
Biomedical subjects
R G DiScipio
Publications and source records attributed to R G DiScipio.
Ultrastructures and interactions of complement factors H and I.
The human complement regulatory protein, factor H, was examined by high resolution transmission electron microscopy. Results of electron microscopy confirm hydrodynamic analysis and indicate that factor H is a monomer of M(r) approximately 155,000. Factor H is an extended flexible molecule with a contour length of 495 A and a cross-sectional diameter of 34 A. Most images of factor H indicate that its polypeptide chain typically folds back on itself with the result that the average length of a factor H molecule is about half its contour length. Only one end of factor H associates with C3b. When bound to C3b, factor H still shows considerable conformational flexibility. Factor I is a bilobal protein of 130 A in length, and its two globular parts have maximal diameters of 54 and 49 A. The results establish that factor I is a two domain protein where the smaller subunit is a protease and the larger one is involved with binding C3b. Factor I binds C3b with a one-to-one stoichiometry in an ionic strength-dependent fashion. In the absence of sodium chloride an affinity constant of 5.7 x 10(5) M-1 was determined for factor I interaction with C3b. Whereas the Scatchard plot of factor I binding to C3b in the absence of factor H is linear, in the presence of factor H a curvilinear graph is obtained. The strong binding sites on C3b for factor I have an affinity at least 15-fold higher in the presence of factor H than in its absence. The results of both electron microscopy and binding studies were combined to compose a scheme envisioning how factors H and I cooperate for the processing of C3b.
Binding Sites↗
Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis.
Complement components C3 and C5 are susceptible to limited proteolysis by an arginine-specific cysteine proteinase isolated from Porphyromonas gingivalis. This bacterium is an anaerobe commonly associated with severe periodontal disease. Infection by P. gingivalis is accompanied by an acute inflammatory response, complete with extensive neutrophil involvement. This prompted us to investigate a possible direct role for complement in periodontitis evoked by P. gingivalis. Exposure of C3 and C5 to the cysteine proteinase at molar ratios between 1:25 and 1:100 (enzyme to substrate ratios) resulted in a time-dependent, limited degradation of each component. C3 was converted in a stepwise manner to C3a-like and C3b-like fragments with evidence of extensive further degradation of the C3a-like portion of the molecule. We were unable to demonstrate C3a activity in the C3 digestion mixtures. C3 degradation appears to involve primarily the alpha-chain. Proteolysis of C5 also progresses in a stepwise manner producing an initial internal cleavage of the alpha-chain to generate 30- and 86-kDa fragments. Further digestion of the 86-kDa amino-terminal fragment of the alpha-chain leads to the release of C5a or a C5a-like fragment that is biologically active for neutrophil activation. The fact that a potent chemotactic factor, i.e. C5a, can be generated from C5 by a proteinase derived from P. gingivalis suggests a recruiting mechanism for attracting neutrophils to the gingival lesion site in periodontal disease.
Adhesins, Bacterial↗
Formation and structure of the C5b-7 complex of the lytic pathway of complement.
The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.
Amino Acid Sequence↗
The relationship between polymerization of complement component C9 and membrane channel formation.
C9 was studied with the objective to clarify the relationship between the process of C9 polymerization and membrane channel formation. Conditions that favor C9 polymerization include low ionic strength and calcium ion in the buffer. Moreover, polymerization is dependent on the concentration of C9. Calcium ion evokes about a threefold increase in the affinity constant for C9 self-association, and at 0 degrees C it imparts reversible amphiphilic properties in the molecule. These were discerned by measuring increases in the degree of reversible nonspecific binding of C9 to hydrophobic (tyramine-zymosan) and hydrophilic (arginyl-glutamyl-zymosan) supports as well as to erythrocytes. At 0 degrees C the hydrophilic-to-amphiphilic alteration of C9 is reversible, but upon incubation at 37 degrees C this transition is rendered permanent with the formation of poly(C9). A functional relationship between C9 polymerization and cytolysis was demonstrated by showing that polymerizing C9 can lyse reduced and alkylated erythrocytes. By studying comparative radiolabeling of tyrosine side chains within thrombin-nicked C9 and its polymerized form, it was demonstrated that upon polymerization the membrane-binding site of C9 becomes exposed. It is concluded that the process of circular polymerization of C9 causes a hydrophilic-to-amphiphilic transition that is required for membrane perforation and channel formation.
Animals↗
DNA polymorphisms and linkage relationship of the human complement component C6, C7, and C9 genes.
In this report we describe the linkage between genes encoding human complement components C6, C7, and C9. Polymorphisms have been described at the DNA level for the C7 and C9 genes. We have studied 20 individuals by Southern blot analysis with four C6 cDNA subclones to detect restriction fragment length polymorphisms (RFLPs). We have found a Taq I polymorphism defined by two alleles of 8.0 (C6 H) and 6.0 (C6 L) kilobases (kb). RFLP segregation for the C6, C7, and C9 loci in informative families allowed us to estimate the maximum Lod scores at a recombination fraction of theta = 0.0 (C6-C7), theta = 0.0 (C7-C9), and theta = 0.0 (C6-C9). Significant linkage disequilibrium was found between C6 and C7 and between C7 and C9 loci in directly determined haplotypes of unrelated parents. Data from this study show that the genes encoding the human terminal complement components C6, C7, and C9 define a cluster in the short arm of chromosome 5. We propose that the clusters involving the C8A and C8B and the C6, C7, and C9 genes be referred to as MACI and MACII, respectively.
Chromosome Mapping↗
The assignment of the genes coding for human complement components C6 and C7 to chromosome 5.
A panel of 19 somatic cell hybrids was tested for the presence of human sequences coding for complement components C6 and C7 by restriction enzyme digestion and Southern blots probed with human C6 and C7 cDNA probes. C7 was also detected by amplifying part of the human gene in hybrid DNA using the polymerase chain reaction. Detection of human C6 and C7 was completely correlated with the presence of chromosome 5.
Animals↗
Hydrodynamic, electron microscopic, and ligand-binding analysis of the Epstein-Barr virus/C3dg receptor (CR2).
The interaction of the Epstein-Barr virus/45-kDa proteolytic fragment of C3 (C3dg) receptor (CR2) with its viral ligand, the Epstein-Barr virus glycoprotein gp350/220, initiates the sequence of events leading to virus internalization and B lymphocyte transformation. Soluble recombinant receptor (rCR2) and gp350/220 as well as the natural ligand, C3dg, were subjected to a number of analytical techniques including gel permeation chromatography, density gradient ultracentrifugation, circular dichroism, and electron microscopy in order to determine their hydrodynamic, structural, and binding properties. Both rCR2 and gp350/220 were found to be highly extended proteins (f/fo = 2.1 and 2.4/2.2, respectively). C3dg, in contrast to the viral ligand, is only somewhat elongated (f/fo = 1.5). Soluble rCR2, visualized by high resolution electron microscopy, was shown to be an extended, highly flexible molecule comprised of ringlet domains, each approximately 24.1 A in length, which likely correspond to the short consensus repeat motif deduced from the CR2 cDNA nucleotide sequence. Ligand-binding studies carried out under physiological conditions indicated that gp350/220 binding to rCR2 was saturable and univalent, with a dissociation constant of 3.2 nM. In contrast, monomeric C3dg did not bind to rCR2 under physiological conditions; however, at reduced ionic strength, monomeric C3dg binding could be measured. These studies indicate that the affinity of the C3dg monomer for rCR2 under physiologic conditions is approximately 10(4)-fold less than that of the viral ligand. The molecular properties of rCR2 revealed in these studies provide essential information for future studies of the biologic functions of the Epstein-Barr virus/C3dg receptor.
Antigens, CD↗
The molecular architecture of human complement component C6.
The molecular architecture of human complement component C6 was elucidated at several levels of structural organization. The entire primary structure of C6 was determined by sequencing C6 cDNA that was cloned from a human liver lambda gt11 library. The polypeptide chain of C6 contains 913 amino acids. The protein is homologous with the other terminal components of complement, C7-C9. Specifically, C6 has 29% of its residues identical with C7, and 55 of the 56 cysteines found in C7 match those in C6. The C6 polypeptide chain is cross-linked by 32 disulfide bonds, and most of the cysteines are located in short (34-77 amino acids) discrete segments that exhibit homology with a wide variety of other proteins such as thrombospondin, the low density lipoprotein receptor, epidermal growth factor, and complement factors H and I. C6 is a glycoprotein, and it has two oligosaccharide groups attached to asparagines located near the amino and the carboxyl termini of the molecule. The organization of secondary structural elements in C6 was elucidated using circular dichroism spectroscopy and an empirical method based on sequence analysis. C6 has an estimated 12% alpha-helix, but is comparatively richer in beta-sheet (29%) and beta-turns (21%). Most of the predicted alpha-helical structure resides in a portion of the polypeptide chain that is free of cysteine and which shares homology with C9 and perforin. The tertiary structure of the C6 molecule was visualized by transmission electron microscopy; it has a sickle shape with dimensions of 144 x 66 A. The combined results are discussed and comparisons made with the other late acting components of complement and perforin.
Amino Acid Sequence↗
The structure of human complement component C7 and the C5b-7 complex.
The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.
Amino Acid Sequence↗
Late-acting components of complement: their molecular biochemistry, role in the host defense, and involvement in pathology.
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Animals↗
The architecture of complement component C9 and poly(C9).
The architecture of human complement component C9 and poly(C9) was investigated by transmission electron microscopy. Monomeric native C9 (Mr = 66,000) exhibits an ellipsoid appearance (70 X 50 A) with a crevice visible on one face. C9 polymerizes spontaneously to form hollow tubular structures consisting of 12-16 monomeric subunits. Poly(C9) is a cylinder (150 A-outer diameters and 90 A-inner diameter) rimmed by a torus (46-A thick) on one end. Electron micrographs of poly(C9) indicate that the torus is formed by radial strands of polypeptide. Each subunit of poly(C9) is apparently tilted relative to the central axis of the cylindrical structure. C9 can be cleaved by alpha-thrombin into two single-chain polypeptide fragments: C9a (Mr = 28,000) and C9b (Mr = 38,000), which are the amino- and carboxyl-terminal segments of the protein, respectively. The cleaved form of the protein, C9a,b, can be induced to polymerize under suitable conditions to form sodium dodecyl sulfate-resistant poly(C9), indicating that the resistance of poly(C9) to denaturation is a collective feature of both C9a and C9b. The C9a and C9b polypeptide regions have been mapped on poly(C9) by immunoelectron microscopy. Determinants for the C9a region were observed about the torus, base, and on the midsection of the poly(C9) cylinder. C9b epitopes are concentrated predominantly about the torus and base, but were rarely observed on the midsection of poly(C9). Thus, the C9a and C9b segments of the C9 polypeptide are not clearly segregated in poly(C9). The locations of oligosaccharide units on poly(C9) were visualized by electron microscopy after labeling of the complex with concanavalin A bound to colloidal gold. The oligosaccharide positions were found on the periphery of the torus and base. In summary, C9 appears to be a single-domain protein. Polymerization involves a major rearrangement. To form a subunit of poly(C9) the polypeptide chain must form at least one major fold parallel to the central axis of the tubule.
Amino Acid Sequence↗
Nucleotide sequence of cDNA and derived amino acid sequence of human complement component C9.
The nucleotide sequence coding for the ninth component of human complement (C9) has been determined and the corresponding amino acid sequence has been derived. A human liver cDNA library was screened by the colony-hybridization technique using two radiolabeled oligonucleotide probes that correspond to known regions of the C9 amino acid sequence. Two recombinant plasmids were isolated and their cDNA inserts were sequenced. The derived protein sequence consists of 537 amino acids in a single polypeptide chain. A profile of the hydropathic index versus sequence number indicates that the amino-terminal half of C9 is predominantly hydrophilic in character whereas the carboxyl-terminal section of this protein is more hydrophobic. The amphipathic organization of the primary structure of C9 is consistent with the known potential of polymerized C9 to penetrate lipid bilayers, causing the formation of transmembrane channels.
Amino Acid Sequence↗
The activation of human complement component C5 by a fluid phase C5 convertase.
Complement component C5 is converted to C5a and C5b by the cobra venom factor-dependent C3/C5 convertase CVF,Bb (EC 3.4.21.47). The C5 convertase produces selective proteolytic cleavage of an arginyl-leucine peptide bond at positions 74-75 in the alpha chain of C5. Circular dichroism studies in both the far and near UV regions provide evidence that a conformational change accompanies the C5 activation process. When C5 is activated by CVF,Bb in the presence of complement component C6, the C5b,6 complex is formed. However, when C6 is added after C5 has been converted to C5b, the C5b,6 complex fails to form. Therefore, the activation of C5 results in a transient binding site for C6. Hydrophobic sites are probably exposed upon C5 activation because C5b undergoes aggregation when C5 is converted to C5b in the absence of C6. Transmission electron micrographs of the C5 molecule indicate a multilobal, irregular ultrastructure with estimated dimensions of 104 X 140 X 168 A. Aggregated C5b has the appearance of globular particles with a diameter range of 350-700 A. Although C5 shares a number of features with the third component of complement, including a similar ultrastructure and partial sequence homology, C5 is devoid of the unusual thiol ester linkage found in C3. It is the labile thiol ester that permits covalent attachment between C3 and nucleophilic acceptors. In contrast, interactions between C5 and C6 or C5 and membranes remain noncovalent.
Amino Acids↗
Purification and structural studies on the complement-system control protein beta 1H (Factor H).
An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.
Amino Acid Sequence↗
The activation of the alternative pathway C3 convertase by human plasma kallikrein.
Human plasma kallikrein can replace factor D for the activation of the alternative pathway C3 convertase of human complement. The factor B cleavage patterns by factor D and kallikrein are indistinguishable. The ability of kallikrein to cleave factor B is influenced by the magnesium ion concentration and the C3b concentration. Factor D is about ten-fold more effective on a molar basis, for the alternative pathway C3 convertase activation than is kallikrein. The physiological role of the action of kallikrein on the alternative pathway C3 convertase is discussed.
Centrifugation, Density Gradient↗
The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.
The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.
Binding, Competitive↗
The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.
The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.
Amines↗