Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen.
The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophysical characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in solution by native mass spectrometry and size-exclusion chromatography. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-saturated IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, the Netherlands.
Guanbo
G
Wang
Genmab, Yalelaan 60, 3584 CM Utrecht, the Netherlands.
Rob N
RN
de Jong
Genmab, Yalelaan 60, 3584 CM Utrecht, the Netherlands.
Ewald T J
ET
van den Bremer
Genmab, Yalelaan 60, 3584 CM Utrecht, the Netherlands.
Frank J
FJ
Beurskens
Genmab, Yalelaan 60, 3584 CM Utrecht, the Netherlands.
Aran F
AF
Labrijn
Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research and Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, the Netherlands.
Deniz
D
Ugurlar
Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research and Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, the Netherlands.
Piet
P
Gros
Genmab, Yalelaan 60, 3584 CM Utrecht, the Netherlands.
Janine
J
Schuurman
Genmab, Yalelaan 60, 3584 CM Utrecht, the Netherlands; Department of Immunohematology and Blood Transfusion, University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands. Electronic address: p.parren@genmab.com.
Paul W H I
PW
Parren
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, the Netherlands. Electronic address: a.j.r.heck@uu.nl.
Albert J R
AJ
Heck
D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003167:Complement Activation; D015922:Complement C1q / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D055550:Protein Stability; D053719:Tandem Mass Spectrometry
D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D015922:Complement C1q