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PubMed1977

Deviated lysis: transfer of complement lytic activity to unsensitized cells. IV. Parital isolation of the activity.

Deviated lysis (d.l.) was previously characterized as the lysis of non-sensitized erythrocytes by activated complement (C) in the presence of EDTA (1, 2, 3). The lytic activity was present in serum fractions of a m.w. in the proximity of 220,000. All the C factors C5 through C9 were found in these fractions and they were all needed for lysis. It is proposed that in d.l. small aggregates of the C components C5 through C9 coexist in the reaction mixture without further interaction. Only when appropriate receptors such as present on target cells surfaces are available, the factors react in a sequential order eventually to result in lysis of the target cell.

Animals↗
PubMed1977

Complement system in human colostrum: presence of nine complement components and factors of alternative pathway in human colostrum.

Evidence has been obtained for the presence in human colostrum of all nine components of complement (C), C1 through C9, and factors of the alternative pathway. Samples of colostrums collected from five women at 1-4 days after normal parturition were assayed for the haemolytic activities of individual components. As compared with normal human sera, the activities of each component ranged from 0.03 to 7% of those in sera. The activities of C4, C7 and C9 were relatively high, while that of C1 was extremely low. In most of the cases, the activities of individual components gradually increased following delivery, when expressed as the activity per unit weight (g) of protein in the colostrum. When the colostrums were treated with cobra venom factor, most of the colostrums showed 10-20% reduction in the C3 activity. This finding indicates the presence of factors such as B and D which are involved in the activation of C through the alternative pathway. The role as a defense factor of the C system in human colostrum and milk is discussed in connection with the ability of secretory IgA to react with C.

Colostrum↗
PubMed1983

Normal bactericidal capacity against Neisseria meningitidis in serum from a patient with a hemolytically inactive complement factor 8 (C8).

The bactericidal capacity of serum against Neisseria meningitidis from a 27-year-old male with two episodes of meningococcal meningitis and C8 deficiency was compared to that of normal human serum (NHS) without demonstrable antibodies against Neisseria. The in vitro bactericidal capacity of the patient serum was found to be equal to that of NHS. Incubation of both sera at 56 degrees C for 30 minutes abolished the bactericidal effect. Rocket-immunoelectrophoresis analysis of molecules immunochemically identifiable as C8 revealed no consumption of these molecules in any of the sera in the bactericidal assay. No hemolytic complement activity was found in the patient serum, whereas the donor serum had normal total hemolytic complement activity with significant consumption of C8.

Adult↗
PubMed1988

The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities.

S-Protein/vitronectin is a serum glycoprotein that inhibits the lytic activity of the membrane attack complex of complement, i.e., of the complex including the proteins C5b, C6, C7, C8, and C9n. We show that intact S-protein/vitronectin or its cyanogen bromide generated fragments also inhibit the hemolysis mediated by perforin from cytotoxic T-cells at 45 and 11 microM, respectively. The glycosaminoglycan binding site of S-protein/vitronectin is responsible for the inhibition, since a synthetic peptide corresponding to a part of this highly basic domain (amino acid residues 348-360) inhibits complement- as well as perforin-mediated cytolysis. In the case of C9, the synthetic peptide binds to the acidic residues occurring in its N-terminal cysteine-rich domain (residues 101-111). Antibodies raised against this particular segment react 25-fold better with the polymerized form of C9 as compared with its monomeric form, indicating that this site becomes exposed only upon the hydrophilic-amphiphilic transition of C9. Since the cysteine-rich domain of C9 has been shown to be highly conserved in C6, C7, and C8 as well as in perforin, the inhibition of the lytic activities of these molecules by S-protein/vitronectin or by peptides corresponding to its heparin binding site may be explained by a similar mechanism.

Animals↗
PubMed1990

Identification of the heterozygotes for deficiency of the beta-subunit of the eighth component of complement by reduced levels of C8 beta and increased amounts of free C8 alpha-gamma.

Sera from obligate heterozygotes for deficiency of the C8 beta subunit of the eighth component of human complement (C8) were analyzed for the molecular composition of C8. The C8 alpha-gamma and C8 beta subunits were separated by SDS-PAGE, visualized by immunoblotting, and the resulting bands were quantitated by laser densitometry. The laser densitometric absorption data were set to 100 arbitrary units (AU) for both subunits of pooled normal human sera. The AU values of individual normal sera ranged from 45 to 150 AU for C8 alpha-gamma (median 99 AU) and from 45 to 140 AU for C8 beta (median 101), whereas the C8 alpha-gamma/C8 beta-ratio varied from 0.7 to 1.4. Sera from C8 beta-deficient heterozygotes differed, as expected, from the normal sera for the markedly reduced levels of C8 beta (20 to 90 AU, median 55 AU) and for the higher C8 alpha-gamma/C8 beta-ratio (1.3 to 3.5). High voltage agarose gel electrophoresis was used to separate free and C8 beta-bound C8 alpha-gamma. The migration of free and C8 beta-bound C8 alpha-gamma subunit was checked by hemolytic overlay gels and by second dimension SDS-PAGE and immunoblotting. Immunochemical evaluation of C8 alpha-gamma using this system revealed about 5-14% free C8 alpha-gamma in sera with normal C8 and higher levels, from 33-71%, in the C8 beta D heterozygous sera. Functional analysis confirmed the substantial increase of free C8 alpha-gamma in the heterozygous group. We conclude that the C8 in C8 beta D heterozygous sera is characterized by increased amounts of free C8 alpha-gamma due to reduced concentrations of the C8 beta subunit. This finding may help to identify individuals heterozygous for C8 beta deficiency.

Complement C8↗
PubMed1992

Enhanced expression of the complement-regulatory factor C8 binding protein (C8bp) on U937 cells after stimulation with IL-1 beta, endotoxin, IFN-gamma, or phorbol ester.

C8 binding protein (C8bp) is a 65-kDa membrane glycoprotein that inhibits complement-mediated lysis by homologous C5b-9. C8bp was first identified on human erythrocytes, but could also be detected on peripheral blood cells, platelets, glomerular cells and synovial fibroblasts. Lack of C8bp as seen in patients with paroxysmal nocturnal hemoglobinuria type III results in enhanced susceptibility of the cells toward C5b-9. We studied C8bp expression on the promonocytic cell line U937. In addition to the membrane-bound C8bp, a cytoplasmic form of C8bp could also be identified by immunofluorescence, blotting, and precipitation. Stimulation of the cells with IL-1 beta, endotoxin, IFN-gamma, or phorbol ester increased C8bp surface expression. Because cycloheximide did not inhibit enhanced surface expression, it was most probably mobilized from cytoplasmic reservoirs. Thus, resistance of nuclear cells to complement attack seems to be based on two events: 1) the removal of the C5b-9 complex from the membrane; and 2) expression of regulatory surface proteins such as C8bp, which inhibit C5b-9-mediated lysis. We propose that the C8bp mobilization by cytokines might provide an additional protection against complement attack by its known interference with the C5b-9 assembly.

Blood Proteins↗
PubMed2006

Schistosoma japonicum and S. mansoni cercariae: different effects of protein in medium, of mechanical stress, and of an intact complement system on in vitro transformation to schistosomula.

The cercariae of Schistosoma japonicum were subjected in vitro to treatments known for Schistosoma mansoni to generate schistosomula-like organisms. As a technical prerequisite to pipette or to otherwise handle the sticky cercariae of S. japonicum, the addition of protein to water or medium was found to abolish the stickiness of cercariae of this species. Shearing forces exerted in vitro by syringe (22 G) passage are known since long to fully transform S. mansoni cercariae, but this treatment was found to be much less efficient with S. japonicum. Thus, even with very narrow needles (27 G), complete transformation of cercariae was not obtained with S. japonicum. Complement, provided by fresh human serum, is also well known to induce rapid transformation of S. mansoni cercariae with subsequent killing of the schistosomula. This treatment of S. japonicum cercariae induced degeneration of the tails and strongly promoted the transformation to schistosomula-like organisms, but at a much slower pace. These effects were absent from sera either heat-inactivated or depleted of factor B or of complement component C8, but were restored after adding the purified respective complement components. The schistosomula-like organisms of S. japonicum were not susceptible to lysis after 1 day of in vitro culture in the presence of 50% fresh human serum, although both cercariae and schistosomula of S. mansoni were killed under these conditions. In conclusion, the dynamics of in vitro transformation of S. japonicum cercariae differ significantly from those of S. mansoni, and complement has a major transformation-promoting activity.

Animals↗
PubMed2006

Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni.

Schistosomes are believed to evade complement-mediated damage by expression of complement inhibitory proteins. Our previous results [Deng, J., Gold, D., LoVerde, P.T., Fishelson, Z., 2003. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect. Immun. 71, 6402-6410.] have demonstrated that paramyosin (Pmy) of the blood fluke S. mansoni binds to the human complement proteins C8 and C9, inhibits complement activation at the terminal stage and protects the parasite from complement-mediated damage. In order to locate the Pmy binding site to C8 and C9, various fragments of Pmy cDNA were PCR-cloned into a pET28a bacterial expression vector. Recombinant His-tagged Pmy fragments were expressed in BL21 Escherichia coli and purified over a nickel-nitrilotriacetic acid column. Binding assays by Western blotting with monoclonal anti-His antibody demonstrated that PmyCC (Pmy amino acids (744)Asp-(866)Met) was the only Pmy fragment that bound to human C8 and C9. Functional analyses demonstrated that PmyCC inhibited hemolysis of rabbit erythrocytes and of antibody-sensitized sheep erythrocytes by human complement. Importantly, PmyCC inhibited in vitro killing of trypsin-sensitized schistosomula of S. mansoni by human complement. In the presence of PmyCC, Zn(2+)-induced C9 polymerization was inhibited. Most of the immunodominant B-cell antigenic epitopes of Pmy are present in the PmyCC region, as antibodies collected from mice immunized with recombinant Pmy bound primarily to PmyCC. Taken together, this study has mapped the complement regulatory domain in Pmy, capable of binding to C8 and C9 and preventing polyC9 formation, to its C-terminal region.

Animals↗
PubMed1985

Biosynthesis of the third (C3), eighth (C8), and ninth (C9) complement components by guinea pig hepatocyte primary cultures.

In the present report guinea pig hepatocyte primary cultures were established in order to study the synthesis of the eighth (C8) and ninth (C9) complement component. As reference-protein, the third complement component (C3) was measured antigenetically and hemolytically. Synthesis of C8 and C9 was determined by means of the hemolytic activity of the culture supernatant harvested every 24 h during a 6-day incubation period in vitro. The data to be reported demonstrated that the hepatocytes are able to synthesize spontaneously and secrete C8 and C9; in their culture medium a hemolytic activity of about 15-25 X 10(8) em/10(6) cells/24 h for C8 and of 25-90 x 10(8) em/10(6) cells/24 h for C9 were found. The same hepatocyte cultures produced 2500-6000 micrograms/10(6) cells/24 h of C3. Hemolytic C3 activity was also found in the culture media. The synthesis of C8 and C9 could be reversibly inhibited by addition of 30-50 micrograms cycloheximide per ml of culture medium. The kinetics of synthesis show a slight decrease after the first day of culture and a recovery in the following days up to a rate that is two- to threefold higher than that of the first day. The data suggest that hepatocytes could contribute to the production of C8 and C9 present in the plasma.

Animals↗
PubMed1982

Interaction of rabbit spermatozoa and serum complement components.

Unheated rabbit and human sera were found to induce acrosomal loss in rabbit spermatozoa, while similar concentrations of heated sera did not. In addition, human serum did not induce acrosomal loss when pretreated with antiserum to complement component C8, suggesting that acrosomal loss in unheated serum is caused by the membrane attack complex of complement. Human serum complement anaphylatoxins did not induce acrosomal loss, although they are known to induce exocytosis of secretory granules in other cell types. When incubated directly in human or rabbit sera, rabbit spermatozoa fixed complement; i.e., reduced the potential hemolytic activity of the sera. Fixation was suppressed by adding EGTA to reduce free calcium. This indicates that rabbit spermatozoa fix complement by initiating the classical pathway to complement activation. Initiation requires the presence of cell-bound immunoglobulins and the subsequent binding of complement component C1q. Immunoglobulins were detected in detergent extracts of washed ejaculated spermatozoa by a solid-phase radioimmunoassay, and the binding of 125 I-human C1q was detected on samples of living ejaculated spermatozoa. Seminal plasma was found to inhibit complement-induced hemolysis of erythrocytes. These results suggest that, in the absence of seminal plasma, spermatozoa may activate complement where it is present in the male or female tract.

Acrosome↗
PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of Salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane.

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.

Blood Bactericidal Activity↗
PubMed2001

Detection of mRNA for the terminal complement components C5, C6, C8 and C9 in human umbilical vein endothelial cells in vitro.

Human umbilical vein endothelial cells (HUVEC) have previously been shown to synthesize the functional terminal pathway of complement based on the detection by radioimmunoassay of the terminal complement complex (TCC) on coincubated agarose beads. In addition, C7 secretion by these cells in amounts comparable to C3, as well as C7 mRNA, has recently been demonstrated. However, it has not been possible to detect C5-6 and C8 in the fluid phase, and only trace amounts of soluble C9. Against this background we examined whether mRNA for the remaining terminal complement factors was present in HUVEC. By the use of reverse transcription (RT)-polymerase chain reaction (PCR) and Northern blot the presence of mRNA for complement factors C5, C6, C8 and C9 was demonstrated.

Base Sequence↗
PubMed1997

Binding of human and rat CD59 to the terminal complement complexes.

CD59-antigen (protectin) is a widely distributed glycolipid-anchored inhibitor of complement lysis. CD59 interacts with complement components C8 and C9 during assembly of the membrane attack complex (MAC). To evaluate species specificity of these interactions we have in the present study examined cross-species binding of isolated human and rat CD59 to the terminal complement components C8 and C9. By using primarily soluble CD59 isolated from urine (CD59U) potentially non-specific binding interactions of the phospholipid portion of the membrane forms of CD59 could be avoided. Sucrose density gradient ultracentrifugation analysis showed that human CD59U bound to both human and rat C8 in the SC5b-8 complexes. Similar binding occurred when rat CD59U was used. The degree of binding did not significantly differ between the heterologous and homologous CD59-C8 combinations. C9 from both species inhibited the binding of CD59 to soluble SC5b-8. In ligand blotting analysis human and rat CD59U bound to human and rat C8 alpha gamma-subunit and C9. Binding of human and rat CD59U was stronger to human than rat C9. In plate binding assays the erythrocyte form of CD59 (CD59E) bound to both human and rat C8. Binding of CD59E to heterologous C9 was considerably weaker than to homologous C9. Our results imply that the reciprocal binding sites between C8 and CD59 and to a lesser degree between CD59 and C9 are conserved between human and rat. Interactions of CD59 with the terminal C components are thus species selective but not 'homologously restricted'.

Animals↗
PubMed2003

Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin.

Larvae and adults of the parasitic blood fluke Schistosoma mansoni are resistant to killing by human complement. An earlier search by Parizade et al. for a schistosome complement inhibitor identified a 94-kDa surface protein which was named SCIP-1 (M. Parizade, R. Arnon, P. J. Lachmann, and Z. Fishelson, J. Exp. Med. 179:1625-1636, 1994). Following partial purification and analysis by mass spectrometry, we have determined SCIP-1 to be a surface-exposed form of the muscle protein paramyosin. As shown by immunofluorescence, anti-paramyosin antibodies label the surface of live schistosomula and adult worms. Like SCIP-1, purified native paramyosin reacts with a polyclonal rabbit anti-human CD59 antiserum, as shown by Western blot analysis. Also, the human complement components C8 and C9 bind to recombinant and native paramyosin. Analysis of paramyosin binding to fragments of C9 generated by thrombin or trypsin has demonstrated that paramyosin binds to C9 at a position located between Gly245 and Arg391. Paramyosin inhibited Zn(2+)-induced C9 polymerization and poly-C9 deposition onto rabbit erythrocytes (E(R)). In addition, paramyosin inhibited lysis of E(R) and of sensitized sheep erythrocytes by human complement. Finally, anti-paramyosin antibodies enhanced in vitro killing of schistosomula by normal and C4-depleted human complement. Taken together, these findings suggest that an exogenous form of S. mansoni paramyosin inhibits activation of the terminal pathway of complement and thus has an important immunomodulatory role in schistosomiasis.

Animals↗
PubMed1999

Phylogenetic analysis of the homologous proteins of the terminal complement complex supports the emergence of C6 and C7 followed by C8 and C9.

The plasma complement system comprises several activation pathways that share a common terminal route involving the assembly of the terminal complement complex (TCC), formed by C5b-C9. The order of emergence of the homologous components of TCC (C6, C7, C8alpha, C8beta, and C9) has been determined by phylogenetic analyses of their amino acid sequences. Using all the sequence data available for C6-C9 proteins, as well as for perforins, the results suggested that these TCC components originated from a single ancestral gene and that C6 and C7 were the earliest to emerge. Our evidence supports the notion that the ancestral gene had a complex modular composition. A series of gene duplications in combination with a tendency to lose modules resulted in successive complement proteins with decreasing modular complexity. C9 and perforin apparently are the result of different selective conditions to acquire pore-forming function. Thus C9 and perforin are examples of evolutionary parallelism.

Amino Acid Sequence↗