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.

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.

Complement activation and white cell sequestration in postischemic skeletal muscle

1990 ◽  
Vol 259 (2) ◽  
pp. H525-H531
Author(s):  
B. B. Rubin ◽  
A. Smith ◽  
S. Liauw ◽  
D. Isenman ◽  
A. D. Romaschin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Complement Activation ◽  
Alternative Pathway ◽  
Ischemia Reperfusion ◽  
White Blood Cells ◽  
Classical Pathway ◽  
Myeloperoxidase Activity ◽  
Factor B ◽  
Complement Proteins ◽  
Muscle Ischemia

After skeletal muscle ischemia, tissue damage is augmented during reperfusion. White blood cells (WBCs) and complement proteins may participate in the reperfusion injury. The purpose of this study was to define the kinetics of classical and alternative pathway complement activation and WBC sequestration by postischemic skeletal muscle during the first 48 h of reperfusion in vivo. The isolated canine gracilis muscle model was used. Systemic levels of the complement proteins factor B (alternative pathway) and C4 (classical pathway) were quantitated by hemolytic assay. WBC sequestration was measured by gracilis arterial-venous WBC differences and tissue myeloperoxidase activity. Reperfusion was associated with an 18% decrease in systemic factor B levels but no consistent change in systemic C4 levels. WBCs were sequestered during the first 4 h of reperfusion, and tissue myeloperoxidase activity was elevated 97-fold after 48 h of reperfusion. These results suggest that skeletal muscle ischemia-reperfusion stimulates 1) activation of the alternative but not the classical complement pathway and 2) an immediate and prolonged sequestration of WBCs.

scholarly journals SCH 48973: a potent, broad-spectrum, antienterovirus compound.

1997 ◽  
Vol 41 (6) ◽  
pp. 1220-1225 ◽  
Author(s):  
P J Buontempo ◽  
S Cox ◽  
J Wright-Minogue ◽  
J L DeMartino ◽  
A M Skelton ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Specific Interaction ◽  
Kinetic Studies ◽  
Human Enterovirus ◽  
Affinity Constant ◽  
Potential Candidate ◽  
Binding Studies ◽  
Competition Binding

SCH 48973 is a novel molecule with potent, selective, antienterovirus activity. In assays of the cytopathic effect against five picornaviruses, SCH 48973 had antiviral activity (50% inhibitory concentrations [IC50s]) of 0.02 to 0.11 microg/ml, with no detectable cytotoxicity at 50 microg/ml. SCH 48973 inhibited 80% of 154 recent human enterovirus isolates at an IC50 of 0.9 microg/ml. The antiviral activity of SCH 48973 is derived from its specific interaction with viral capsid, as confirmed by competition binding studies. The affinity constant (Ki) for SCH 48973 binding to poliovirus was 8.85 x 10(-8) M. In kinetic studies, a maximum of approximately 44 molecules of SCH 48973 were bound to poliovirus capsid. SCH 48973 demonstrated efficacy in a murine poliovirus model of enterovirus disease. SCH 48973 increased the survival of infected mice when it was administered orally at dosages of 3 to 20 mg/kg of body weight/day. Oral administration of SCH 48973 also reduced viral titers in the brains of infected mice. On the basis of its in vitro and in vivo profiles, SCH 48973 represents a potential candidate for therapeutic intervention against enterovirus infections.

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.

Electrochemical characterization of Cu(II) complexes of brain-related tau peptides

2021 ◽  
Vol 99 (7) ◽  
pp. 628-636
Author(s):  
Camilla Golec ◽  
Jose O. Esteves-Villanueva ◽  
Sanela Martic
Keyword(s):  
Metal Ions ◽  
Tau Protein ◽  
Metal Ion ◽  
Differential Pulse ◽  
Redox Couple ◽  
Peptide Sequence ◽  
Binding Studies ◽  
Biologically Relevant ◽  
Redox Active ◽  
Competition Binding

Metal ion dyshomeostasis plays an important role in diseases, including neurodegeneration. Tau protein is a known neurodegeneration biomarker, but its interactions with biologically relevant metal ions, such as Cu(II), are not fully understood. Herein, the Cu(II) complexes of four tau R peptides, based on the tau repeat domains, R1, R2, R3, and R4, were characterized by electrochemical methods, including cyclic voltammetry, square-wave voltammetry, and differential pulse voltammetry in solution under aerobic conditions. The current and potential associated with Cu(II)/(I) redox couple was modulated as a function of R peptide sequence and concentration. All R peptides coordinated Cu(II) resulting in a dramatic decrease in the current associated with free Cu(II), and the appearance of a new redox couple due to metallo–peptide complex. The metallo–peptide complexes were characterized by the irreversible redox couple at more positive potentials and slower electron-transfer rates compared with the free Cu(II). The competition binding studies between R peptides with Cu(II) indicated that the strongest binding affinity was observed for the R3 peptide, which contained 2 His and 1 Cys residues. The formation of complexes was also evaluated as a function of peptide concentration and in the presence of competing Zn(II) ions. Data indicate that all metallo–peptides remain redox active pointing to the potential importance of the interactions between tau protein with metal ions in a biological setting.

scholarly journals The purification and properties of the second component of human complement

1978 ◽  
Vol 171 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M A Kerr ◽  
R R Porter
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Factor ◽  
Human Complement ◽  
Terminal Amino Acid ◽  
Factor B ◽  
Antigenic Properties ◽  
Terminal Amino

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.

scholarly journals PAR recognition by multiple reader domains of PARP1 allosterically regulates the DNA-dependent activities and independently stimulates the catalytic activity of PARP1

2021 ◽  
Author(s):  
Waghela Deeksha ◽  
Suman Abhishek ◽  
Eerappa Rajakumara
Keyword(s):  
Catalytic Activity ◽  
Repair Process ◽  
Single Strand ◽  
Genomic Integrity ◽  
Post Translational Modification ◽  
Dna Breaks ◽  
Binding Studies ◽  
Competition Binding ◽  
Multiple Reader ◽  
Stimulation Of

Poly(ADP-ribosyl)ation is a post translational modification, predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single strand (SSB) and double strand (DSB) DNA breaks are bonafide stimulators of PARP1 activity. We identified that, in addition, single strand (ss) DNA also binds and stimulates the PARP1 activity. Poly(ADP-ribose) (PAR) is chemically similar to ssDNA. However, PAR mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT and WGR, hitherto uncharacterized, as PAR-specific reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared to PAR, but do not bind to DNA. Conversely, N-terminal domains, ZnF1 and ZnF2, of PARP1 recognize DNA but not PAR. Further competition binding studies suggest that PAR binding, allosterically releases DNA from PARP1. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampers the DNA dependent stimulation. Altogether, our work discovers dedicated PAR and DNA reader domains of the PARP1, and uncovers a novel mechanism of allosteric stimulation of the catalytic activity of PARP1 but retardation of DNA-dependent activities of PARP1 by its catalytic product PAR.

scholarly journals The alternative complement pathway is activated in protoporphyria patients during sun exposure

2020 ◽  
Author(s):  
Francesca Granata ◽  
Lorena Duca ◽  
Valentina Brancaleoni ◽  
Silvia Fustinoni ◽  
Giacomo De Luca ◽  
...  
Keyword(s):  
Light Exposure ◽  
Alternative Pathway ◽  
Global Radiation ◽  
Protoporphyrin Ix ◽  
Sun Exposure ◽  
Factor H ◽  
Factor B ◽  
Alternative Complement ◽  
Differential Involvement ◽  
The Complement System

ABSTRACTThe homeostasis of tissues in chronic disease is an important function of the alternative pathway (AP) of the complement system (CS). However, if not controlled, it may also be detrimental to healthy cells.Protoporphyria (PP) is a rare disease that causes photosensitivity at the visible light due to the accumulation of Protoporphyrin-IX in the dermis. The aim of this study was to deep the knowledge about the involvement of AP in PP photoreaction.Global radiation and UV data were provided from regional agency of environmental protection (ARPA). Properdin, Factor H (FH) and C5 levels were assessed in the serum collected during winter and summer from 19 PP patients and 13 controls..Properdin in winter and summer reflected a positive increase compared to controls. The values in summer were higher than winter. The C5 results were altered only in summer. The outcome was reversed for FH: in the winter, it was higher compared to the summer. A positive correlation was reported between properdin and C3 in summer; a negative tendency between Factor B (FB) and FH was detected.This study substantiated the differential involvement of AP depending on the increase in light exposure during the season, which was demonstrated with ARPA data. The enhanced systemic response could justify the malaise sensation of patients after long light exposure and can be exploited to elucidate the new therapeutic approach.

Complement in the Newborn Infant

PEDIATRICS ◽  
1979 ◽  
Vol 64 (5) ◽  
pp. 781-786
Author(s):  
Richard B. Johnston ◽  
Karl M. Altenburger ◽  
Alva W. Atkinson ◽  
Robert H. Curry
Keyword(s):  
Newborn Infant ◽  
Gestational Age ◽  
Hemolytic Activity ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Term Infants ◽  
Complement Components ◽  
Factor B ◽  
Serious Infection ◽  
Dual Pathway

Classical pathway activities and component concentrations in sera from newborns can be compared more realistically to normal adult values than to maternal values since activities and components are increased in maternal sera. Whole complement hemolytic activity appears to be subnormal in approximately half of term infants, with mean activity being about 70% to 90% of normal. Concentrations of Clq, C4, C2, C3, and C7 have been 60% to 100% of adult concentrations in term infants and somewhat less in preterm infants. Younger gestational age has been correlated with lower levels of total hemolytic activity, Clq, C4, and C3. Activity of the alternative pathway appears to be more frequently subnormal in newborn sera than does activity of the classical pathway. Factor B and properdin concentrations have varied from about 35% to 60% and 35% to 70% of adult values, respectively. Opsonization and hemolysis mediated by the alternative pathway have been subnormal in 15% to 75% of term infants, depending upon the assay. Gestational age appears to correlate with alternative pathway hernobytic activity and properdin concentration but not with concentration of factor B. Reductions such as these in single complement components and functions probably would not predispose an otherwise normal individual to infection. However, it seems likely that the multicomponent and dual pathway deficiencies found in neonates, especially in conjunction with the decreased phagocyte function known to exist in that population, could increase the likelihood of serious infection. Predicted infections with this configuration of abnormalities would be extracellular bacterial. Whether the newborn infant is actually predisposed to infection because of the complement deficiencies summarized here remains to be proved. This and other unanswered questions lead us to conclude that understanding of the complement system in the newborn is, pardon the expression, still in its infancy.

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.

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