Biomedical subjects

Peter F Zipfel

Publications and source records attributed to Peter F Zipfel.

At least 19 recordsLinked to original sources

PubMed2007

The C-terminus of complement factor H is essential for host cell protection.

Complement is a powerful self-amplifying system of innate immune defense with the capacity to eliminate microbes directly. Factor H is a central regulator in plasma which protects host tissue from complement mediated damage. Here we characterize the relevance of surface attached factor H, and study the regulatory activity of factor H on endothelial cells. Although these cells expressed membrane bound regulators, cell bound factor H contributed substantially to complement regulatory activity at the cell surface. Blockade of the C-terminus of factor H with monoclonal antibodies inhibited cell binding of this soluble regulator and resulted in enhanced complement activation on the cells. In the absence of factor H, increased deposition and slower inactivation of C3b resulted in higher amount of membrane attack complexes on the cell surface. When the membrane regulators CD55 and CD59 were removed by enzymatic treatment, complement mediated cell lysis was enhanced in the absence of factor H. Importantly, inhibition of the C-terminus did not compromise the regulatory function of factor H in fluid phase. Altogether these data point to a highly relevant, yet so far underestimated role of factor H for complement control at cellular surfaces, and reveal a decisive role of the factor H C-terminus in host cell recognition and protection.

Antibodies, Monoclonal↗

PubMed2007

Hemolytic uremic syndrome: a factor H mutation (E1172Stop) causes defective complement control at the surface of endothelial cells.

Defective complement regulation results in hemolytic uremic syndrome (HUS), a disease that is characterized by microangiopathy, thrombocytopenia, and acute renal failure and that causes endothelial cell damage. For characterization of how defective complement regulation relates to the pathophysiology, the role of the complement regulator factor H and also of a mutant factor H protein was studied on the surface of human umbilical vein endothelial cells. The mutant 145-kD factor H protein was purified to homogeneity, from plasma of a patient with HUS, who is heterozygous for a factor H gene mutation G3587T, which introduces a stop codon at position 1172. Functional analyses show that the lack of the most C-terminal domain short consensus repeats 20 severely affected recognition functions (i.e., binding to heparin, C3b, C3d, and the surface of endothelial cells). Wild-type factor H as well as the mutant protein formed dimers in solution as shown by cross-linking studies and mass spectroscopy. When assayed in fluid phase, the complement regulatory activity of the mutant protein was normal and comparable to wild-type factor H. However, on the surface of endothelial cells, the mutant factor H protein showed severely reduced regulatory activities and lacked protective functions. Similarly, with the use of sheep erythrocytes, the mutant protein lacked the protective activity and caused increased hemolysis when it was added to factor H-depleted plasma. This study shows how a mutation that affects the C-terminal region of the factor H protein leads to defective complement control on cell surfaces and damage to endothelial cells in patients with HUS. These effects explain how mutant factor H causes defective complement control and in HUS-particularly under condition of inflammation and complement activation-causes endothelial cell damage.

Cells, Cultured↗

PubMed2007

The interactive Factor H-atypical hemolytic uremic syndrome mutation database and website: update and integration of membrane cofactor protein and Factor I mutations with structural models.

Atypical hemolytic uremic syndrome (aHUS) is a disease of hemolytic anemia, thrombocytopenia, and renal failure associated with defective alternative pathway (AP) complement control. Previously, we presented a database (www.FH-HUS.org) focusing on aHUS mutations in the Factor H gene (CFH). Here, new aHUS mutations are reported for the complement regulatory proteins Factor H (FH), Factor I (FI), and membrane cofactor protein (MCP). Additional mutations or polymorphisms within CFH have been associated with membranoproliferative glomerulonephritis (MPGN) and age-related macular degeneration (AMD). Accordingly, the database now includes substitutions that predispose to aHUS, MPGN, and AMD. For this, structural models for the domains in MCP and FI were developed using homology modeling. With this new database, patients with mutations in more than one gene can be displayed and interpreted in a coherent manner. The database also includes SNP polymorphisms in CFH, MCP, and IF. There are now a total of 167 genetic alterations, including 100 in CFH, 43 in MCP, and 24 in IF. The mutations characterize clinical outcomes that vary from several AMD-associated polymorphisms to those associated with aHUS, MPGN, or FI deficiency. A consensus short complement regulator (SCR) domain structure facilitated the interpretations of aHUS mutations. Specific locations within this consensus domain often correlate with the occurrence of clinical phenotypes. The AMD Tyr402His polymorphism is structurally located at a hotspot for several aHUS mutations. The database emphasizes the causative role of the alternative pathway of complement in disease and provides a repository of knowledge to assist future diagnosis and novel therapeutic approaches.

Amino Acid Sequence↗

PubMed2006

A fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in Escherichia coli.

Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.

Endopeptidases↗

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

MPGN II--genetically determined by defective complement regulation?

MPGN II is a rare disease which is characterized by complement containing deposits within the GBM. The disease is characterized by functional impairment of the GBM causing progressive loss of renal function eventually resulting in end stage renal disease. It now becomes evident that in addition to C3NeF, which inhibits the inactivation of the alternative C3 convertase C3bBb, different genetically determined factors are also involved in the pathogenesis of MPGN II. These factors though different from C3NeF also result in defective complement regulation acting either through separate pathways or synergistically with C3NeF. Following the finding of MPGN II in Factor H deficient animals, patients with MPGN II were identified presenting with an activated complement system caused by Factor H deficiency. Factor H gene mutations result in a lack of plasma Factor H or in a functional defect of Factor H protein. Loss of Factor H function can also be caused by inactivating Factor H autoantibodies, C3 mutations preventing interaction between C3 and Factor H, or autoantibodies against C3. Identification of patients with MPGN II caused by defective complement control may allow treatment by replacement of the missing factor via plasma infusion, thus possibly preventing or at least delaying disease progress.

Animals↗

PubMed2006

Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and acute renal failure. Lack of complement inactivating factor H predisposes to the development of atypical HUS. Little is known about mechanisms linking complement activation with loss of erythrocyte integrity during HUS. Recent studies disclosed that increased cytosolic Ca2+ activity and cellular ceramide trigger programmed erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. In the present study, we investigated whether eryptosis occurs during the course of HUS. To this end, erythrocytes from healthy volunteers were exposed to plasma from a patient with severe idiopathic recurrent HUS secondary to factor H depletion. Phosphatidylserine exposure (Annexin binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation [anti-ceramide antibody and enzymatic (diacylgycerol kinase) analysis] were determined. Exposure of erythrocytes to plasma from the patient, but not to plasma from healthy individuals, triggered Annexin binding. The effect of plasma on erythrocyte Annexin binding was abolished by plasmapheresis or filtration at 30 kDa. It was paralleled by formation of ceramide and increase of cytosolic Ca2+ activity. Enhanced Annexin binding of erythrocytes from healthy individuals was observed after exposure to plasma from three other patients with HUS. The proeryptotic effect of patient plasma was mimicked by exposure to the Ca2+ ionophore ionomycin, and eryptosis was potentiated in the presence of cell membrane-permeable C6-ceramide. Furthermore, in vitro complement activation similarly triggered erythrocyte phosphatidylserine exposure, an effect which was blunted by the addition of factor H. In conclusion, our present observations disclose a novel, pathophysiological, factor-H dependent mechanism leading to injury of erythrocytes during the course of hemolytic uremic syndrome.

Aged↗

PubMed2006

Synthesis and antileprosy activity of some dialkyldithiocarbamates.

OBJECTIVES: To investigate the antileprosy potential of a set of original compounds with antimycobacterial activity. METHODS: We developed a facile synthesis of 2-chloro-3-cyano-5-nitropyridine and synthesized a series of 3-cyano-2-dialkyldithiocarbamoyl-5-nitropyridine derivatives. In vivo therapeutic efficacy against Mycobacterium leprae was assessed in the infected mouse footpad model. RESULTS: The compounds were active in vitro against Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium vaccae and Mycobacterium fortuitum, with MICs generally in the range of 0.4-6.25 mg/L. Reduction of the bacterial load in vivo in the mouse footpad and toxic side effects were dependent on the individual structure of the compounds and on the doses applied. Compounds 2a, 3a and 3b reduced the number of M. leprae by two orders of magnitude, comparable to the effect of dapsone. Co-administration of compounds 2a and 3a with dapsone synergistically enhanced the activity. In addition, these compounds were well tolerated over the treatment period of 7.5 months. CONCLUSIONS: Individual synthetic dithiocarbamate derivatives have promising antileprosy activity.

Animals↗

PubMed2006

Structure-function mapping of BbCRASP-1, the key complement factor H and FHL-1 binding protein of Borrelia burgdorferi.

Borrelia burgdorferi, a spirochaete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease, the most frequent vector-borne disease in Eurasia and North America. Sporadically Lyme disease develops into a chronic, multisystemic disorder. Serum-resistant B. burgdorferi strains bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochaete surface. This binding is dependent on the expression of proteins termed complement-regulator acquiring surface proteins (CRASPs). The atomic structure of BbCRASP-1, the key FHL-1/FH-binding protein of B. burgdorferi, has recently been determined. Our analysis indicates that its protein topology apparently evolved to provide a high affinity interaction site for FH/FHL-1 and leads to an atomic-level hypothesis for the functioning of BbCRASP-1. This work demonstrates that pathogens interact with complement regulators in ways that are distinct from the mechanisms used by the host and are thus obvious targets for drug design.

Amino Acid Sequence↗

PubMed2006

An interactive web database of factor H-associated hemolytic uremic syndrome mutations: insights into the structural consequences of disease-associated mutations.

Factor H (FH) is a central complement regulator comprised of 20 short complement repeat (SCR) domains. Nucleotide changes within this gene (CFH) have been observed in patients with hemolytic uremic syndrome (HUS), and also membranoproliferative glomerulonephritis and age-related macular degeneration. All parts of FH are affected, but many mutations are clustered in the C-terminal part of FH. Up to now, structural analyses of HUS have been based on SCR-20, a domain that is involved in FH interactions with C3b, heparin, and endothelial cells. In order to identify the structural and functional consequence of HUS mutations, further disease-associated mutations were analyzed in terms of homology and nuclear magnetic resonance (NMR) models for factor H SCR domains. An interactive web database of 54 human HUS-associated mutations and others was created from the literature (www.FH-HUS.org). This has comprehensive search and analysis tools, integrating phenotypic and genetic data with structural analysis. Each mutation can be highlighted on the SCR structure together with the patient FH and C3 levels where available. Two new insights were obtained from our collection of data. First, phenotypic data on FH clarify our previously-proposed classification of Type I and Type II disorders that both lead to HUS, where Type I affects FH secretion and folding, and Type II leads to expressed protein in plasma that is functionally defective. Second, the new mutations show more clearly that SCR domains from SCR-16 to SCR-19 are important for the ligand binding activities of FH as well as SCR-20. This FH web database will facilitate the interpretation of new mutations and polymorphisms when these are identified in patients, and it will clarify the functional role of FH.

Amino Acid Sequence↗

PubMed2006

De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome.

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.

Animals↗

PubMed2006

Binding of human complement regulators FHL-1 and factor H to CRASP-1 orthologs of Borrelia burgdorferi.

The complement regulator-acquiring surface protein (CRASP)-1 is a member of the paralogous gene family gbb54 and the dominant FHL-1 and factor H binding protein of Borrelia burgdorferi sensu stricto (s.s.). It was shown recently that expression of BbCRASP-1 directly correlates with serum resistance of B. burgdorferi s.s. isolates. In the present study we have elucidated the putative potential of other members of the gbb54 paralogous family, including orthologs ZSA66, ZSA69, ZSA70, ZSA71, ZSA72 and ZSA73 of the European B. burgdorferi s.s. strain ZS7, to bind human FHL-1 and factor H. In spite of their overall similarity in protein sequence, between 47% and 67%, and the fact that the C-terminal region of ZSA69 shows 70% similarity with BbCRASP-1, none of the orthologous proteins was able to bind human FHL-1 and/or factor H. BbCRASP-1 is the only member of the paralogous gene family gbb54 that binds to human complement regulators, supporting the notion that BbCRASP-1 plays a critical role in evasion of complement by B. burgdorferi s.s. and thus may be helpful in the development of novel therapeutic strategies against Lyme borreliosis.

Bacterial Proteins↗

PubMed2006

Complement and diseases: defective alternative pathway control results in kidney and eye diseases.

The complement system is a central part of innate immunity and in its normal setting aimed to recognize and eliminate microbes. For elimination toxic activation products are generated locally and are reported directly of the surface of the invading microbe. A deregulation of the alternative pathway results in defective recognition and toxic activation products can be formed on the surface of host tissues and structures. Recent studies have shown that mutated or defective regulators of the alternative pathway of complement are associated with auto immune diseases of the kidney, including the atypical form of hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (MPGN) and also of the eye, such as age-related macular degeneration (ARMD). Current research provides clues how mutations occurring in genes coding for single complement components or the inactivation of single regulators lead to defective alternative pathway amplification, via the convertase C3bBb. These scenarios explain how defects of a single regulator lead to local, organ specific damage.

Animals↗

PubMed2006

The role of defective complement control in hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (HUS) is a severe disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Recent evidence has shown that defective complement activation and defective complement control is a cause of HUS. So far, mutations in single genes coding for the cofactor and complement regulator factor H, the membrane cofactor protein (MCP/CD46), the serine protease factor I, and autoantibodies to factor H have been linked to HUS. All of these proteins affect the same enzyme the alternative pathway convertase C3bBb. This article explains how alternative pathway activation proceeds and how defective control increases activation, which ultimately leads to endothelial cell damage.

Complement Factor H↗

PubMed2006

Complement dysfunction in hemolytic uremic syndrome.

PURPOSE OF REVIEW: Hemolytic uremic syndrome is a rare disease of microangiopathic hemolytic anemia, low platelet count and is associated with renal impairment. The atypical form, which occurs in adult patients, is associated with defective complement control. RECENT FINDINGS: Recent data show that atypical hemolytic uremic syndrome is a genetic disease and gene mutations have been reported for factor H, membrane cofactor protein/CD46 and factor I. All corresponding gene products act in concert and control the activity of the complement convertase C3bBb. This enzyme initiates the alternative pathway as well as amplification of the complement system. Similar to genetic defects, autoantibodies which bind to factor H have been linked to the disease. Defective complement control resulting in hemolytic uremic syndrome explains the disease mechanism and allows improved diagnosis and therapy. SUMMARY: The atypical form of hemolytic uremic syndrome is associated with defective complement control and inappropriate protein function and may influence disease progression and provide new ways for treatment. Positive effects were reported upon substitution of a defective protein by plasma exchange or plasmaphoreses. The disease recurrence rate for renal transplants depends on the type of gene mutated; patients with mutations in the membrane cofactor protein gene have a better prognosis than patients with mutations in other genes.

Adult↗

PubMed2006

The vesicle transport protein Vac1p is required for virulence of Candida albicans.

The putative vesicle transport protein Vac1p of the human pathogenic yeast Candida albicans plays an important role in virulence. To determine the cellular functions of Vac1p, a null mutant was generated by sequential disruption of both alleles. The vac1 null mutant strain showed defective endosomal vesicle transport, demonstrating a role of Vac1p in protein transport to the vacuole. Vac1p also contributes to resistance to metal ions, as the null mutant strain was hypersensitive to Cu(2+), Zn(2+) and Ni(2+). In addition, the loss of Vac1p affected several virulence factors of C. albicans. In particular, the vac1 null mutant strain showed defective hyphal growth, even when hyphal formation was induced via different pathways. Furthermore, Vac1p affects chlamydospore formation, adherence to human vaginal epithelial cells, and the secretion of aspartyl proteinases (Saps). Avirulence in a mouse model of systemic infection of the vac1 null mutant strongly suggests that Vac1p of C. albicans is essential for pathogenicity. In summary, the Vac1p protein is required for several cellular pathways, in particular those that control virulence and pathogenicity. Consequently, Vac1p is a novel and interesting target for antifungal drugs.

Animals↗

PubMed2006

Functional characterization of BbCRASP-2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL-1.

Borrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H-like protein-1 (FHL-1) allows spirochetes to resist complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP-2. BbCRASP-2 is distinct from the four previously identified factor H/FHL-1-binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP-2, cspZ, is located on the linear plasmid lp28-3. BbCRASP-2 is highly divergent from the factor H/FHL-1-binding protein BbCRASP-1 and from members of the factor H-binding Erp (OspE/F-related) protein family. Peptide mapping analysis revealed that the factor H/FHL-1 binding site is discontinuous and it was found that C-terminal truncations abrogate factor H and FHL-1 binding. The predominant BbCRASP-2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL-1 bound to BbCRASP-2 maintain cofactor activity for factor I-mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP-2 in serum-sensitive B. burgdorferi mutant B313 increased resistance to complement-mediated lysis. The characterization of BbCRASP-2 now provides a complete picture of the three diverse complement regulator-binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.

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↗