Figure 16.

Native-MS-assisted analysis of the initial steps in complement activation: stepwise reconstitution of the 2.1 MDa C1:IgG:Ag complexes, comprising 40 protein subunits, monitored by native MS. (A) Native MS analysis of reconstituted C1, C1:IgG, and C1:IgG:Ag complexes. The signals shown in dashed boxes are color-coded according to the stoichiometries of C1 assembly, as specified to the left of the corresponding spectra. The bottom spectrum demonstrates the successful reconstitution of C1, containing C1q, two copies of C1r, and two copies of C1s. The middle and top spectra show that the latter assembly successfully binds to (IgG)₆ and subsequently to 12 copies of the antigen (Ag). (B) Model summarizing molecular determinants contributing to IgG-mediated activation of the classical complement pathway. The assembly of the complement initiation complex is proposed to be dependent on (1) the availability of specific antigens recognized by IgG antibodies, (2) Ag compatibility with clustering at the cell surface or in solution, (3) the Fc-domain conformation, (4) the high-avidity binding site for hexavalent C1q, (5) the composition of the IgG glycan chains, (6) the presence of Fab arms, and (7) Ag binding by the Fab arms. Reproduced with permission from ref (293). Copyright 2016 Elsevier Inc.