Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC)

Sánchez-Corral, Pilar; Antón, Luis C.; Alcolea, JoséM.; Marqués, Guillermo; Sánchez, Angel; Vivanco, Fernando

doi:10.1016/0022-1759(89)90340-2

Cited by 26 publications

(20 citation statements)

References 25 publications

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“…C3b was generated by limited digestion with trypsin as previously described (24). The monoclonal anti-human C3b antibody, C3-30, has been reported previously (25).…”

Section: Methodsmentioning

confidence: 99%

Complement Factor H Binds to Denatured Rather than to Native Pentameric C-reactive Protein

Hakobyan

Harris

Berg

et al. 2008

Journal of Biological Chemistry

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Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca 2؉ to maintain its native fold and pentameric subunit assembly or by specific Ca 2؉ -dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca 2؉ , conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein.We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.

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“…C3b was generated by limited digestion with trypsin as previously described (24). The monoclonal anti-human C3b antibody, C3-30, has been reported previously (25).…”

Section: Methodsmentioning

confidence: 99%

Complement Factor H Binds to Denatured Rather than to Native Pentameric C-reactive Protein

Hakobyan

Harris

Berg

et al. 2008

Journal of Biological Chemistry

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“…A 1/600 dilution of this broth was realized in 60% of NHS (Normal Human Serum) or hiNHS (heat-inactivated Normal Human Serum) and incubated at 37°C. Normal Human Serum AB-type (PAA Laboratories) used was handled in a manner to preserve complement activity [34] or heat-inactivated at 56°C during 30 min. The percentage of bacteria surviving at 30 min was determined.…”

Section: Methodsmentioning

confidence: 99%

A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation

et al. 2017

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Neisseria meningitidis is a commensal of human nasopharynx. In some circumstances, this bacteria can invade the bloodstream and, after crossing the blood brain barrier, the meninges. A filamentous phage, designated MDAΦ for Meningococcal Disease Associated, has been associated with invasive disease. In this work we show that the prophage is not associated with a higher virulence during the bloodstream phase of the disease. However, looking at the interaction of N. meningitidis with epithelial cells, a step essential for colonization of the nasopharynx, we demonstrate that the presence of the prophage, via the production of viruses, increases colonization of encapsulated meningococci onto monolayers of epithelial cells. The analysis of the biomass covering the epithelial cells revealed that meningococci are bound to the apical surface of host cells by few layers of heavily piliated bacteria, whereas, in the upper layers, bacteria are non-piliated but surrounded by phage particles which (i) form bundles of filaments, and/or (ii) are in some places associated with bacteria. The latter are likely to correspond to growing bacteriophages during their extrusion through the outer membrane. These data suggest that, as the biomass increases, the loss of piliation in the upper layers of the biomass does not allow type IV pilus bacterial aggregation, but is compensated by a large production of phage particles that promote bacterial aggregation via the formation of bundles of phage filaments linked to the bacterial cell walls. We propose that MDAΦ by increasing bacterial colonization in the mucosa at the site-of-entry, increase the occurrence of diseases.

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“…Secondary antibodies were goat anti-rabbit (GAR) or goat anti-mouse (GAM) conjugated to HRP (Dako) for immunoblotting experiments or to Alexa Fluor cytochromes (Molecular probes) or Cy5-conjugated (Millipore) for confocal microscopy experiments. Human serum AB (PAA) was handled in a manner to preserve complement activity [35].…”

Section: Methodsmentioning

confidence: 99%

Nucleolin, a Shuttle Protein Promoting Infection of Human Monocytes by Francisella tularensis

2010

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Background Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells.Methodology/Principal FindingsHere, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant.Conclusions/SignificanceWe herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.

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Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC)

Cited by 26 publications

References 25 publications

Complement Factor H Binds to Denatured Rather than to Native Pentameric C-reactive Protein

Complement Factor H Binds to Denatured Rather than to Native Pentameric C-reactive Protein

A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation

Nucleolin, a Shuttle Protein Promoting Infection of Human Monocytes by Francisella tularensis

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