scholarly journals Analysis of the interaction between properdin and factor B, components of the alternative-pathway C3 convertase of complement

1988 ◽  
Vol 253 (3) ◽  
pp. 667-675 ◽  
Author(s):  
T C Farries ◽  
P J Lachmann ◽  
R A Harrison
Keyword(s):  
Alternative Pathway ◽  
Direct Interaction ◽  
Fluid Phase ◽  
Cross Linking ◽  
Binding Studies ◽  
Factor B ◽  
Activation Products ◽  
Initial Interaction ◽  
High Concentrations ◽  
A Site

The interactions between Factor B (B), its activation products Ba and Bb, properdin (P) and C3i or C3b, components that together form the alternative-pathway C3 convertase enzyme of human complement, have been analysed. Fluid-phase complexes of the purified components C3i, B and P were probed with the homobifunctional cross-linking reagent disuccinimidyl tartarate, and efficient cross-linking of B to P was observed. The 140 kDa B-P conjugate formed was cleaved by Factor D to yield a single product of 85 kDa. This is consistent with a Ba-P heterodimer, and suggests that the initial interaction of B and P includes an interaction of P with the Ba domain of intact B. (The Ba fragment is not retained in the active P-stabilized complex, C3bBbP). By contrast, no cross-linking of P to the Bb domain of B could be demonstrated. Binding studies on cellular intermediates also provided evidence for a site of interaction between B and P, with high concentrations of B inhibiting P binding to EAC3b (sheep erythrocytes coated with antibody and C3b). Neither isolated Ba nor Bb had any effect on the P-EAC3b interaction. High concentrations of B also accelerated the decay of the functional EAC3bBbP complex. These data indicate that the positive co-operativity of binding to C3i or to C3b between B and P is mediated, at least in part, through a direct interaction between B and P.

scholarly journals Modulation of complement activation on hemodialysis membranes by immobilized heparin.

1992 ◽  
Vol 2 (8) ◽  
pp. 1328-1337
Author(s):  
A K Cheung ◽  
C J Parker ◽  
J Janatova ◽  
E Brynda
Keyword(s):  
Complement Activation ◽  
Alternative Pathway ◽  
Membrane Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluid Phase ◽  
Factor H ◽  
Binding Studies ◽  
Factor B ◽  
Pathway Activity

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

scholarly journals The binding of human complement proteins C5, factor B, β1H and properdin to complement fragment C3b on zymosan

1981 ◽  
Vol 199 (3) ◽  
pp. 485-496 ◽  
Author(s):  
R G DiScipio
Keyword(s):  
Protein Modification ◽  
Alternative Pathway ◽  
Covalent Binding ◽  
Association Constants ◽  
Affinity Constant ◽  
Distinct Region ◽  
Binding Studies ◽  
Factor B ◽  
Complement Proteins ◽  
Competition Binding

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.

Interaction of C3b2–IgG complexes with complement proteins properdin, factor B and factor H: implications for amplification

2000 ◽  
Vol 349 (1) ◽  
pp. 217-223
Author(s):  
Emiliana JELEZAROVA ◽  
Anna VOGT ◽  
Hans U. LUTZ
Keyword(s):  
Present Report ◽  
Alternative Pathway ◽  
Fluid Phase ◽  
Factor H ◽  
Complement Protein ◽  
Factor B ◽  
Physiological Conditions ◽  
Amplification Loop ◽  
Target Molecules ◽  
Properdin Factor B

Nascent C3b can form ester bonds with various target molecules on the cell surface and in the fluid phase. Previously, we showed that C3b2-IgG complexes represent the major covalent product of C3 activation in serum [Lutz, Stammler, Jelezarova, Nater and Späth (1996) Blood 88, 184-193]. In the present report, binding of alternative pathway proteins to purified C3b2-IgG complexes was studied in the fluid phase by using biotinylated IgG for C3b2-IgG generation and avidin-coated plates to capture complexes. Up to seven moles of properdin ‘monomer’ bound per mole of C3b2-IgG at physiological conditions in the absence of any other complement protein. At low properdin/C3b2-IgG ratios bivalent binding was preferred. Neither factor H nor factor B affected properdin binding. On the other hand, properdin strongly stimulated factor B binding. Interactions of all three proteins with C3b2-IgG exhibited pH optima. An ionic strength optimum was most pronounced for properdin, while factor B binding was largely independent of the salt concentration. C3b2-IgG complexes were powerful precursors of the alternative pathway C3 convertase. In the presence of properdin, C3 convertase generated from C3b2-IgG cleaved about sevenfold more C3 than the enzyme generated on C3b. C3b2-IgG complexes could therefore maintain the amplification loop of complement longer than free C3b.

scholarly journals The Human Complement Regulator Factor H Binds Pneumococcal Surface Protein PspC via Short Consensus Repeats 13 to 15

2002 ◽  
Vol 70 (10) ◽  
pp. 5604-5611 ◽  
Author(s):  
Thomas G. Duthy ◽  
Rebecca J. Ormsby ◽  
Eleni Giannakis ◽  
A. David Ogunniyi ◽  
Uwe H. Stroeher ◽  
...  
Keyword(s):  
Alternative Pathway ◽  
Regulatory System ◽  
Surface Protein ◽  
Fluid Phase ◽  
Direct Consequence ◽  
Factor H ◽  
Negative Regulator ◽  
Short Consensus Repeats ◽  
High Concentrations ◽  
Complement Regulator

ABSTRACT The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCΔPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.

scholarly journals Complement receptor is an inhibitor of the complement cascade.

1981 ◽  
Vol 153 (5) ◽  
pp. 1138-1150 ◽  
Author(s):  
K Iida ◽  
V Nussenzweig
Keyword(s):  
Alternative Pathway ◽  
Protein A ◽  
Fluid Phase ◽  
Local Concentration ◽  
Classical Pathway ◽  
Strong Inhibitor ◽  
Complement Components ◽  
Serum Inhibitor ◽  
Irreversible Effects ◽  
High Concentrations

A glycoprotein from the membrane of human erythrocytes has been identified as a receptor for C3b (CR1). It promotes the dissociation of the alternative pathway C3 convertase C3b,Bb and the cleavage of C3b by C3b/C4b inactivator. We find that CR1 also inactivates the C3 and C5 convertases of the classical pathway. CR1 inhibits the consumption of C3 by C3 convertase EAC142 and enhances the decay of C4b,2a sites. On a weight basis, CR1 is approximately 5-10 times more active than C4 binding protein, a serum inhibitor of C4b,2a. The binding of 125I-CR1 to EAC14 cells is inhibited by C2. Therefore, it is likely that CR1 and C2 compete for a site on C4b. CR1 inhibited C5 convertase even more effectively, but had no effect on the assembly of the late complement components. At high concentrations, CR1 alone has no irreversible effects on cell-bound C4b. In the fluid phase, CR1 can function as a cofactor for the cleavage of the alpha' chain of C4b by C3b/C4b inactivator. A well-known function of CR1 is to promote adherence of microbes or immune complexes bearing C3b and C4b to cells. This interaction could result in a microenvironment damaging to the plasma membrane of the responding cell because the extrinsic C3b and C4b fragments can serve as additional sites of assembly of enzymes of the cascade. We therefore wish to propose that CR1 on the surface of cells supplies an increased local concentration of a strong inhibitor of the amplifying enzymes of the complement system and provides cells with a mechanism for circumventing damage when they bind C3b- and C4b-bearing substrates.

Prothrombin Activation By Factor B (Bb) Of The Alternative Pathway Of Complement

1981 ◽  
Author(s):  
D S Fair ◽  
J S Sundsmo ◽  
B S Schwartz ◽  
T S Edgington ◽  
H J Müller-Eberhard
Keyword(s):  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Fluid Phase ◽  
Classical Pathway ◽  
Complement Factor ◽  
Factor X ◽  
Serine Esterase ◽  
Complement Pathway ◽  
Factor B

It has been reported that complement activation in serum by inulin, endotoxin, staphlococcal protein A, or heat aggregated γ-globulin results in consumption of prothrombin (Zimmerman and Müller-Eberhard, 1971, J. Exp. Med., 124;1601). The availability of purified complement proteins and coagulation factors prompted the study of complement factor(s) mediating prothrombin consumption. The ability of the classical and alternative pathway C3 convertase to cleave prothrombin was investigated by incubating 125I-labeled prothrombin (30min./37°C) with erythrocytes (E) bearing intermediate complexes of complement. Cleavage of prothrombin was analyzed by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. 1) EC3b, Bb, NF; 2) EAC4oxy2,3b, Bb; and 3) Factor Xa were found to cleave prothrombin 43.2%; 20.2%; and 100% respectively. Prothrombin cleavage products generated by complement were similar to those produced by Factor Xa. The controls 1) EC3b; 2) EC3b, B; and 3) Edid not cleave prothrombin. Cells bearing the classical pathway C3-convertase EAC4oxy2 cleaved prothrombin at a slower rate (1.2%). Fluid phase C3-convertase formed with purified cobra venom factor (CVF), CVF, Bb, also cleaved prothrombin (28.7%). The precursor proteins of the enzyme, (CVF, B, or D) were incapable of cleaving prothrombin, whereas purified Bb, alone was active. Cleavage of prothrombin required Ca++ and Mg++. Cleavage by EC3b, Bb was inhibited by affinity purified goat anti-B immunoglobulin and by Trasylol. Both reagents were also found to inhibit Factor B hemolytic activity. These results suggest that activated Factor B (Bb), the central serine esterase of the alternative complement pathway, may cleave prothrombin directly without the intervention of Factor X. The observation of a complement pathway prothrombinase emphasized the integration of mediation pathways and suggests that previously unrecognized routes for initiating the coagulaion pathways may still exist.

scholarly journals Analysis of the interactions between properdin, the third component of complement (C3), and its physiological activation products

1988 ◽  
Vol 252 (1) ◽  
pp. 47-54 ◽  
Author(s):  
T C Farries ◽  
P J Lachmann ◽  
R A Harrison
Keyword(s):  
Ionic Strength ◽  
Alternative Pathway ◽  
Fluid Phase ◽  
Complement C3 ◽  
Initial Product ◽  
Native Form ◽  
Factor I ◽  
The Third ◽  
Activation Products ◽  
Third Component Of Complement

The interactions of properdin with both surface-bound and fluid-phase C3 (the third component of complement) and its activation products have been investigated by using a purified preparation of the ‘native’ form. At physiological ionic strength, a weak interaction with cell-bound C3b (the larger activation fragment of C3) could be demonstrated. In the presence of Factor B this interaction was enhanced, and further enhancement was seen when C3bBb sites were formed on the erythrocytes. The avidities of properdin for cell-bound iC3b (the initial product of Factors I and H action on C3b) and C3b were compared at low ionic strength, with that measured for iC3b being less than that for C3b. In contrast, the affinities of properdin for fluid-phase C3b, iC3b and C3c (the larger product of Factors I and H or CR1 (the C3b receptor) action on iC3b) were all very similar, and apparently much weaker than that for cell-bound C3b. No interaction with either native C3 or, more surprisingly, C3i (haemolytically inactive C3) could be detected. Properdin also inhibited Factor I binding to, and action upon, cell-bound C3b, but did not inhibit Factor I action on fluid-phase C3b. These data permit a more detailed description of the roles of properdin in the alternative pathway of complement activation, emphasizing its importance in concentrating activation at the activating surface.

scholarly journals Solubilization of immune precipitates by six isolated alternative pathway proteins.

1981 ◽  
Vol 154 (6) ◽  
pp. 1743-1751 ◽  
Author(s):  
T Fujita ◽  
Y Takata ◽  
N Tamura
Keyword(s):  
Alternative Pathway ◽  
Fluid Phase ◽  
Molar Ratio ◽  
Phase Reaction ◽  
Total Consumption ◽  
Antibody Molecule ◽  
Factor B ◽  
Alternative Pathway Of Complement ◽  
Antigen Antibody ◽  
Solubilization Of Immune Complexes

Immune precipitates were solubilized by the alternative pathway of complement assembled from isolated proteins, i.e., C3, factor B, factor D, properdin, C3b inactivator (C3bINA), and beta 1H. The kinetic curves of solubilization in the isolated system and in EGTA-serum were virtually indistinguishable. No requirement of other factors was apparent. Deletion of C3bINA and beta 1H from the complete mixture caused total consumption of C3 in the fluid phase and resulted in neither C3 binding to the complexes nor solubilization. Thus, the presence of a regulated fluid-phase reaction is essential for efficient fixation of C3 and the consequent solubilization. In addition, properdin plays an essential role in the complement-mediated solubilization in the presence of the two regulators. A large amount of C3 was incorporated into the antigen-antibody lattice. Solubilization of immune complexes started after the binding of one C3 molecule to one antibody molecule in the complexes, and the molar ratio of C3:antibody in the solubilized complexes also is approximately 1.

Abstract WMP45: Targeted Inhibition of the Alternative Complement Pathway Enhances Rehabilitation-Induced Cognitive and Motor Recovery in a Murine Model of Chronic Stroke

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Ali Alawieh ◽  
Meredith Andersen ◽  
DeAnna L Adkins ◽  
Stephen Tomlinson
Keyword(s):  
Cognitive Performance ◽  
Infarct Volume ◽  
Alternative Pathway ◽  
Artery Occlusion ◽  
Motor Recovery ◽  
Acute Injury ◽  
Neuroprotective Effects ◽  
Recovery Processes ◽  
Targeted Inhibition ◽  
A Site

Introduction: Activation of inflammatory cascades after stroke exacerbate acute injury and limit response to rehabilitation (rehab). The complement (C) system is a prominent component of inflammatory injury after stroke, but can also contribute to neuroprotective recovery processes. We investigated the neuroprotective effects of CR2fH, a site-targeted C inhibitor that specifically inhibits the alternative pathway and prevents amplification of the C cascade. We assessed how CR2fH affects chronic outcomes, and how acute C inhibition impacts the response to rehab therapy. Methods: Mice were subjected to 1hr middle cerebral artery occlusion and 15 days of reperfusion. Mice were treated with CR2fH or vehicle and then randomized to normal housing or an enriched environment (EE) to model cognitive and motor rehab. Animals were assessed for infarct volume using MRI at days 4 and 14 after injury, and for their performance on several motor and cognitive tasks. Expression of inflammatory markers in the brain was assessed using high throughput Nanostring analysis and immunostaining. Results: Compared to vehicle or EE only, treatment with CR2fH 90 mins after cerebral ischemia significantly reduced infarct volume and improved motor and cognitive performance up to 15 days, as assessed with mNSS scores, corner task, locomotor activity, pasta handling, and passive avoidance tasks (p’s<0.01). Less prominent, although still significant improvement was also achieved when CR2fH was administered 6 or 12 hrs. after ischemia (p’s<0.05). EE alone did not significantly reduce infarct volume or improve performance on motor tasks, but resulted in a significant improvement in cognitive performance compared to vehicle (p’s<0.05). A combination of EE and CR2fH therapy resulted in a significant potentiation of cognitive and motor recovery compared to either single intervention (p’s<0.05). CR2fH related behavioral improvements correlated with reduction in C deposition and inflammatory microglial activation during recovery. Conclusions: Since a humanized version of CR2fH, TT30, was shown to be safe, tolerated and nonimmunogenic in humans, targeted inhibition of alternative C pathway is a candidate therapy to promote recovery and potentiate response to rehab after stroke.

Sign in / Sign up

Export Citation Format

Share Document