scholarly journals Localization of the complement-component-C3b-binding site and the cofactor activity for factor I in the 38kDa tryptic fragment of factor H

1984 ◽  
Vol 224 (2) ◽  
pp. 389-398 ◽  
Author(s):  
J Alsenz ◽  
J D Lambris ◽  
T F Schulz ◽  
M P Dierich
Keyword(s):  
Binding Site ◽  
Gel Filtration ◽  
Fluid Phase ◽  
Factor H ◽  
Tryptic Fragment ◽  
Factor I ◽  
Close Proximity ◽  
Cofactor Activity ◽  
A Site ◽  
Sepharose 4B

Trypsin treatment of human factor H (H160) [enzyme/substrate ratio 1:100 (w/w), 30 min, 37 degrees C] generated a 38 kDa (H38) and a 142 kDa (H142) fragment linked by disulphide bonds (H38/142). The fragments were purified by reduction with 2-mercapto-ethanol, gel filtration on a Sephadex G-200 column and affinity chromatography with monoclonal anti-(factor H) antibody coupled to Sepharose 4B. This monoclonal antibody bound to a site in the 38 kDa fragment. To localize the C3b binding site in factor H we used two enzyme-linked immunosorbent assays (e.l.i.s.a.). For the first test, e.l.i.s.a. plates were coated with C3b; H160, H38/142, H38 and H142 were added, and their binding was monitored by goat anti-(factor H) and peroxidase-labelled rabbit anti-goat antibodies. Only intact factor H bound to the C3b-coated plates. For the second test, e.l.i.s.a. plates were coated with comparable amounts of factor H or its fragments, and C3b was offered at several dilutions. In contrast with the results from the first assay, C3b bound to intact factor H, H38/142 and H38 but not to H142, thus characterizing H38 as the fragment carrying the C3b-binding site. To identify the fragment responsible for the cofactor activity of factor H (cleavage of fluid-phase C3b by factor I), 125I-C3b was incubated with either H38 or H142 and factor I. H142 had no cofactor activity, whereas H38 had the same cofactor function as intact H. To further investigate the relationship between the C3b-binding site and the site of factor H essential for its cofactor activity, we made use of monoclonal antibodies directed against the H38. Those antibodies inhibiting the binding of C3b to H160 also inhibited the cofactor function, whereas those without effect on the C3b binding also did not interfere with the cofactor activity. This suggests that the C3b-binding site and the site essential for the cofactor activity of factor H are both localized in the 38 kDa tryptic fragment of factor H in close proximity or are identical.

scholarly journals Functional properties of membrane cofactor protein of complement

1989 ◽  
Vol 264 (2) ◽  
pp. 581-588 ◽  
Author(s):  
T Seya ◽  
J P Atkinson
Keyword(s):  
Complement System ◽  
Fluid Phase ◽  
Factor H ◽  
Classical Pathway ◽  
Membrane Cofactor Protein ◽  
Cleavage Reaction ◽  
Factor I ◽  
Thioester Bond ◽  
Cofactor Activity ◽  
The Complement System

Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.

scholarly journals Putative Coiled-Coil Structural Elements of the BBA68 Protein of Lyme Disease Spirochetes Are Required for Formation of Its Factor H Binding Site

2005 ◽  
Vol 187 (4) ◽  
pp. 1317-1323 ◽  
Author(s):  
John V. McDowell ◽  
Matthew E. Harlin ◽  
Elizabeth A. Rogers ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Binding Site ◽  
Immune Evasion ◽  
Coiled Coil ◽  
Factor H ◽  
Regulatory Proteins ◽  
Structural Elements ◽  
Binding Ability ◽  
Complement Regulatory Proteins ◽  
Factor I

ABSTRACT Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.

BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
K Suzuki ◽  
J Nishioka ◽  
H Kusumoto ◽  
Y Deyashiki
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Disulfide Bonds ◽  
Protein C ◽  
Binding Protein ◽  
Gel Filtration ◽  
Activated Protein C ◽  
Protein S ◽  
Cofactor Activity ◽  
Carboxyl Terminal

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

2003 ◽  
Vol 418 (2) ◽  
pp. 108-118 ◽  
Author(s):  
Anna M Blom ◽  
Lena Kask ◽  
Bala Ramesh ◽  
Andreas Hillarp
Keyword(s):  
Binding Protein ◽  
Factor H ◽  
Factor I ◽  
Cofactor Activity

scholarly journals Characterization of the lymphocyte membrane receptor for factor H (β1H- globulin) with an antibody to anti-factor H idiotype

1982 ◽  
Vol 155 (5) ◽  
pp. 1400-1411 ◽  
Author(s):  
JD Lambris ◽  
GD Ross
Keyword(s):  
B Cells ◽  
B Cell ◽  
Binding Site ◽  
B Lymphocyte ◽  
Sodium Dodecyl ◽  
Chain Structure ◽  
Factor H ◽  
Antigenic Structure ◽  
Lymphocyte Membrane ◽  
Factor I

Antibody to the binding site (idiotype) of anti-factor H was shown to have specificity for both B lymphocyte membrane H receptors and C3b. Goat F(ab')(2) anti-human H was purified by absorption and elution from H agarose and used for rabbit immunization to produce anti-anti-H (aaH). After absorption with nonimmune goat IgG, (125)I-labeled aaH bound to B lymphocytes and to sheep erythrocytes coated with C3b (EC3b) but did not bind to T lymphocytes or to EC3d. All B cell- and C3b-specific activities of the aaH were removed and subsequently recovered by absorption and elution of the antibody from either C3-agarose or goat-anti-H-agarose. This indicated that the aaH probably recognized a single common antigenic structure that was shared by anti-H, C3b, and the membranes of B cells. Affinity-purified aaH resembled H in that it bound to B cells, blocked the uptake of H onto B cell H receptors, and triggered B cells to release endogenous factor I (C3b inactivator). In addition, aaH functioned with factor I as either a cofactor for cleavage of fluid-phase C3b or a potentiator for cleavage of bound C3b. This same spectrum of C3 binding functions could not be demonstrated with either sheep anti-C3b or rabbit-anti-C3c. Analysis by sodium dodecyl sulfate- polyacrylamide get electrophoresis of the [(3)H]leucine intrinsically labeled B cell proteins reactive with the purified aaH revealed proteins of 100,000 M(r) and 50,000 M(r) without reduction, and after complete reduction of disulfide bonds, a single protein band of 50,000 M(r). This same protein molecular weight profile was also demonstrated with labeled B cell proteins that were absorbed and eluted from H-agarose. Thus, aaH is apparently specific for both B cell H receptors and C3b. However, because parallel analysis of C3b confirmed its known 115,000- and 75,000-M(r) polypeptide chain structure, the H receptor is probably not C3b and shares only the structure of the H binding site with C3b.

scholarly journals Prediction from sequence comparisons of residues of factor H involved in the interaction with complement component C3b

1996 ◽  
Vol 315 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Candida J. SOAMES ◽  
Antony J. DAY ◽  
Robert B. SIM
Keyword(s):  
Amino Acids ◽  
Human Factor ◽  
Factor H ◽  
Cdna Clones ◽  
Sequence Alignments ◽  
Sequence Comparisons ◽  
Factor I ◽  
Cofactor Activity ◽  
Human And Mouse ◽  
Bovine Factor

The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2–12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2–5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2–5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu-17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys-18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact.

scholarly journals Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies.

1982 ◽  
Vol 156 (1) ◽  
pp. 205-216 ◽  
Author(s):  
P J Lachmann ◽  
M K Pangburn ◽  
R G Oldroyd
Keyword(s):  
Monoclonal Antibodies ◽  
Complement Activation ◽  
Antigenic Determinant ◽  
Fluid Phase ◽  
Factor H ◽  
Alpha Chain ◽  
Factor I ◽  
Activation Products ◽  
Do So

The physiological breakdown of C3 has been studied using monoclonal anti-C3 antibodies, and it has been found that the later stages of this process--the breakdown of C3bi--is more complex than had previously been recognized. C3bi is the reaction product produced from C3b by the action of factor I which, in the presence of factor H, produces a double cleavage in the alpha chain of C3b. It is here reported that, both on cells and in the fluid phase, the breakdown of C3bi in serum gives rise to two products: C3c and the product previously described as alpha 2D, which we now propose to designate C3d,g. Alpha 2D differs from C3d in that it contains an additional fragment of approximately 8,000 mol wt that carries the antigenic determinant for the clone 9 monoclonal anti-C3 antibody. C3g cannot be precipitated by anti-C3 antisera and therefore behaves as a uni- or bideterminant antigen. The cleavage of C3d,g to C3d and C3g does not occur in sterile serum. It is also still uncertain what enzyme cleaves C3bi to C3c and C3d,g in plasma. Plasmin can do so in vitro, but plasminogen-depleted serum can still produce the cleavage. The antigenic determinant recognized by clone 9 in C3 is not exposed in C3 or C3b, but appears as a neoantigen in C3bi (and in C3d,g). Anti-C3g therefore is a potentially useful ligand for detecting complement-activation products. C3g represents a new, highly anionic C3 fragment and seems not to be identical with the C3e fragment described by others.

scholarly journals The amino-terminal module of the C4b-binding protein α-chain is crucial for C4b binding and factor I-cofactor function

1997 ◽  
Vol 323 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Ylva HÄRDIG ◽  
Andreas HILLARP ◽  
Björn DAHLBÄCK
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Binding Protein ◽  
Protein S ◽  
Chimeric Proteins ◽  
Amino Terminal ◽  
Factor I ◽  
Cofactor Activity ◽  
Α Chain ◽  
Β Chain

C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (α-chains) and one unique one (β-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the α-chain contains eight CCPs and the β-chain three. Each α-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the α-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP α-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP β-chain. Furthermore, seven different monoclonal antibodies were raised against C4BP and characterized using the recombinant chimeric proteins. Whereas all three recombinant chimeras bind protein S with the same affinity as plasma-purified C4BP, none of them bound to C4b. Three of the antibodies, which were found to bind to α-chain CCP 1 and CCP 2, completely inhibited the binding of plasma-purified C4BP to immobilized C4b. In addition, two of these antibodies totally blocked the factor I-cofactor activity of C4BP in a C4b-degradation assay. The binding site for one of the monoclonal antibodies was also studied using electron microscopy where it was confirmed that this antibody bound to the amino-terminal tip of the α-chain. These results show that the amino-terminal CCP of the C4BP α-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.

scholarly journals Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.

1983 ◽  
Vol 157 (6) ◽  
pp. 1971-1980 ◽  
Author(s):  
M K Pangburn ◽  
R D Schreiber ◽  
J S Trombold ◽  
H J Müller-Eberhard
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Fluid Phase ◽  
Factor H ◽  
Human Erythrocytes ◽  
Cell Sorter ◽  
Factor I ◽  
Distinct Cell ◽  
Normal Human ◽  
Initial Deposition ◽  
Functional Deficiency

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.

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