Mapping of the properdin-binding site in the third component of complement.
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ABSTRACT:
SUBMITTER: Lambris JD
PROVIDER: S-EPMC1153213 | biostudies-other | 1984 Jan
REPOSITORIES: biostudies-other
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Project description:The interactions of properdin with both surface-bound and fluid-phase C3 (the third component of complement) and its activation products have been investigated by using a purified preparation of the 'native' form. At physiological ionic strength, a weak interaction with cell-bound C3b (the larger activation fragment of C3) could be demonstrated. In the presence of Factor B this interaction was enhanced, and further enhancement was seen when C3bBb sites were formed on the erythrocytes. The avidities of properdin for cell-bound iC3b (the initial product of Factors I and H action on C3b) and C3b were compared at low ionic strength, with that measured for iC3b being less than that for C3b. In contrast, the affinities of properdin for fluid-phase C3b, iC3b and C3c (the larger product of Factors I and H or CR1 (the C3b receptor) action on iC3b) were all very similar, and apparently much weaker than that for cell-bound C3b. No interaction with either native C3 or, more surprisingly, C3i (haemolytically inactive C3) could be detected. Properdin also inhibited Factor I binding to, and action upon, cell-bound C3b, but did not inhibit Factor I action on fluid-phase C3b. These data permit a more detailed description of the roles of properdin in the alternative pathway of complement activation, emphasizing its importance in concentrating activation at the activating surface.
| S-EPMC1149105 | biostudies-other
Project description:The retinal pigment epithelium (RPE) maintains visual function and preserves structural integrity of the retina. Chronic dysfunction of the RPE is associated with retinal degeneration, including age-related macular degeneration (AMD). The AMD pathogenesis includes both increased oxidative stress and complement dysregulation. Physiological sources of oxidative stress in the retina are well known, while complement sources and regulation are still under debate. Using human primary RPE (hpRPE) cells, we have established a model to investigate complement component expression on transcript and protein level in AMD-risk and non-risk hpRPE cells. We evaluated the effect of properdin, a complement stabilizer, on the hpRPE cell-dependent complement profile exposed to oxidative stress. hpRPE cells expressed complement components, receptors and regulators. Complement proteins were also stored and secreted by hpRPE cells. We associated AMD-risk single nucleotide polymorphisms with an increased secretion of complement factors D (CFD) and I (CFI). Furthermore, we detected hpRPE cell-associated complement activation products (C3a, C5a) independent of any extracellularly added complement system. Exogenous properdin increased the mRNA expression of CFI and CFD, but decreased levels of complement components (C1Q, C3), receptors (C3AR, C5AR1, CD11B) and inflammation-associated transcripts (NLRP3, IL1B) in hpRPE cells exposed to oxidative stress. This properdin effect was time-dependently counter regulated. In conclusion, our data unveiled a local, genotype-associated complement component production in hpRPE cells, regulated by exogenous properdin. The local complement production and activation via blood-independent mechanisms can be a new therapeutic target for AMD.
| S-EPMC7555107 | biostudies-literature
Project description:Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.
| S-EPMC5278493 | biostudies-literature
Project description:BACKGROUND:Trichinella spiralis is a tissue-dwelling parasite has developed the ability to evade the host immune attack to establish parasitism in a host. One of the strategies evolved by the nematode is to produce proteins that immunomodulate the host immune system. TsPmy is a paramyosin secreted by T. spiralis on the surface of larvae and adult worms that can interact with complement components C1q and C8/C9 to compromise their activation and functions. To better understand the mechanism of TsPmy involved in the C1q inactivation and immune evasion, the C1q-binding site on TsPmy was investigated. METHODS:The TsPmy C1q-binding site was investigated by sequential narrow-down fragment expression in bacteria and peptide binding screening. C1q binding activity was identified by Far-Western blotting and ELISA assays. RESULTS:After several runs of sequential fragment expression, the C1q binding site was narrowed down to fragments of N-terminal TsPmy226-280aa and TsPmy231-315aa, suggesting the final C1q binding site is probably located to TsPmy231-280aa. A total of nine peptides covering different amino acid sequences within TsPmy231-280aa were synthesized. The binding assay to C1q determined that only P2 peptide covering TsPmy241-280aa binds to C1q, indicating that the C1q binding domain may need both the linearized sequence and conformational structure required for binding to C1q. The binding of peptide P2 to C1q significantly inhibited both C1q-initiated complement classical activation and C1q-induced macrophage chemotaxis. CONCLUSIONS:This study identifies the C1q binding site within TsPmy which provides helpful information for developing a vaccine against trichinellosis by targeting the C1q-binding activity of TsPmy.
| S-EPMC6307294 | biostudies-literature
Project description:Properdin is a plasma protein and is also released from neutrophil granules following stimulation. At inflammatory sites it can bind bacteria and apoptotic bodies to trigger alternative pathway (AP) activation. Principles governing properdin homeostasis are unknown. We monitored properdin during AP activation and in complement-deficient mice. There was a >90% reduction of properdin in the Crry single-knockout mice (Crry SKO). These membrane complement regulatory protein-deficient mice feature accelerated AP turnover, leading to reduced C3 and fB. Injecting cobra venom factor into wild-type mice activated the AP and led to the consumption of C3, fB, and properdin. However, and unexpectedly, properdin was also deficient in C3(-/-), fB(-/-), and fD(-/-) mice. It was present in C1q(-/-), C4(-/-), and C5(-/-) mice. These findings implicate AP turnover in the maintenance of basal levels of properdin in the blood. To explore the mechanism, classical pathway-activating immune complexes were infused. Within 10 min, properdin was partially restored in fB(-/-) but not in C3(-/-) mice. Markedly reduced properdin in mice deficient in an AP component and its partial restoration by activating C3 suggest a requirement for continuous C3 activation via AP tickover to maintain properdin homeostasis. The mechanism underlying this C3-dependent process was not identified. Engagement of C3a and C5a receptors was ruled out. These findings represent an instructive example of how a positive regulator of an innate immune recognition and effector pathway is controlled. A rationale for such a means to supply properdin for immune reactions is proposed.
| S-EPMC2984156 | biostudies-literature
Project description:Apoptotic cells must be rapidly eliminated to avoid harmful inflammatory and autoimmune reactions. Innate immunity is designed/poised to identify dying cells by their unique surface-associated molecular patterns. Here we demonstrate for the first time, to our knowledge, that the human complement protein properdin binds to early apoptotic T cells and initiates complement activation, leading to C3b opsonization and ingestion by phagocytic cells. Properdin binding was facilitated by the glycosaminoglycan chains of surface proteoglycans. Properdin released by activated neutrophils was particularly effective at recognition of apoptotic T cells, whereas the binding activity of properdin in the serum appeared to be inhibited. "Properdin tagging" of apoptotic T cells also induced their uptake by phagocytes independent of complement activation or other complement proteins. Although our findings were made primarily with apoptotic T cells, they suggest that properdin could play a similar role during apoptosis of other cell types.
| S-EPMC2449358 | biostudies-literature
Project description:The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.
| S-EPMC4010523 | biostudies-literature
Project description:The sodium pump, or Na,K-ATPase, exports three intracellular sodium ions in exchange for two extracellular potassium ions. In the high resolution structure of the related calcium pump, two cation-binding sites have been identified. The two corresponding sites in the sodium pump are expected to be alternatively occupied by sodium and potassium. The position of a third sodium-specific site is still hypothetical. Here, we report the large effects of single residue substitutions on the voltage-dependent kinetics of the release of sodium to the extracellular side of the membrane. These mutations also alter the apparent affinity for intracellular sodium while one of them does not affect the intrinsic affinity for potassium. These results enable us to locate the third sodium-specific site of the sodium pump in a space between the fifth, sixth, and ninth transmembrane helices of the alpha-subunit and provide an experimental validation of the model proposed by Ogawa and Toyoshima [Ogawa, H. & Toyoshima, C. (2002) Proc. Natl. Acad. Sci. USA 99, 15977-15982].
| S-EPMC1200292 | biostudies-literature
Project description:Background Properdin (P) is a positive regulator of the alternative pathway of complement activation. Although P inhibition is expected and has been shown to ameliorate the alternative pathway of complement-mediated tissue injury in several disease models, it unexpectedly exacerbated renal injury in a murine model of C3 glomerulopathy. The role of P in atypical hemolytic uremic syndrome (aHUS) is uncertain.Methods We blocked P function by genetic deletion or mAb-mediated inhibition in mice carrying a factor H (FH) point mutation, W1206R (FHR/R), that causes aHUS and systemic thrombophilia with high mortality.Results P deficiency completely rescued FHR/R mice from premature death and prevented thrombocytopenia, hemolytic anemia, and renal disease. It also eliminated macrovessel thrombi that were prevalent in FHR/R mice. All mice that received a function-blocking anti-P mAb for 8 weeks survived the experimental period and appeared grossly healthy. Platelet counts and hemoglobin levels were significantly improved in FHR/R mice after 4 weeks of anti-P mAb treatment. One half of the FHR/R mice treated with an isotype control mAb but none of the anti-P mAb-treated mice developed stroke-related neurologic disease. Anti-P mAb-treated FHR/R mice showed largely normal renal histology, and residual liver thrombi were detected in only three of 15 treated mice.Conclusions These results contrast with the detrimental effect of P inhibition observed in a murine model of C3 glomerulopathy and suggest that P contributes critically to aHUS pathogenesis. Inhibition of P in aHUS may be of therapeutic benefit.
| S-EPMC6050941 | biostudies-literature
Project description:The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.
| S-EPMC1163402 | biostudies-other