Biomedical subjects

J Hellwage

Publications and source records attributed to J Hellwage.

At least 19 recordsLinked to original sources

PubMed2003

Release of endogenous anti-inflammatory complement regulators FHL-1 and factor H protects synovial fibroblasts during rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease of unknown aetiology predominantly affecting cells and tissues of synovial joints. Here we show that the two important complement regulators FHL-1 and factor H play a protective anti-inflammatory role in rheumatoid arthritis. Expression analyses at the mRNA- and protein level show in vitro expression and secretion of both regulators by synovial fibroblasts derived from patients with rheumatoid arthritis. Similarly the two regulators are synthesized in vivo in diseased synovial tissue, and in particular synovial lining cells express high levels of FHL-1. The anti-inflammatory role of these regulators in rheumatoid arthritis is highlighted by their induction with IFN-gamma and dexamethasone, whilst the pro-inflammatory cytokine TNF-alpha had no effect. Transient transfection experiments with various FHL-1/factor H promoter-luciferase reporter constructs into cells of distinct origin show independent cell and tissue specific promoter regulated transcription of these two regulators. The inducible expression, specifically of FHL-1 has physiological consequences. By binding directly to surfaces the released proteins protect cells from inflammatory damage and complement-mediated cell lysis. This study shows a novel protective and anti-inflammatory role of the two important complement regulators FHL-1 and factor H in rheumatoid arthritis and suggests a disease controlling role of the two proteins.

Arthritis, Rheumatoid↗

PubMed2002

Factor H family proteins: on complement, microbes and human diseases.

At present, the human Factor H protein family represents seven multidomain, multifunctional serum proteins. This group includes the complement and immune regulators Factor H, the Factor H-like protein 1 (FHL-1) and five Factor H-related proteins proteins (FHR-1, -2, -3, -4 and -5). Each is exclusively composed of individually folded protein domains, termed short consensus repeats (SCRs) or complement control modules. Structure-function analyses allowed the localization of the complement regulatory domain of Factor H and FHL-1 in the N-terminal region within SCRs 1-4. In addition, multiple binding sites for C3b, heparin and microbial surface proteins were localized in the N-terminus, within the middle region and also in the C-terminus of Factor H and FHL-1. Recent results show a central role for the C-terminus of Factor H, i.e. SCRs 19-20. These particular domains are conserved in all FHRs identified so far, include contact points for C3b, heparin and microbial surface proteins and represent a 'hot-spot' for gene mutations in patients that suffer from the Factor H-associated form of haemolytic uraemic syndrome.

Borrelia↗

PubMed2002

The yeast Candida albicans binds complement regulators factor H and FHL-1.

The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.

Binding Sites↗

PubMed2001

Complement-regulator factor H and related proteins in otitis media with effusion.

Otitis media with effusion (OME) is a common disease in childhood. It is characterized by chronic inflammation in which the proinflammatory activity of the complement (C) system is one of the underlying factors. The C system becomes strongly activated in the middle ear effusion (MEE) fluid, but the reasons for this are not known. Here we demonstrate by using complement Bb fragment ELISA that MEE specimens strongly activate the alternative C pathway (AP) in normal human serum (NHS). Some of the MEEs were also found to promote lysis of rabbit erythrocytes by NHS. These findings indicated a disturbance in the fluid-phase regulation of the AP in MEE. The main regulator of the AP, factor H (FH), and proteins structurally related to it (FHL-1, FHR-1, -2, -3, and -4) were present in the MEE fluids of OME patients. Relative to serum, the FHR proteins were more abundant in the MEEs. In addition, we detected the recently discovered 65-kDa FH-related protein FHR-5 in the MEE. The FHR proteins share binding sites with FH in the C3d region of C3b. Thus they may compete with FH in binding to C3b and interfere with the regulatory activity of FH. Consequently, a disturbance in AP control in the MEE may lead to an ongoing excessive C activation and inflammation in OME.

Apolipoproteins↗

PubMed2001

Complement C3b interactions studied with surface plasmon resonance technique.

The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.

Binding Sites↗

PubMed2000

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi.

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.

Antigens, Bacterial↗

PubMed2000

Each of the three binding sites on complement factor H interacts with a distinct site on C3b.

Factor H (fH) restricts activation of the alternative pathway of complement at the level of C3, both in the fluid phase and on self-structures, but allows the activation to proceed on foreign structures. To study the interactions between fH and C3b we used surface plasmon resonance analysis (Biacore(R)) and eight recombinantly expressed fH constructs containing fragments of the 20 short consensus repeat domains (SCRs) of fH. We analyzed the binding of these constructs to C3b and its cleavage products C3c and C3d. Three binding sites for C3b were found on fH. Site 1 was localized to the five amino-terminal SCRs (SCR1-5), and its reciprocal binding site on C3b was found to be lost upon the cleavage of C3b to C3c and C3d. Site 2 on fH was localized by exclusion probably within or near SCRs 12-14 (fragment SCR8-20 bound to C3b, C3c, and C3d; SCR8-11 did not bind to C3b at all; and SCR15-20 bound only to the C3d part of C3b). Site 3 on fH for C3b was localized to the carboxyl-terminal SCRs 19-20, and its reciprocal binding site was mapped to the C3d part of C3b. In conclusion, we confirmed and mapped three binding sites on fH for C3b and demonstrated that the three binding sites on fH interact with distinct sites on C3b. Multiple reciprocal interactions between C3b and fH can provide a basis for the different reactivity of the alternative pathway with different target structures.

Binding Sites↗

PubMed2000

Different regulation of factor H and FHL-1/reconectin by inflammatory mediators and expression of the two proteins in rheumatoid arthritis (RA).

Factor H and the FHL-1/reconectin protein are two human plasma proteins that act as important regulators of the alternative complement pathway. Each protein is encoded by a unique transcript, but both mRNAs are derived from the factor H gene by means of alternative processing. In order to address potential functional differences between the two proteins we analysed their expression in hepatic and non-hepatic cells and studied their regulation by inflammatory mediators. We demonstrate that factor H and FHL-1/reconectin transcripts which are regulated by the same gene promoter and are initiated at the same transcription start site are differently expressed. Expression of the molecules is induced and regulated by the inflammatory mediators interferon-gamma (IFN-gamma) and the anti-inflammatory glucocorticoid dexamethasone. Both factor H and FHL-1/reconectin are expressed and secreted by synovial fibroblasts and are present in synovial fluid derived from patients suffering from rheumatoid or reactive arthritis. The local synthesis in synovial fibroblasts and their induction by IFN-gamma and dexamethasone, but not by tumour necrosis factor-alpha, suggests for each of the two complement regulators a protective role in RA.

Alternative Splicing↗

PubMed1999

Functional properties of complement factor H-related proteins FHR-3 and FHR-4: binding to the C3d region of C3b and differential regulation by heparin.

The human factor H-related proteins FHR-3 and FHR4 are members of a family of proteins related to the complement factor H. Here, we report that the two proteins bind to the C3d region of complement C3b. The apparent K(A) values for the interactions of FHR-3 and FHR-4 with C3b are 7.5 x 10(6) M(-1) and 2.9 x 10(6) M(-1), respectively. Binding studies performed with C3b-coated pneumococci confirmed the results obtained with the biosensor system. A C-terminal construct of factor H showed similar binding characteristics. The interaction of FHR-3, but not of FHR4, with opsonised pneumococci was inhibited by heparin.

Apolipoproteins↗

PubMed1999

Regulation of complement activation by C-reactive protein: targeting the complement inhibitory activity of factor H by an interaction with short consensus repeat domains 7 and 8-11.

C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8-11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.

Binding Sites↗

PubMed1999

The factor H protein family.

The factor H gene family provides a prime example of a multidomain multifunctional protein family whose individual members are defined by conserved common structural elements and display diverse but often overlapping functions. The six identified members of this protein family represent secreted plasma proteins that are primarily synthesized in the liver. Here, we summarize the current understanding of the function of these proteins and suggest a common role in complement control. Factor H is the best characterized member and acts as a complement regulator. The protein displays cofactor activity for factor I in the degradation of the central complement component C3b, acts as a decay accelerating factor for the C3 convertase, C3bBb and is a competitor for factor B binding to C3b. Factor H is a multifunctional protein and displays functions outside the complement system: it binds to the cellular integrin receptor (CD11b/CD18), interacts with cell surface glycosaminoglycans and also binds to the surface of certain pathogenic microorganisms. In addition, factor H has several binding sites for the C3 protein. The factor H-like protein 1 (FHL-1) or reconectin shares the complement regulatory functions with factor H and interacts with heparin. The protein displays cell spreading activity and binds to the N-terminus of the streptococcal M protein. The function of the factor H-related proteins (FHR-1 to FHR-4) is currently under investigation. These proteins are differently distributed. Three proteins (FHR-1, FHR-2 and FHR-4) are constituents of lipoproteins, while FHR-3 interacts with heparin. Binding to C3b and C3d has been demonstrated for FHR-3 and FHR-4 and the two proteins display a cofactor related activity.

Complement Factor H↗

PubMed1998

Role of the hypervariable region in streptococcal M proteins: binding of a human complement inhibitor.

Antigenic variation allows pathogenic microorganisms to evade the immune system of the infected host. The variable structure must play an important role in pathogenesis, but its function is in most cases unknown. Here, we identify a function for the surface-exposed hypervariable region of streptococcal M5 protein, a virulence factor that inhibits phagocytosis. The hypervariable region of M5 was found to bind the human complement inhibitor FHL-1 (factor H-like protein 1), a 42-kDa plasma protein. Plasma absorption experiments with M5-expressing bacteria showed that the interaction with FHL-1 occurs also under physiologic conditions. Studies of another extensively characterized M protein, M6, indicated that this protein also has a binding site for FHL-1 in the hypervariable region. The complement-inhibitory function of FHL-1 was retained after binding to streptococci, suggesting that bound FHL-1 protects bacteria against complement attack. All available data now indicate that FHL-1, or another human complement inhibitor, binds to the hypervariable region of M proteins. These findings provide insights into the forces that drive antigenic variation and may explain why the hypervariable region of M protein is essential for phagocytosis resistance. Moreover, these data add to a growing body of evidence that human complement inhibitors are major targets for pathogenic microorganisms.

Antigenic Variation↗

PubMed1998

The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins.

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR18 (factor H) and SCR3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.

Animals↗

PubMed1998

Identification of the second heparin-binding domain in human complement factor H.

Complement factor H (fH) regulates activation of the alternative pathway of C, reducing the amount of C3b deposited on sialic acid-rich surfaces. Heparin binding has been used as a model for examining the sialic acid-binding characteristics of fH. We have previously shown that of the 20 short consensus repeat (SCR) modules of fH, SCR 7 contains an important heparin binding site, but other SCRs also play a role in heparin binding. To localize the other sites, we prepared recombinant truncated and SCR deletion mutants of fH and tested them by heparin-agarose affinity chromatography. The 5 C-terminal SCRs were found to contain a heparin binding site as an SCR 7 deletion mutant of the N terminal 15 SCRs did not bind heparin, but a construct consisting of SCRs 16-20 was shown to bind heparin. Double deletion of SCRs 7 and 20 from fH abrogated binding to heparin, indicating that SCR 20 contains a heparin binding site. This finding was confirmed with the observation that attachment of SCR 20 to a group of nonbinding SCRs produced a heparin-binding protein. A protein consisting of SCRs 19 and 20 did not bind heparin, whereas SCRs 18-20 did, indicating that, although SCR 20 contains a heparin binding site, at least two nonspecific adjacent SCRs are required. fH-related protein-3 (FHR-3) possesses an SCR homologous to SCR 7 of fH and bound heparin, whereas FHR-4, which lacks such an SCR, did not. Thus, fH contains two separate heparin binding sites, which are located in SCRs 7 and 20.

Apolipoproteins↗

PubMed1998

Identification of a domain in human factor H and factor H-like protein-1 required for the interaction with streptococcal M proteins.

The plasma protein factor H (FH) inhibits the alternative pathway of complement activation. Previous work has shown that FH binds to group A streptococci and that the interaction does not interfere with the complement-inhibitory capacity of FH. In this work, we report a molecular analysis of this interaction. In absorption experiments with human plasma, M protein-expressing group A streptococci bound both FH and FH-like protein-1 (FHL-1), an active 42-kDa splice product of the FH-gene transcript comprising the first 7 of its 20 short consensus repeat (SCR) domains. rFHL-1 also bound to M protein-expressing streptococci, but rFH fragments containing SCR 1-5 or SCR 1-6 did not. rFHL-1 bound to purified M5 protein with an affinity that was higher than the value calculated for the interaction between FH and M5 protein. The binding of radiolabeled rFHL-1 to immobilized M5 was blocked completely by unlabeled rFHL-1, but was inhibited only partially by SCR 1-6, emphasizing the importance of SCR 7 for the interaction. In experiments with the FH-related proteins FHR-3 and FHR-4, only the former bound to M protein-expressing streptococci, again pointing to an involvement of SCR 7, since FHR-3, but not FHR-4, contains a domain that is similar to SCR 7. Finally, the interaction between rFHL-1 and purified M5 protein was inhibited by heparin, which binds FH via SCR 7. Together, these data indicate that the interaction between streptococcal M proteins and FH or FHL-1 requires SCR 7.

Absorption↗

PubMed1997

The human complement regulatory factor-H-like protein 1, which represents a truncated form of factor H, displays cell-attachment activity.

Complement factor H (FH) and factor-H-like protein 1 (FHL-1) are human plasma proteins with regulatory functions in the alternative pathway of complement activation. FH and FHL-1 are organized in repetitive elements termed short consensus repeats (SCRs) and the seven SCRs of FHL-1 are identical with the N-terminal domain of the 20 SCRs of FH. The fourth SCR of both proteins (SCR 4) includes the sequence Arg-Gly-Asp (RGD), a motif that is responsible for the major adhesive activity of matrix proteins like fibronectin. A synthetic hexapeptide with the sequence ERGDAV derived from the RGD domain of FH/FHL-1 interferes with cell attachment to a fibronectin matrix. Although the identical motif is present in both FH and FHL-1, only FHL-1 acts as a matrix for cell spreading and attachment, thus the two proteins differ in function. The adhesive activity of FHL-1 is localized to the RGD-containing SCR 4 by the use of recombinant fragments. All three analysed anchorage-dependent cell lines (CCl64, C32 and MRC-5) adhere to an FHL-1 matrix. The use of synthetic peptides in competition assays, on either FHL-1-derived or fibronectin matrices, shows that the cellular receptors binding to the FH/FHL-1-derived RGD motif are related to or identical with integrin receptors which interact with fibronectin. The identification of a functional adhesive domain in the FH/FHL-1 sequence demonstrates, at least for FHL-1, a role in cell attachment and adhesion.

Animals↗

PubMed1997

The human factor H-related protein 4 (FHR-4). A novel short consensus repeat-containing protein is associated with human triglyceride-rich lipoproteins.

A novel apoprotein of an apparent molecular mass of 86 kDa in its unreduced form was identified in human triglyceride-rich lipoproteins. This protein was purified and the amino acid sequence of six proteolytic fragments was found to overlap with that of the factor H-related proteins. In parallel we identified the cDNA encoding a new complement factor H-related protein, termed FHR-4. The sequences of the new apoprotein overlapped with that of the FHR-4 protein. Similar to the previously described factor H-related proteins, FHR-4 contains a hydrophobic signal sequence followed by a stretch of five repetitive elements termed short consensus repeats. Recombinant FHR-4 protein was expressed in the baculovirus system and has an apparent molecular mass of 42 kDa. In addition a 84-kDa dimeric form of the recombinant FHR-4 was detected. Using an immunoaffinity column with antibodies raised against the recombinant FHR-4, we isolated a 86-kDa protein from human plasma. The different molecular mass of the recombinant FHR-4 and the dimeric FHR-4 in plasma is due to different carbohydrate moieties. The 86-kDa plasma protein and the novel apolipoprotein had identical mobility on SDS-polyacrylamide gel electrophoresis analysis and reacted with antisera raised against the reFHR-4 and the purified apoprotein. In conclusion, we have identified a novel factor H-related protein, FHR-4, in human plasma and demonstrate that this protein is present in triglyceride-rich lipoproteins in a dimeric form. This observation provides an intriguing new aspect on possible function(s) of this novel protein and the other factor H-related proteins.

Amino Acid Sequence↗

PubMed1997

Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector.

We have made use of the plasmid vector pBSV-8His recently established for baculovirus-mediated expression of His-tagged proteins for the production of a truncated soluble complement regulator protein. The protein comprised the N-terminal part, i.e. short consensus repeats (SCRs) 1-4, of the B-cell membrane protein complement receptor type two (CR2; CD21) and contained the functional epitopes which mediate the binding of the complement component C3 fragments C3dg and iC3b. This recombinant protein, termed rsCR2.1-4, was furnished with a C-terminal histidine-tag for easy purification from insect cell supernatant. The yield of > 90% pure rsCR2.1-4 was 3 micrograms/ml supernatant at day eight p.i. RsCR2.1-4 was expressed as two proteins with a M(r) of 29 and 31 representing differentially glycosylated forms. Both reacted specifically with anti-CR2 mAb HB5 directed against SCRs 3-4, but not with anti-CR2 mAbs recognizing SCRs beyond SCR 4. RsCR2.1-4 was able to bind C3dg and to block binding of C3dg-coated beads to Raji cells. Used as antigen for immunization, it allowed the efficient and well-aimed generation of antisera which specifically blocked attachment of C3dg-coated beads to Raji B cells. Thus, insect cell derived rsCR2.1-4 has proved a valuable tool to study the functional domain of CR2 and its immunoregulatory capacity in B-lymphocytes.

Animals↗