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PubMedDate not supplied

Domain structure, functional activity, and polymerization of trout complement protein C9.

The 3' region of trout C9 has been resequenced and found to differ from the previously published sequence (Stanley and Herz, EMBO J. 6:1951; 1987). In contrast to other sequenced C9 molecules, but in common with the other terminal complement components, trout C9 was found to contain an additional carboxy terminal thrombospondin domain. This domain does not restrict polymerization, as has been previously suggested (Stanley and Luzio, Nature 334:475; 1988), since alternative pathway activation of trout complement by rabbit erythrocytes lead to the formation of circular membrane attack complement lesions on the erythrocyte membrane. Although the trout C9 molecule is larger than human C9, the diameters of circular trout membrane attack complexes were approximately 30% smaller than their human counterparts. No lysis of erythrocytes bearing human C5b-7 or C5b-8 complexes was detected following incubation with trout serum containing EDTA, which suggests that trout C8 and C9 are unable to bind to human C7 and C8, respectively. Finally, trout and human serum were equally effective at killing the human serum-sensitive strain Salmonella minnesota Re595.

Amino Acid Sequence↗
PubMed1986

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore.

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.

Animals↗
PubMed1997

Presence of immunoglobulins, C3 and cytolytic C5b-9 complement components on the surface of erythrocytes from patients with beta-thalassaemia/HbE disease.

The occurrence of IgG, IgM, IgA, C3 and C5b-9 complement complexes on erythrocytes from 43 patients with beta-thalassaemia HbE disease was investigated. Indirect immunoradiometric assays using radioiodinated protein A were employed to quantify the individual components. We confirmed that circulating erythrocytes from thalassaemic patients contained elevated amounts of IgG, and small but significant amounts of C3. In addition, small but significant amounts of C5b-9 were detected. Levels of cell-bound IgG, C3 and C5b-9 were higher in splenectomized versus non-splenectomized patients. The presence of C5b-9 on circulating cells from five splenectomized patients was confirmed by an ELISA employing a monoclonal antibody specific for a C5b-9 neoantigen. When C5b-9 positive cells from two patients were solubilized with detergent and subjected to sucrose density gradient centrifugation, the terminal complexes sedimented as 25-40S macromolecules, thus behaving as membrane C5b-9 complexes. The presence of C8 and C9 in these high molecular weight fractions was directly demonstrated by Western blotting. These results constitute the first demonstration that circulating diseased erythrocytes may carry low numbers of potentially cytolytic C5b-9 complement complexes which may be partly responsible for the known ionic disturbances found in thalassaemic cells. Both bound C3 and C5b-9 could promote removal of diseased cells in the reticuloendothelial system.

Adult↗
PubMed1993

Activation of the alternative complement pathway and production of factor H by skeletal myotubes.

Skeletal muscle myotubes from neonatal rats were used to study the interaction of skeletal muscle with complement. Serum from guinea pig, rabbit, and human, in the absence of muscle-specific antibody, caused creatine phosphokinase release, which required activation of the terminal complement cascade. Cleavage of serum C3 and Factor B in the presence of myotubes was dependent on Mg2+, but not Ca2+, and C3 cleavage occurred only in the presence of Factor B. Rat myotubes caused significant consumption of C8 and C9 in rat serum, which also required Mg2+, but not Ca2+. All of these findings are typical of a tissue capable of activating the alternative pathway. In addition, the C2 myotube cell line was shown to produce Factor H, an inhibitory protein of the alternative pathway, as demonstrated by Factor H mRNA expression and immunoprecipitation of the protein.

Animals↗
PubMed1992

Complement and pregnancy: new insights into the immunobiology of the fetomaternal relationship.

Recent studies have revealed that human trophoblast expresses three membrane-bound proteins which function specifically to regulate the activity of complement. These proteins are already known to be widely distributed in normal adult tissues where they protect host cells from damage resulting from the fortuitous deposition of activated complement components. Their activities are focused at two distinct steps in the complement pathway. Decay accelerating factor (DAF, CD55) and membrane co-factor protein (MCP, CD46) act at the level of the C3 convertase enzymes which activate C3 to C3b. A further protein, CD59, directly regulates the formation and function of the terminal cytolytic membrane attack complex (MAC) by specifically interacting with C8 and C9. These proteins appear to play an important role in the maintenance of normal human pregnancy. DAF, MCP and CD59 are all expressed where trophoblast surfaces are in contact with maternal blood and tissues and expression occurs from at least 6 weeks of gestation. The semi-allogeneic human conceptus therefore appears to be effectively protected from maternal complement-mediated damage arising either from alternative or classical pathway activation or in a bystander fashion following a response to microbial infection in the mother. Complement regulatory protein deficiency disorders with clinically demonstrable consequences especially in terms of haemolytic disease are known to exist and have proved valuable in establishing the biological role of these proteins in vivo. The demonstration of this new family of immunoregulatory proteins on trophoblast raises important questions about the potential involvement of these products in pregnancy pathologies.

Complement C3↗
PubMed1986

The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy.

Using a model of rat membranous nephropathy (MN), we examined the relationship between the development of glomerular epithelial cell injury and the formation and stability of the membrane attack complex (MAC) of complement. Isolated rat kidneys were perfused with buffered bovine albumin (BSA) or various plasmas (complement source). Kidneys containing nephritogenic amounts of complement-fixing sheep antibody to glomerular epithelial antigens (aFx1A) perfused with BSA (n = 5), and normal kidneys perfused with normal human plasma in BSA (50% v/v, n = 6) excreted 0.30 +/- 0.02 mg protein/min/g during 90 min perfusion (control groups). When normal plasma was added to the perfusate of aFx1A kidneys at concentrations of 12.5, 25, and 50% v/v, protein excretion rose in a time- and concentration-dependent manner. Perfusions with 25% plasma resulted in baseline proteinuria from 0 to 20 min that increased to 2.8 +/- 0.9 mg/min/g at 20 to 40 min and 8.6 +/- 2.1 at 40 to 60 min (n = 4, p less than 0.01 vs control groups). Removal of plasma at 20 min did not prevent this rise in protein excretion (3.9 +/- 2.4 and 5.8 +/- 2.6 mg/min/g at 30 to 40 and 55 to 65 min respectively, p less than 0.01, n = 4). Perfusion of aFx1A kidneys with C8-deficient (C8D) human plasma (25% v/v, n = 4) or C6D rabbit serum (25% v/v, n = 2) independently produced low levels of proteinuria comparable with BSA, but in combination, the two reagents restored enhanced protein excretion (n = 2). In aFx1A kidneys containing C5b-7, addition of C8 and C9 (C6D serum) after intervals of 20, 60, or 90 min immediately reconstituted heavy proteinuria. Thus, the magnitude of MAC-induced glomerular epithelial injury in rat MN is related to the complement dose. Altered glomerular permeability is delayed with respect to the onset of complement activation. Once sufficient C5b-9 is formed, proteinuria can develop despite cessation of new MAC assembly, implying that C5b-9 persists after formation. Moreover, the C5b-7 MAC intermediate is not eliminated rapidly in this model.

Animals↗
PubMed1994

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Amino Acid Sequence↗
PubMed1991

Nuclease S1-mediated enhancement of the 32P-postlabeling assay for aromatic carcinogen-DNA adducts.

Treatment of DNA digests with nuclease P1 prior to 32P-labeling of adducts has previously been shown to enhance the sensitivity of the 32P-postlabeling assay for the detection of aromatic carcinogen-DNA adducts. The enhancement was based on the ability of nuclease P1 to remove the 3'-phosphate from normal nucleotides but not the corresponding phosphate from most aromatic adducted nucleotides. We investigated the utility of another 3'-dephosphorylating enzyme, nuclease S1, for this purpose, and found it to be as effective as nuclease P1. The recovery of DNA adducts derived from benzo[a]-pyrene (B[a]P), benzoquinone (BQ) and 2-acetylaminofluorene (AAF) was comparable after enhancement with either enzyme. Some differences were, however, observed. Recovery of a minor B[a]P adduct was 1.5 times higher by the S1 procedure. Among minor adducts of BQ, two showed higher values (2.8- and 6.1-fold) by the S1 procedure and one by the P1 procedure (2.4-fold). The major AAF adduct, deoxyguanosine-C8-AF, exhibited poorer recovery (1-11%) by either procedure, while the minor adducts, deoxyguanosine-N2-AAF and deoxyguanosine-C8-AAF, showed better recovery (2-3 times) than by the enhancement procedure involving extraction of adducts into butanol. Our results show that the nuclease S1 assay can complement the nuclease P1 assay, with improved recoveries for some adducts. Considering the complexity of the postlabeling assay, this additional variant may prove useful in unequivocal detection of DNA adducts.

2-Acetylaminofluorene↗
PubMed1986

In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

Animals↗
PubMed1978

Assembly of the cytolytic alternative pathway of complement from 11 isolated plasma proteins.

The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.

Cell-Free System↗
PubMed1987

Formation of transmural complement pores in serum-sensitive Escherichia coli.

The binding of C9 at 0 and 37 degrees C to viable Escherichia coli K-12 cells carrying C5b-8 complexes was quantified. At low temperature, limited average binding of only 1 to 1.4 molecules of C9 per C8 molecule occurred, whereas 6 to 8 C9 molecules were bound per C8 molecule at 37 degrees C. Despite incorporation of C9 into C5b-9 complexes at 0 degrees C, these terminal complexes caused no loss of bacterial viability even when present in very large numbers (1,000 to 1,500 per CFU) on the bacterial cells. In contrast, generation of 50 to 100 C5b-9 complexes carrying multiple C9 molecules per CFU caused loss of viability. The failure of C5b-81C91 complexes to generate transmural pores was confirmed by measurements of o-nitrophenyl-beta-D-galactoside influx into the cells. Whereas treatment of C5b-8-laden cells with C9 at 32 degrees C caused virtually instantaneous influx of the marker, almost no influx was registered in cells receiving C9 at 0 degrees C. When cells carrying C5b-7 were brought into the stationary phase and given C8 and C9 at 32 degrees C, a C9-dependent disruption of the outer membrane permeability barrier immediately occurred as demonstrated by cleavage of a chromogenic substrate by periplasmic beta-lactamase. In sharp contrast, o-nitrophenyl-beta-D-galactoside influx was markedly retarded over a prolonged period, with abrupt permeability increases of the inner membrane toward this molecule being noted just before bacterial cell division occurred. We conclude that killing of E. coli requires binding of C5b-9 complexes containing C9 oligomers to the outer membrane and suggest that formation of pores in the inner membrane occurs when these complexes are "hit" by transiently forming zones of bioadhesion. Formation of the latter may be a dynamic process that is accentuated during cell division and quiescent during the stationary phase.

Blood Bactericidal Activity↗
PubMed1992

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.

Animals↗
PubMed1996

Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8).

The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered.

Base Sequence↗
PubMed1985

Opsonophagocytosis of Neisseria gonorrhoeae: interaction of local and disseminated isolates with complement and neutrophils.

The phagocytosis of serum-sensitive (SS) and serum-resistant (SR) gonococci by neutrophils was examined. SS strains were more rapidly and completely ingested and killed than were SR strains (8.8% +/- 3.4% vs. 64.4% +/- 7.7% survival at 30 min [P less than .005]) in C8-deficient serum or C8-depleted normal serum. Opsonic requirements of the two types of isolates differed. Heat-labile and -stable serum factors played an important role in the phagocytosis of SS but not SR strains. Indeed, killing of SR strains by polymorphonuclear neutrophils did not vary over a 1,000-fold change in serum concentration. SS strains consumed and fixed C3 more rapidly and in greater amounts than did SR strains (83.3% +/- 17.4% vs. 20.8% +/- 5.0% at 10 min [P less than .01]). However, this difference in C3 consumption and fixation did not completely account for the difference in phagocytosis because killing of SS strains was still greater than that of SR strains under conditions of equal C3 fixation.

Blood Bactericidal Activity↗
PubMed1987

[Hereditary angioedema: changes in serum levels of C4 in response to danazol].

12 patients (7 males and 5 females) suffering from the common form of HAE were included in the study protocol. All patients were older than 18 years. They were evaluated Cl INH, C4, Clq, Cls, C3, C5, C8, Bf, CH 50% and CIC. For the purpose of the study they were only considered Cl INH, C4, CH 50%, CIs and CIC levels. The rest of the complement components were among normal values. Data were recorded at day 0 and after 10 days on treatment with 400 mg/day of Danazol. Results were as follows: 50% of the patients had CIC when CH 50% values were below 120 U/ml., after treatment CIC disappeared and CH 50, Cls, C4 and Cl INH increased significatively. C4 seemed to increase more and quicker than Cl INH in terms of absolute values. We postulate that in the group of patients with CIC, the primary cause of the disease may be an alteration in C4 synthesis and that Cl INH may be lowered because of its consumption. We postulate that Danazol could act in these cases stimulating C4 synthesis independently or in accordance with Cl INH.

Adult↗
PubMed2000

Production and functional characterization of a soluble recombinant form of mouse CD59.

This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using purified terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.

Animals↗
PubMed1981

How complement kills E. coli. II. The apparent two-hit nature of the lethal event.

We have studied the nature of complement (C) action on red blood cells and E. coli with respect to the number of "hits" required for membrane damage. Our method of analysis involves adding various amounts of purified C7 or C8 to serum preparations immunochemically depleted of C7 or C8, respectively, in order to construct dose-response curves for the action of C's terminal complex. The shape of the dose-response curves reflects the single or multiple-hit nature of C action. Our method confirms that C acts on red cells by a 1-hit mechanism, whether measured by lysis or by the permeation of a small molecule. In contrast, we find with E. coli that C-mediated outer membrane damage, inner membrane damage, and killing all appear to require more than 1 hit. We have also discovered a property of E. coli that displays a nonlethal 1-hit response to C that is particularly useful in the analysis of multiple-hit dose-response curves. Simultaneous measurements of this single-hit phenomenon and the multiple-hit killing of E. coli allow us to make direct comparisons of the amount of C needed for each response. On the basis of the midpoints of the single and multiple-hit curves, C-mediated membrane damage and killing of E. coli appear to be a 2-hit process.

Animals↗