Biomedical subjects

Jens Hellwage

Publications and source records attributed to Jens Hellwage.

14 recordsLinked to original sources

PubMed2006

Two factor H-related proteins from the mouse: expression analysis and functional characterization.

Complement factor H-related (FHR) proteins display structural and functional similarities to each other and to the complement regulator factor H (FH). FHRs have been identified in various species, including human, rat, and the fish barred sand bass. As mice provide a useful model system to study the physiological role of FHRs in vivo, we aimed at characterizing murine FHR proteins. Two putative FHRs of approximately 100 and 38 kDa were detected in mouse plasma using FH-specific antiserum. In a liver cDNA library, three murine FHR-encoding transcripts were identified. Two clones code for related FHR proteins termed FHR-C and FHR-C_v1, which in secreted form are composed of 14 and 13 short consensus repeat (SCR) domains, homologous to SCRs 6-17 and 19-20 of FH. The third transcript, FHR-B, is derived from a separate gene and codes for a secreted protein composed of five SCR domains. FHR-B displays homology to SCRs 5-7 and 19-20 of FH. Expression of FHR-B in various tissues was analyzed by real-time polymerase chain reaction and was identified at high levels in liver, kidney and heart. In liver, FHR-B transcript level was even higher than that of FH. In addition, FHR-B was expressed as a recombinant 37-kDa protein, and this recombinant FHR-B interacted with the ligands heparin and human C3b. Using mouse plasma, the native presumptive FHR proteins were also analyzed in binding assays. In summary, we identify two FHR proteins in mice and for the first time characterize a murine FHR as a heparin- and C3b-binding protein.

Amino Acid Sequence↗

PubMed2006

LfhA, a novel factor H-binding protein of Leptospira interrogans.

The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

Animals↗

PubMed2005

Comparison of surface recognition and C3b binding properties of mouse and human complement factor H.

Factor H (FH) is a central complement regulator both in plasma and on certain cellular and acellular surfaces that are in contact with plasma. Although FH deficiency has been shown to lead to similar diseases in man and mice (membranoproliferative glomerulonephritis or dense deposit disease) little is known about the similarity between the human and murine FH functions. We here characterize the interactions of murine FH (mFH) with C3b, glycosaminoglycans, and endothelial cells and compare these interactions with those of human FH (hFH). To achieve this we purified mFH and murine C3 from plasma, prepared murine C3b, and expressed recombinant mFH constructs containing domains 1-5 and 18-20 (mFH1-5 and mFH18-20). For comparisons, hFH, human C3b, and recombinant hFH1-5 and hFH18-20 were used. We demonstrate that mFH and mFH1-5 do act as cofactors for factor I-mediated cleavage of human C3b. Surface plasmon resonance analysis showed binding of mFH18-20 to murine C3b and weak binding to human C3b. The mFH18-20 construct bound to heparin in a manner comparable to hFH18-20. It was demonstrated by flow cytometry that mFH and mFH18-20 bind to human endothelial cells in a similar manner to hFH and hFH18-20. Taken together, locations of the key functions of mFH, i.e. complement regulation and surface recognition, are comparable to hFH. Recently, mutations in the carboxy-terminal end of hFH have been found to be associated with atypical hemolytic uremic syndrome (aHUS). Based on the results in this report it is conceptually attractive to establish a murine model for aHUS.

Animals↗

PubMed2005

Complement factor H as a marker for detection of bladder cancer.

BACKGROUND: The BTA TRAK and BTA stat tests for bladder cancer use monoclonal antibodies (mAbs) X13.2 and X52.1 to detect factor H (FH)-related material in urine. The exact ligands remain unknown. METHODS: Western blot analyses of purified FH, recombinant factor H-related protein 1 (FHR-1), and serum and urine samples were used to identify the ligands of X13.2 and X52.1. Recombinant FH constructs were used to identify the target sites of X13.2 and X52.1. To analyze whether natural ligands of FH could compete with its recognition by the capture mAb X52.1, we used surface plasmon resonance analysis. The role of the ligands of X52.1 in the BTA TRAK assay was tested with use of purified proteins and FH-depleted samples. RESULTS: X13.2 bound to domain 3 of FH and FH-like protein 1, whereas X52.1 bound to domain 18 of FH and to FHR-1. Using specific FH depletion from a bladder cancer patient's urine and purified FH, we demonstrated that FH is the ligand recognized by the BTA TRAK test. By contrast, FHR-1 in urine reduced the FH-dependent test signal. CONCLUSIONS: FH is a tumor marker for bladder cancer. To reveal the presence of bladder cancer, the BTA TRAK assay detects FH, whereas FHR-1 is able to partly inhibit this detection. This indicates a special mechanism for a diagnostic immunoassay based on the combined effect of simultaneous positive and negative signals in a single sample.

Antibodies, Monoclonal↗

PubMed2005

Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.

Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.

Amino Acid Substitution↗

PubMed2004

The group B streptococcal beta and pneumococcal Hic proteins are structurally related immune evasion molecules that bind the complement inhibitor factor H in an analogous fashion.

Complement evasion by different mechanisms is important for microbial virulence and survival in the host. One strategy used by pathogenic bacteria is to bind the soluble complement inhibitor factor H (fH) to their surfaces. In group B streptococci and pneumococci, fH binding has been shown to be mediated by the surface proteins beta and Hic, respectively. We showed previously that Hic binds to the middle region of fH and protects the pneumococcus from opsonophagocytosis. As the beta protein and Hic are structurally closely related, we wanted to compare the fH binding characteristics of these two proteins. By using direct binding assays with radiolabeled proteins and surface plasmon resonance analysis we show that both beta and Hic bind to the short consensus repeats 8-11 and 12-14 in the middle region of fH. Peptide mapping analysis suggested that the fH-binding sites on beta and Hic were composed of discontinuous and partially homologous sequences. Thus, the bacterial virulence proteins use multiple binding sites on fH to secure high avidity. Also, the functionally active sites on fH are thereby left free to inhibit C3b deposition and opsonophagocytosis. These results reveal the evolutionary conservation of an analogous immune evasion strategy in different types of pathogenic streptococci. Importantly, the respective virulence factors could be exploited in the development of protein-based vaccines against these pathogens.

Amino Acid Sequence↗

PubMed2004

Immunological characterization of the complement regulator factor H-binding CRASP and Erp proteins of Borrelia burgdorferi.

Complement activation plays an important role in the elimination of invading microorganisms. Borrelia (B.) burgdorferi sensu lato the etiological agent of Lyme borreliosis, can resist complement-mediated killing. The mechanism of complement resistance of B. burgdorferi sensu stricto apparently depends on the expression of several outer surface proteins described as CRASPs (complement regulator-acquiring surface proteins). These borrelial surface proteins are able to bind components of the complement regulatory system, factor H and/or factor H-like protein 1 (FHL-1), two crucial fluid-phase negative regulators of the alternative pathway of complement. It was previously demonstrated that one CRASP is encoded by a member of the erp gene family. The purpose of the study was to use a set of monoclonal antibodies (mAb) and polyclonal antisera to characterize the relatedness of factor H-binding CRASP and Erp proteins among several B. burgdorferi sensu stricto and B. afzelii strains. Based on the observed cross-reactivities between B. burgdorferi sensu stricto strains LW2 and PKa-1, it is concluded that BbCRASP-3 is similar to ErpP, BbCRASP-4 is structurally related to ErpC, and BbCRASP-5 is similar to ErpA. The BaCRASP-2 and BaCRASP-4 proteins of B. afzelii strain EB1 reacted with both anti-ErpA and anti-ErpP antibodies whereas BaCRASP-5 of B. afzelii strain FEM1-D15 exclusively reacted with BbCRASP-3/ErpP specific antibodies. Together, these data indicate that most of the factor H-binding CRASPs are members of the Erp protein family, which represents a polymorphic class of proteins with similar or identical immunological reactivities.

Antigens, Bacterial↗

PubMed2003

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

Amino Acid Sequence↗

PubMed2003

Pig complement regulator factor H: molecular cloning and functional characterization.

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1-4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15-20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1-7 and SCR 15-20 proteins, but not the SCR 1-4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.

Amino Acid Sequence↗

PubMed2003

Immune evasion of Borrelia burgdorferi: mapping of a complement-inhibitor factor H-binding site of BbCRASP-3, a novel member of the Erp protein family.

The causative agents of Lyme disease, Borrelia burgdorferi s.s., B. garinii, and B. afzelii, differ in their susceptibility to complement-mediated lysis. This phenomenon apparently depends on the expression of proteins termed complement regulator-acquiring surface proteins (CRASP) and their binding to the inhibitory plasma proteins factor H and FHL-1. To characterize these bacterial proteins in more detail we have now isolated from a B. burgdorferi expression library a novel factor H-binding protein. In accordance with our previous studies this protein was termed BbCRASP-3 and represents a novel member of the polymorphic Erp (OspE/F-related) protein family. On the basis of protease accessibility assays using intact spirochetes, BbCRASP-3 is identified as a surface-exposed protein and binds the C-terminal short consensus repeats of factor H. Applying deletion mutants of BbCRASP-3, the factor H-binding site was mapped to the nine-amino-acid motif LEVLKKNLK localized at the C-terminal end of BbCRASP-3. Factor H bound to BbCRASP-3 maintains its cofactor activity in factor I-mediated C3b inactivation. Binding of BbCRASP-3 to factor H can be inhibited by heparin, a physiological ligand of the complement regulator factor H. Blocking of factor-H-binding by soluble BbCRASP-3 leads to an increase of complement deposition on intermediate serum-resistant strain ZS7. In conclusion, BbCRASP-3 has been identified as a novel factor H-binding protein on B. burgdorferi which by conferring complement resistance to the pathogen may contribute to its persistence in the mammalian host.

Amino Acid Sequence↗

PubMed2003

Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Recent studies have identified a factor H-associated form of HUS, caused by gene mutations that cluster in the C-terminal region of the complement regulator factor H. Here we report how three mutations (E1172Stop, R1210C, and R1215G; each of the latter two identified in three independent cases from different, unrelated families) affect protein function. All three mutations cause reduced binding to the central complement component C3b/C3d to heparin, as well as to endothelial cells. These defective features of the mutant factor H proteins explain progression of endothelial cell and microvascular damage in factor H-associated genetic HUS and indicate a protective role of factor H for tissue integrity during thrombus formation.

Animals↗

PubMed2002

Complement C3b/C3d and cell surface polyanions are recognized by overlapping binding sites on the most carboxyl-terminal domain of complement factor H.

Factor H (FH) is a potent suppressor of the alternative pathway of C in plasma and when bound to sialic acid- or glycosaminoglycan-rich surfaces. Of the three interaction sites on FH for C3b, one interacts with the C3d part of C3b. In this study, we generated recombinant constructs of FH and FH-related proteins (FHR) to define the sites required for binding to C3d. In FH, the C3d-binding site was localized by surface plasmon resonance analysis to the most C-terminal short consensus repeat domain (SCR) 20. To identify amino acids of FH involved in binding to C3d and heparin, we compared the sequences of FH and FHRs and constructed a homology-based molecular model of SCR19-20 of FH. Subsequently, we created an SCR15-20 mutant with substitutions in five amino acids that were predicted to be involved in the binding interactions. These mutations reduced binding of the SCR15-20 construct to both C3b/C3d and heparin. Binding of the wild-type SCR15-20, but not the residual binding of the mutated SCR15-20, to C3d was inhibited by heparin. This indicates that the heparin- and C3d-binding sites are overlapping. Our results suggest that a region in the most C-terminal domain of FH is involved in target recognition by binding to C3b and surface polyanions. Mutations in this region, as recently reported in patients with familial hemolytic uremic syndrome, may lead to indiscriminatory C attack against self cells.

Amino Acid Sequence↗

PubMed2002

Onchocerca volvulus microfilariae avoid complement attack by direct binding of factor H.

The filarial parasite Onchocerca volvulus is the causative agent of river blindness. The adult worms produce microfilariae (mf), which are responsible for the disease pathogenesis; mf activate the complement system, but the activation stops before the formation of terminal complement complexes. Because of the arrest of complement activation, this study analyzed binding of the main alternative pathway regulator, factor H (fH), to the mf. The mf bound fH after incubation in nonimmune human serum or with purified radiolabeled fH. In the presence of factor I, mf-bound fH promoted the cleavage of complement 3 molecule b (C3b) to iC3b. An analysis with recombinant constructs of fH showed that the C-terminal short consensus repeats (SCRs) 8-20 of fH bound to mf, whereas the N-terminal SCRs 1-7 containing the complement-regulatory domains in SCRs 1-5 did not. Thus, mf of the nematode O. volvulus may evade human complement by binding fH and by promoting inactivation of C3b into iC3b.

Animals↗

PubMed2002

Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-encoded Hic protein that binds to short consensus repeats 8-11 of factor H.

Streptococcus pneumoniae is an important cause of upper and lower respiratory tract infections, meningitis, peritonitis, bacterial arthritis, and sepsis. Here we have studied a novel immune evasion mechanism of serotype 3 pneumococci, which are particularly resistant to phagocytosis. On their surfaces the bacteria express the factor H-binding inhibitor of complement (Hic), a protein of the pneumococcal surface protein C family. Using radioligand binding, microtiter plate assays, surface plasmon resonance analysis, and recombinant constructs of factor H, we located the binding site of Hic to short consensus repeats (SCRs) 8-11 in the middle part of factor H. This represents a novel microbial interaction region on factor H. The only other ligand known so far for SCRs 8-11 of factor H is C-reactive protein (CRP), an acute phase protein that binds to the pneumococcal C-polysaccharide. The binding sites of Hic and CRP within the SCR8-11 region were different, however, because CRP did not inhibit the binding of Hic and required calcium for binding. Binding of factor H to Hic-expressing pneumococci promoted factor I-mediated cleavage of C3b and restricted phagocytosis of pneumococci. Thus, virulent pneumococci avoid complement attack and opsonophagocytosis by recruiting functionally active factor H with the Hic surface protein. Hic binds to a previously unrecognized microbial interaction site in the middle part of factor H.

Bacterial Proteins↗