IgG molecules that bind antigen on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centered on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.
Keywords: C1q; CL; high-speed atomic force microscopy; immunoglobulin G; nuclear magnetic resonance.
© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology.