Production of mouse monoclonal antibodies that detect distinct neoantigenic epitopes on bound C3b and iC3b but not on the corresponding soluble fragments

Nilsson, Bo; Svensson, K.-E.; Borwell, P.; Nilsson, Ulf

doi:10.1016/0161-5890(87)90023-x

Cited by 39 publications

(24 citation statements)

References 15 publications

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“…4 -7 indicate that there is a fundamental difference between the control of soluble C3b and that of cellbound C3b. Nilsson et al (67) describe antigenic differences between bound and soluble C3b. Conformational neo-epitopes were suggested to be present on surface-bound C3b, which are not present on soluble C3b, although these epitopes have not been characterized.…”

Section: Discussionmentioning

confidence: 99%

Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces

Ferreira

Herbert

Hocking

et al. 2006

The Journal of Immunology

133

109

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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.

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Section: Discussionmentioning

confidence: 99%

Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces

Ferreira

Herbert

Hocking

et al. 2006

The Journal of Immunology

133

109

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“…Several studies have indicated that C3 undergoes conformational changes upon its conversion to C3(H 2 O), but the regions involved in these changes are largely unknown (1,3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Using HDX and mass spectrometry, we have localized changes in solvent accessibility between native C3 and C3(H 2 O).…”

Section: Discussionmentioning

confidence: 99%

Solvent Accessibility of Native and Hydrolyzed Human Complement Protein 3 Analyzed by Hydrogen/Deuterium Exchange and Mass Spectrometry

Winters

Spellman

Lambris

2005

The Journal of Immunology

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Complement protein C3 is a 187-kDa (1641-aa) protein that plays a key role in complement activation and immune responses. Its hydrolyzed form, C3(H2O), is responsible for the initiation of the activation of alternative complement pathway. Previous analyses using mAbs, anilinonaphthalenesulfonate dyes, and functional studies have suggested that C3 is conformationally different from C3(H2O). We have used amide hydrogen/deuterium exchange and MALDI-TOF mass spectrometry to identify and localize structural differences between native C3 and C3(H2O). Both proteins were incubated in D2O for varying amounts of time, digested with pepsin, and then subjected to mass-spectrometric analysis. Of 111 C3 peptides identified in the MALDI-TOF analysis, 31 had well-resolved isotopic mass envelopes in both C3 and C3(H2O) spectra. Following the conversion of native C3 to C3(H2O), 17 of these 31 peptides exhibited a change in deuterium incorporation, suggesting a conformational change in these regions. Among the identified peptides, hydrogen/deuterium exchange data were obtained for peptides 944–967, 1211–1228, 1211–1231, 1259–1270, 1259–1273, 1295–1318, and 1319–1330, which span the factor H binding site on C3d and factor I cleavage sites, and peptides 1034–1048, 1049–1058, 1069–1080, 1130–1143, 1130–1145, 1211–1228, 1211–1231, 1259–1270, and 1259–1273, spanning 30% of the C3d region of C3. Our results suggest that hydrolysis may produce a looser (more open) structure in the C3d region, in which some of the changes affect the conversion of helical segments into coil segments facilitating interactions with factors I and H. This study represents the first detailed study mapping the regions of C3 involved in conformational transition when hydrolyzed to C3(H2O).

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“…AT and C3 were purified from human plasma as described previously. 31,32 The anti-C3 mAb 26.1 against a neo-epitope in C3d,g was produced and characterized according to Nilsson et al, 33 and polyclonal anti-C3c was bought from Dako A/S, Glostrup, Denmark. The purified proteins were diluted in veronal-buffered saline (VB þþ ; 5 mM barbiturate, pH 7.4; 145 mM NaCl; 0.15 mM Ca 2þ ; 0.5 mM Mg 2þ ).…”

Section: Methodsmentioning

confidence: 99%

Absence of conformational change in complement factor 3 and factor XII adsorbed to acrylate polymers is related to a high degree of polymer backbone flexibility

et al. 2017

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In previous investigations, the authors have examined the adsorption of albumin, immunoglobulin, and fibrinogen to a series of acrylate polymers with different backbone and side-group flexibility. The authors showed that protein adsorption to acrylates with high flexibility, such as poly(lauryl methacrylate) (PLMA), tends to preserve native conformation. In the present study, the authors have continued this work by examining the conformational changes that occur during the binding of complement factor 3 (C3) and coagulation factor XII (FXII). Native C3 adsorbed readily to all solid surfaces tested, including a series of acrylate surfaces of varying backbone flexibility. However, a monoclonal antibody recognizing a “hidden” epitope of C3 (only exposed during C3 activation or denaturation) bound to the C3 on the rigid acrylate surfaces or on polystyrene (also rigid), but not to C3 on the flexible PLMA, indicating that varying degrees of conformational change had occurred with binding to different surfaces. Similarly, FXII was activated only on the rigid poly(butyl methacrylate) surface, as assessed by the formation of FXIIa-antithrombin (AT) complexes; in contrast, it remained in its native form on the flexible PLMA surface. The authors also found that water wettability hysteresis, defined as the difference between the advancing and receding contact angles, was highest for the PLMA surface, indicating that a dynamic change in the interface polymer structure may help protect the adsorbed protein from conformational changes and denaturation.

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Production of mouse monoclonal antibodies that detect distinct neoantigenic epitopes on bound C3b and iC3b but not on the corresponding soluble fragments

Cited by 39 publications

References 15 publications

Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces

Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces

Solvent Accessibility of Native and Hydrolyzed Human Complement Protein 3 Analyzed by Hydrogen/Deuterium Exchange and Mass Spectrometry

Absence of conformational change in complement factor 3 and factor XII adsorbed to acrylate polymers is related to a high degree of polymer backbone flexibility

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