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Michael K Pangburn

Publications and source records attributed to Michael K Pangburn.

11 recordsLinked to original sources
PubMed2006

Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces.

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19-20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19-20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19-20 (rH 19-20). Direct competition with the N-terminal site is unlikely to explain this because regulation of fluid phase C3b is unaffected by domains 19-20. Finally, we show that addition of isolated rH 19-20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken together, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19-20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue.

Animals↗
PubMed2006

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase.

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.

Animals↗
PubMed2006

Disease-associated sequence variations congregate in a polyanion recognition patch on human factor H revealed in three-dimensional structure.

Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

Amino Acid Sequence↗
PubMed2006

Herpes simplex virus type 1 and 2 glycoprotein C prevents complement-mediated neutralization induced by natural immunoglobulin M antibody.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.

Animals↗
PubMed2005

In vivo binding of complement regulator factor H by Streptococcus pneumoniae.

Pneumococcal surface protein C (PspC) binds to the complement regulatory protein factor H (FH), which inhibits alternative pathway activation. In the present study, using a mouse model of systemic infection and flow-cytometric analyses, we demonstrated an in vivo interaction between FH and pneumococci and showed differential FH binding during bacteremia. Flow-cytometric analyses of pneumococci harvested after intraperitoneal (ip) challenge demonstrated increased binding of FH, compared with that after intravenous (iv) challenge. Real-time polymerase chain reaction analyses of PspC mRNA showed that, relative to pneumococci grown in vitro, those recovered from the blood of mice 24 h after iv challenge exhibited 23-fold higher mRNA levels; however, after ip challenge, PspC mRNA induction was increased 870-fold. A subsequent increase in PspC expression was detected by flow cytometry using a monoclonal antibody against PspC. Furthermore, pneumococci with FH bound to complement before exposure had increased proliferation, compared with pneumococci not pretreated with FH. These results suggest that the interaction between PspC and FH contributes to pneumococcal virulence.

Animals↗
PubMed2004

Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H.

Streptococcus pneumoniae, also known as the pneumococcus, contains several surface proteins that along with the polysaccharide capsule function in antiphagocytic activities and evasion of the host immune system. These pneumococcal proteins interact with the host immune system in various ways and possess a wide range of biological activities that suggests that they may be involved at different stages of pneumococcal infection. PspC, also known as CbpA and SpsA, is one of several pneumococcal surface proteins that binds host proteins, including factor H (FH) and secretory IgA (sIgA) via the secretory component. Previous work by our laboratory has demonstrated that PspC on the surface of live pneumococcal cells binds FH. This paper provides evidence that FH activity is maintained in the presence of PspC and that the PspC binding site is located in the short consensus repeat 6-10 region of FH. We also report for the first time that although both FH and sIgA binding has been localized to the alpha-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the alpha-helical region of PspC.

Bacterial Proteins↗
PubMed2004

A novel vector for the expression of SCR domains in insect cells.

Exploitation of recombinant technology to study proteins containing strings of short consensus repeat (SCR) domains largely depends on expression vectors. In this paper, we describe a vector for cloning and constitutive expression of single or multiple SCR domains. The recombinant vector has unique additive features over commercially available vectors that make it a universal cloning vector for SCR domains as well as a vector suitable for expressing any protein fragment beginning and ending with cysteine residues. As a demonstration of its usefulness, the constitutive extracellular expression of five SCR-containing proteins derived from complement factor H is presented.

Animals↗
PubMed2004

Interaction of human factor H with PspC of Streptococcus pneumoniae.

BACKGROUND & OBJECTIVES: Streptococcus pneumoniae has acquired virulence factors such as the polysaccharide capsule and various surface proteins, which prevent opsonization mediated by the complement system. PspC is one of the multi-functional pneumococcal surface proteins capable of eliciting an antibody response in mice. Our study further explores the role of pneumococcal surface proteins in resistance to complement mediated opsonophagocytosis by providing evidence that PspC binds human Factor H (FH), a regulatory protein of the alternative complement pathway. The present study was carried out to map the binding regions on PspC and FH, and to assess the functional activity of FH upon binding to PspC. METHODS: FH binding to D39 and other pneumococcal strains was observed by flow cytometry. A series of FH truncated and deletion mutants and PspC mutants were used to localize binding regions within these molecules. The functional activity of FH upon binding to PspC was measured by a haemolysis assay. RESULTS: FH binding to D39 and not to TRE108 (PspC-) cells was demonstrated by flow cytometry. Pneumococcal isolates of 14 different strains varied in their ability to bind FH. The binding region of FH within PspC to the first 225 amino acids of the alpha-helical domain was localized. The corresponding binding site for PspC is located within the SCR 6-10 region of FH. Haemolysis of rabbit red blood cells was inhibited by FH even in the presence of PspC. INTERPRETATION & CONCLUSION: FH binding is specific to PspC on the pneumococcal cell surface. The binding region on PspC mapped to the non-conserved N-terminal region of the alpha-helical domain. The binding site on FH to PspC is different from the active site that functions in degradation of C3b. A haemolysis assay provided evidence that the functional activity of FH was maintained upon binding to PspC. Thus, binding of FH to PspC might be an important mechanism by which S. pneumoniae resist complement activation and opsonophagocytosis.

Bacterial Proteins↗
PubMed2003

Formation of high affinity C5 convertase of the classical pathway of complement.

C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.

Animals↗
PubMed2002

Cutting edge: localization of the host recognition functions of complement factor H at the carboxyl-terminal: implications for hemolytic uremic syndrome.

Incidents of hemolytic uremic syndrome (HUS) include a subset of patients that exhibit mutations in C factor H. These mutations cluster in the C-terminal domains of factor H where previous reports have identified polyanion and C3b-binding sites. In this study, we show that recombinant human factor H with deletions at the C-terminal end of the protein loses the ability to control the spontaneous activation of the alternative C pathway on host-like surfaces. For the pathology of HUS, the findings imply that mutations that disrupt the normal functions of the C-terminal domains prevent host polyanion recognition. The resulting uncontrolled activation of complement on susceptible host tissues appears to be the initiating event behind the acute renal failure of familial HUS patients.

Animals↗