Biomedical subjects

Peter F Zipfel

Publications and source records attributed to Peter F Zipfel.

At least 19 recordsLinked to original sources

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

Successful (?) therapy of hemolytic-uremic syndrome with factor H abnormality.

We report a patient with continuously recurring hemolytic-uremic syndrome due to factor H deficiency. First at the age of 3 months he showed signs of hemolytic anemia, thrombocytopenia and renal insufficiency, often recurring concomitantly with respiratory tract infections, despite weekly to twice weekly plasma substitution (20 ml/kg body weight). Now at the age of 3.5 years glomerular filtration rate is approximately 50 ml/min/1.73 m(2) and psychomotoric development is normal. Since factor H is mainly synthesized in the liver, hepatic transplantation has been proposed as curative treatment. Before justification of liver transplantation as the ultimate treatment for these patients, an international registry should be developed to optimize and standardize therapeutic alternatives.

Child, Preschool↗

PubMed2003

Isolation and characterization of the regulatory subunit of cAMP-dependent protein kinase from the filarial parasite Onchocerca volvulus.

Protein kinases exert major regulatory effects in eukaryotic signaling events. As these proteins play central regulatory and sensory functions they are interesting targets for antiparasitic drug development and serve as vaccine candidates. A cDNA with an open reading frame of 1122 bp coding for the regulatory subunit of the cAMP-dependent protein kinase (Ov-pka-r) of the pathogenic human nematode Onchocerca volvulus has been isolated. The predicted protein displays 84% homology to the corresponding protein of Caenorhabditis elegans and 71% to the human homologue. The O. volvulus protein has unique features, it includes six cysteine residues, as compared to four residues in mammals. Ov-PKA-r was recombinantly expressed as His-tagged protein and under reducing conditions showed a molecular mass of 52 kDa. In sera from O. volvulus patients IgG antibodies were found that strongly reacted with the recombinant Ov-PKA-r. Using rabbit antisera raised against the recombinant protein for immunohistology allowed the localization of the native Ov-PKA-r within the nervous system and sensory organs of adult O. volvulus worms and of microfilariae. The predominant expression in the nervous system and sensory organs as well as the unique structural features identify this signaling molecule of O. volvulus as a new and interesting target for drug or vaccine development.

Amino Acid Sequence↗

PubMed2003

Early growth response proteins (EGR) and nuclear factors of activated T cells (NFAT) form heterodimers and regulate proinflammatory cytokine gene expression.

Activation of transcription factors by receptor mediated signaling is an essential step for T lymphocyte effector function. Following antigenic stimulation of T cells the two central cytokines IL-2 and TNFalpha are co-expressed and co-regulated. Two important transcription factors, i.e., early growth response (EGR) protein EGR-1 and nuclear factors of activated T cells (NFAT) protein NFATc, regulate transcription of the human IL-2 cytokine and the same combination of EGR and NFAT proteins seems relevant for coordinated cytokine expression. Here we demonstrate that the zinc finger protein EGR-1 and two members of the NFAT protein family bind simultaneously to adjacent elements position -168 to -150 within the TNFalpha promoter. Both promoter sites are important for TNFalpha gene transcription as shown by transfection assays having the IL-2 and TNFalpha promoters linked to a luciferase reporter. The use of promoter deletion constructs with the zinc finger protein (ZIP), the NFAT binding element or a combination of both deleted show a functional cooperation of these elements and of their binding factors. These experiments demonstrate that EGR-1 as well as EGR-4 functionally cooperate with NFAT proteins and induce expression of both cytokine genes. Using tagged NFATc and NFATp in glutathione S-transferase pull down assays showed interaction and physical complex formation of each NFAT protein with recombinant, as well as native, EGR-1 and EGR-4 proteins. Thus EGR-NFAT interaction and complex formation seems essential for human cytokine expression as adjacent ZIP and NFAT elements are conserved in the IL-2 and TNFalpha gene promoters. Binding of regulatory EGR and NFAT factors to these sites and the functional interaction and formation of stable heterodimeric complexes indicate an important role of these factors for gene transcription.

Base 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

Genetic screening in haemolytic uraemic syndrome.

PURPOSE OF REVIEW: Haemolytic uraemic syndrome (HUS) is a disease of diverse origin. The last year has witnessed the identification of a novel genetic marker of this disease, the description of the frequency of the factor H associated form of HUS in a registry of over 100 patients and a better understanding of the pathophysiology of the disease. RECENT FINDINGS: In patients with atypical HUS, heterozygous mutations in the gene coding for the soluble complement regulator factor H are reported and most of the mutations cluster in the C-terminal recognition domain of the protein. A novel genetic marker for HUS has also been identified. Mutations occurring in the gene of the von Willebrand factor cleaving protease, ADAMTS13, which were previously linked to thrombotic thrombocytopenic purpura have now been identified in HUS patients. The frequency of factor H-associated HUS was established as 14% in a registry of German speaking countries and also 16 novel disease associated mutations were reported. The pathophysiology of factor H-associated HUS was analysed. Three analysed mutant proteins show normal complement regulatory activities but display defective recognition functions: reduced binding to surface attached C3b, to heparin/polyanions and to endothelial cells. SUMMARY: The identification of effector molecules of the complement as well as the coagulation cascade as disease associated molecules indicate a regulatory protein network, which maintains integrity of endothelial cells during stress or infection. Defining the individual components and how their functional interaction causes microangiopathies will identify additional disease markers and will allow the design of proper diagnostic and therapeutic approaches.

Complement Factor H↗

PubMed2003

Recruitment of complement factor H-like protein 1 promotes intracellular invasion by group A streptococci.

Numerous microbial pathogens exploit complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FHL-1 and FH binding protein expressed on the surface of the human pathogenic bacterium, Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. In the present study, we demonstrate that Fba and FHL-1 work in concert to promote invasion of epithelial cells by S. pyogenes. Fba fragments were expressed as recombinant proteins and assayed for binding of FHL-1 and FH by Western blotting, enzyme-linked immunosorbent assay, and surface plasmon resonance. A binding site for FHL-1 and FH was localized to the N-terminal half of Fba, a region predicted to contain a coiled-coil domain. Deletion of this coiled-coil domain greatly reduced FHL-1 and FH binding. PepSpot analyses identified a 16-amino-acid segment of Fba which overlaps the coiled-coil domain that binds both FHL-1 and FH. To localize the Fba binding site in FHL-1 and FH, surface plasmon resonance was used to assess the interactions between the streptococcal protein and a series of recombinant FH deletion constructs. The Fba binding site was localized to short consensus repeat 7 (SCR 7), a domain common to FHL-1 and FH. SCR 7 contains a heparin binding site, and heparin was found to inhibit FHL-1 binding to Fba. FHL-1 promoted entry of Fba(+) group A streptococci into epithelial cells in a dose-dependent manner but did not affect invasion by an isogenic fba mutant. To our knowledge, this is the first report of a bacterial pathogen exploiting a soluble complement regulatory protein for entry into host cells.

Bacterial Proteins↗

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↗

PubMed2003

Mapping of the adrenomedullin-binding domains in human complement factor H.

Adrenomedullin (AM) is a multifunctional peptide involved in roles as varied as blood pressure regulation, growth, neurotransmission, and inflammation control, among others. We previously identified complement factor H as a serum binding protein for AM and showed that factor H regulates AM functions and vice versa. Here we searched for the specific binding sites for AM by using recombinant fragments of factor H and a non-radioactive binding assay with fluorescein-tagged AM. By this methodology, two specific binding sites for AM were found in factor H. One of them shows a high affinity for AM and is located at the carboxy terminal end of factor H, comprising short consensus repeats (SCR) 15-20. Smaller fragments of this region did not bind to AM efficiently, suggesting that the high affinity binding site of factor H requires a complex three-dimensional structure to recognize AM. Another binding site with lower affinity for AM was found in the middle of the factor H molecule, at SCR 8-11. Antibodies against factor H prevented AM binding altogether, but the main binding partner of factor H, C3b, did not, indicating that C3b and AM bind to different regions of factor H. These structure-function data support previous biochemical observations. Our understanding of the binding between AM and factor H may help in the development of new treatments for diseases in which these molecules play active roles.

Adrenomedullin↗

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

Complement regulator-acquiring surface proteins of Borrelia burgdorferi: a new protein family involved in complement resistance.

The innate immune system, particularly the host complement system, plays an important role in the elimination of invading pathogens. Borrelia burgdorferi, like other human pathogens, has developed strategies to prevent complement-mediated bacteriolysis. It is now well established that Borrelia differ in their complement resistance. In general terms, B. afzelii isolates are mainly resistant to complement-mediated bacteriolysis, whereas the majority of B. burgdorferi s.s. isolates display an intermediate complement-resistant phenotype. Most of the B. garinii isolates, in contrast, are efficiently killed by complement and, therefore, are classified as complement-sensitive. Complement resistance of B. afzelii and B. burgdorferi s.s. isolates correlates directly with the acquisition of the fluid-phase human complement regulators FHL-1/reconectin and factor H. To date, five distinct complement regulator-acquiring surface proteins (CRASPs) have been identified in B. afzelii and B. burgdorferi s.s. isolates. The individual CRASPs can be differentiated according to their size and their binding properties to FHL-1/reconectin and factor H. The domains that interact with CRASPs are localized at the C-terminal ends of these complement regulators. Thus, CRASPs represent a family of functional proteins involved in complement resistance of Borrelia. Furthermore, an alterable pattern of gene expression was observed for three CRASPs of B. afzelii: BaCRASP-1, BaCRASP-2, and BaCRASP-5 are up-regulated at 37 degrees C and down-regulated at 20 degrees C. The continued characterization of CRASPs at the molecular level is expected to identify new virulence factors and potential vaccine candidates.

Animals↗

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↗

PubMed2002

Upstream conserved sequences of mouse leukemia viruses are important for high transgene expression in lymphoid and hematopoietic cells.

Highly conserved enhancer sequences located in the upstream part of the long terminal repeat (LTR) of murine leukemia retroviruses (MLV) were reported to compromise viral gene expression in multipotent embryonic cells in vitro and to reduce the likelihood for maintenance of retroviral gene expression in hematopoietic cells in vivo. We show that deletion of these sequences (nucleotides +37 to +95) attenuates rather than increases the transcriptional activity of retroviral vectors in hematopoietic cells almost independently of the developmental lineage (erythroid, myeloid, or lymphoid). Expression rates of modified vectors were reduced by as much as 34-65%, although the strong enhancer array located in the direct repeat of the LTR was preserved. Sequence analysis and electrophoretic mobility shift assays revealed the presence of a highly conserved binding site for NFAT (nuclear factor of activated T cells) proteins that immediately neighbors a known binding site for the transcription factor Yin-Yang1 (YY1) [corrected]. Specific inactivation of the NFAT site reduced transgene expression in all cell types investigated and had a similar effect as the destruction of a neighboring SP1 motif. Combined destruction of individual motifs for NFAT, SP1, and E twenty-six transcription factors (ETS) resulted in a severe attenuation (by 40-60%) of the retroviral enhancer. These results provide novel clues for the manipulation of retrovirus replication and vector tropism.

5' Flanking Region↗

PubMed2002

Hemolytic uremic syndrome due to an altered factor H triggered by neonatal pertussis.

We report on a previously healthy newborn suffering from severe Bordetella pertussis infection who developed hemolytic uremic syndrome (HUS) a few weeks after the onset of whooping cough, with a fatal outcome. A factor H protein with abnormal mobility was found in the serum of the patient as analyzed by Western blotting, indicating that B. pertussis infection might have triggered HUS in a genetically predisposed patient.

Bordetella pertussis↗

PubMed2002

The molecular basis for hereditary porcine membranoproliferative glomerulonephritis type II: point mutations in the factor H coding sequence block protein secretion.

Porcine membranoproliferative glomerulonephritis type II in piglets of the Norwegian Yorkshire breed is considered the first animal model of human dense deposit disease. Porcine dense deposit disease is caused by the absence of the complement regulator factor H in plasma. Here we report the molecular basis for this absence. Single nucleotide exchanges at position C1590G and T3610G in the coding region of the factor H gene result in amino acid exchanges at nonframework residues L493V and I1166R that are located within SCR 9 and SCR 20, respectively. Apparently the L493V mutation represents a polymorphism whereas the I1166R causes the physiological consequences a block in protein secretion. Expression analysis shows comparable mRNA levels for factor H in liver tissue derived from both affected and healthy animals. In affected piglets, factor H protein is detected in increased amounts in liver cells. Factor H accumulates inside the hepatocytes and is not released as shown by Western blot analysis and immunohistochemistry. These data demonstrate that single amino acid exchanges of two nonframework amino acids either alone or in combination block protein secretion of factor H. This observation is also of interest for other human diseases in which factor H is involved, such as human factor H-associated form of hemolytic uremic syndrome.

Animals↗

PubMed2002

Immune evasion of Borrelia burgdorferi: insufficient killing of the pathogens by complement and antibody.

The innate immune system and, in particular, the complement system play a key role in the elimination of micro-organisms after entrance in the human host. Like other pathogens, borreliae must develop strategies to inactivate host defence mechanisms. By investigating serum (NHS)-susceptibility of borreliae, we found that mainly B. afzelii isolates are serum-resistant, whereas the majority of B. burgdorferi s. s. isolates display an intermediate serum-sensitive phenotype. In contrast, B. garinii isolates are killed effectively by complement and therefore are classified as serum-sensitive. Up to now, we have identified two distinct proteins of 27.5 kDa and 20.7 kDa expressed on the outer surface of borreliae, which interact directly with FHL-1/reconectin and factor H, the two major regulators of the alternative complement pathway. These borrelial proteins are termed CRASPs (complement regulator-acquiring surface proteins). CRASPs are detectable only on serum-resistant borreliae and, accordingly, binding of FHL-1/reconectin and factor H only occur with serum-resistant borrelial isolates. We conclude from these results that the control of complement activation on the borrelial surface is due to the interaction of borrelial CRASPs with host complement regulatory proteins. Thus, CRASPs represent an important mechanism of immune evasion on the part of borrelial isolates belonging mostly to the genospecies B. afzelii. By analysing the humoral adaptive immune response of patients, we detected sera that killed NHS-resistant borreliae. Borreliacidal activity is observed most frequently with sera of patients at stage III of the disease. The killing of NHS-resistant isolates by these immune sera always requires the combination of antibodies and complement. Bactericidal activity, however, is not detected in all immune sera at the different disease stages, although specific anti-Borrelia antibodies are present according to serological test results. This observation suggests that not all borrelial antigens are able to induce a borreliacidal immune response. In an extensive analysis of 24 immune sera, we identified up to 12 borrelial antigens, including OspC, which possess the greatest potential for the induction of borreliacidal antibody. The borreliacidal potential of anti-OspC antibodies was tested directly on an OspC-expressing borrelial wild-type isolate and a corresponding variant lacking OspC. In these studies, only the wild-type isolate expressing OspC on its surface proved positive for the lytic complement complex, thereby indicating the great importance of this antigen for the control of the infection. Additional studies are required to identify further "protective" antigens among these 12 proteins, all of which are candidates for infection control according to our studies involving patient immune sera. These antigens may include the recently detected CRASPs.

Antibodies, Bacterial↗