Binding sites involved in the formation of the C3(H2O)-factor B complex of the alternative pathway of complement

1994 ◽  
Vol 22 (1) ◽  
pp. 2S-2S ◽  
Author(s):  
SAMANTHA C. WILLIAMS ◽  
ROBERT B. SIM
Keyword(s):  
Binding Sites ◽  
Alternative Pathway ◽  
Factor B ◽  
Alternative Pathway Of Complement

Complement factor B and the alternative pathway of complement activation in bovine milk

2002 ◽  
Vol 69 (1) ◽  
pp. 1-12 ◽  
Author(s):  
PASCAL RAINARD
Keyword(s):  
Heat Treatment ◽  
Complement Activation ◽  
Alternative Pathway ◽  
Bovine Milk ◽  
Limiting Factor ◽  
Complement Factor ◽  
Factor B ◽  
Mild Heat ◽  
Alternative Pathway Of Complement ◽  
The Mean

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2·06 μg/ml (±0·18, SEM), 0·57% of the mean value found in serum (360 μg/ml). This proportion was comparable to that of serum albumin (0·63% of serum value) but less than the proportion of C3 in milk (2·71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 °C for 3 min or 50 °C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 °C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.

scholarly journals Amino acid sequence of the Bb fragment from human complement Factor B. Alignment of the cyanogen bromide-cleavage peptides

1983 ◽  
Vol 209 (1) ◽  
pp. 51-60 ◽  
Author(s):  
J Gagnon ◽  
D L Christie
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Human Factor ◽  
Alternative Pathway ◽  
Amino Acid Sequences ◽  
Complement Factor ◽  
Factor B ◽  
Arginine Residues ◽  
Alternative Pathway Of Complement ◽  
Cnbr Cleavage

The alignment of all the CNBr-cleavage peptides of fragment Bb from human Factor B (a component of the alternative pathway of complement) was determined. This was derived from cleavage of the fragment Bb at arginine residues by using trypsin and clostripain. Details of the isolation and amino acid sequences of these peptides are given. Together with previously published N-terminal sequences of the CNBr-cleavage peptides [Christie & Gagnon (1982) Biochem. J. 201, 555-567], this provides the amino acid sequence of the N-terminal half of fragment Bb.

scholarly journals Role of Complement in Protection against Cryptococcus gattii Infection

2008 ◽  
Vol 77 (3) ◽  
pp. 1061-1070 ◽  
Author(s):  
Kileen L. Mershon ◽  
Alex Vasuthasawat ◽  
Gregory W. Lawson ◽  
Sherie L. Morrison ◽  
David O. Beenhouwer
Keyword(s):  
Complement Activation ◽  
Alternative Pathway ◽  
Complement Pathway ◽  
Wild Type ◽  
Complement Components ◽  
Factor B ◽  
Alternative Pathway Of Complement ◽  
Deficient Mice

ABSTRACT Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.

scholarly journals The induction of macrophage spreading by factor B of the properdin system.

1979 ◽  
Vol 149 (2) ◽  
pp. 372-386 ◽  
Author(s):  
O Götze ◽  
C Bianco ◽  
Z A Cohn
Keyword(s):  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Serine Proteinase ◽  
Cell Spreading ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocytes ◽  
Mouse Serum ◽  
Factor B ◽  
Mouse Macrophages ◽  
Alternative Pathway Of Complement

Unstimulated mouse peritoneal macrophages attached to a glass substratum responded to activated human factor B (Bb) of the properdin system but not to native factor B with rapid spreading and a concomitant increase in their apparent surface area. Excellent correlation of the distribution of Bb protein and cell-spreading activity was found upon purification of Bb by ion-exchange and molecular seive chromatography and alkaline polyacrylamide gel electrophoresis. 1.6 microgram of purified Bb was sufficient to induce spreading in 50% of 5 x 10(4) glass attached macrophages within 1-2 h at 37 degrees C. Treatment of Bb with di-isopropyl-fluorophosphate indicated that the intact catalytic site of the serine-proteinase Bb was required for the initiation of macrophage spreading. The involvement of factor B in the induction of rapid cell spreading could also be indirectly demonstrated in an autologous system in which F(ab')2 fragments of an antiserum to mouse B prevented mouse macrophages from spreading in response to complement-activated mouse serum. These experiments suggest a role for factor B and the alternative pathway of complement fixation in the localization of mononuclear phagocytes to areas of inflammation.

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

1978 ◽  
Vol 148 (6) ◽  
pp. 1722-1727 ◽  
Author(s):  
R D Schreiber ◽  
H J Müller-Eberhard
Keyword(s):  
Dose Response ◽  
Hemolytic Activity ◽  
Plasma Proteins ◽  
Alternative Pathway ◽  
Serum Concentrations ◽  
Response Curves ◽  
Component Mixture ◽  
Factor B ◽  
Sheep Erythrocytes ◽  
Alternative Pathway Of Complement

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.

Complement-Dependent Activation Of Plasminogen By Activated Factor B (Bb) Of The Alternative Pathway Of Complement

1981 ◽  
Author(s):  
J S Sundsmo ◽  
L M Wood
Keyword(s):  
Plasminogen Activator ◽  
Alternative Pathway ◽  
Human Monocytes ◽  
Serine Esterase ◽  
Cellular Mechanisms ◽  
Human Fibrinogen ◽  
Factor B ◽  
Molecular Weights ◽  
Alternative Pathway Of Complement ◽  
Native Factor

Activated Factor B (Bb) of the alternative pathway of complement (APC) induces human monocytes and murine macrophages to spread on a glass substrate (Sundsmo and Gątze (1980), Cell. Immunol., 52:1). In studies designed to investigate the effects of Bb on plasminogen activator secretion by human monocytes, it became apparent that Bb possesses innate plasminogen activator (PA) activity. PA activity of Factor Bb was determined by release of radiolabeled fibrin peptides (modified fibrin plate assay) over 4 hrs. at 37°C using purified human plasminogen, 125I-labeled human fibrinogen, and 20% fresh normal human serum as a source of thrombin. Native Factor B did not express significant PA activity, however, purified Bb, or Bb in complex with cobra venom factor released, 52±8% and 79±4%, respectively, of the 125I- fibrin that is released by urokinase. Intermediate complement complexes on rabbit erythrocytes (Er) were tested for their PA activity: ErC3b, Bb released 76±7% of 125I-fibrin, and control Er, C3, and B released <15%. Factor D was without activity. Cleavage of plasminogen by Factor B was investigated by incubating cellular complement intermediates (ErC3b, Bb; EsC3b, Bb, NF) 30 min./37°C with 125iI-labeled plasminogen; and, conducting SDS-PAGE (reducing conditions) to separate native plasminogen from the heavy and light chains of plasmin. It was found that Bb cleaves predominantly the Lys-form of plasminogen to generate fragments with apparent molecular weights of 72,000 and 30,000. As a control, 125I-labeled plasminogen was incubated with Er, C3, or D, and <10% cleavage was observed; EsC3b or native Factor B cleaved <5%. Affinity-purified goat anti-B Ig inhibited Factor Bb- dependent plasminogen cleavage by 99%. These results suggest that activated Factor B, the central serine esterase of the APC, can serve as a plasminogen activator and raise the possibility that Factor B may play a role in initiation of fibrinolysis as well as in complement-dependent humoral and cellular mechanisms of immunity.

scholarly journals Interaction of beta1H globulin with cell-bound C3b: quantitative analysis of binding and influence of alternative pathway components on binding.

1978 ◽  
Vol 147 (6) ◽  
pp. 1792-1805 ◽  
Author(s):  
D H Conrad ◽  
J R Carlo ◽  
S Ruddy
Keyword(s):  
Quantitative Analysis ◽  
Binding Sites ◽  
Alternative Pathway ◽  
Radioactive Tracer ◽  
Scatchard Analysis ◽  
Negative Cooperativity ◽  
Factor B ◽  
Tracer Techniques ◽  
Dose Dependent Increase ◽  
Dose Dependent

Purified beta1H globulin (beta1H) was shown to bind to C3b coated cells by both immunofluorescent and radioactive tracer techniques. With EAC43, the amount of beta1H bound was directly proportional to the amount of C3 used to prepare the cells; EA, EAC14 and EAC14oxy2 bound very small amounts of beta1H. The C3b binding site on beta1H was labile in that not all of the purified 125I-beta1H was capable of binding to C3b, even when an excess of cell-bound C3b was present. Scatchard analysis of binding of beta1H to C3b-coated cells indicated an equilibrium constant of 10(9) L/M. Deviations from linearity were regularly found on Scatchard analyses. This was consistent with the hypothesis that the beta1H binding sites exhibit negative cooperativity in that as more sites become occupied, it becomes more difficult to fill the remaining sites. The stoichiometry of the reaction between C3b and beta1H was examined using EAC14oxy23 prepared with 131I-C3 and beta1H labeled with 125I. Between 0.5--0.8 beta1H molecules were bound per C3b molecule. Other alternative pathway components influenced the binding of 125I-beta1H to cell bound C3b. Both C3b and native C3 inhibited binding of labeled beta1H at an efficiency approximately 1/1,000 that of unlabeled beta1H. Factor B inhibited binding with 1/280 the efficiency of unlabeled beta1H. Properdin caused a dose-dependent increase in the binding of beta1H; this enhancement was abrogated if B was also present in the reaction mixture. Scatchard analysis indicated that the enhancement of beta1H binding by P resulted in an increased number of available binding sites rather than an increase in the affinity of binding.

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