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Updated: Oct 2, 2025
Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells
Published on: January 29, 2014
Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1
Saeko Yanaka1,2, Shigetaka Nishiguchi1, Rina Yogo1,2
1Exploratory Research Center on Life and Living Systems (ExCELLS), Institute for Molecular Science (IMS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
Insights
Researchers visualized how Immunoglobulin G (IgG) interacts with the C1 complex, revealing a secondary binding site on IgG. This discovery offers insights into the complement pathway and potential therapeutic antibody development.
Area of Science:
- Immunology
- Structural Biology
- Biochemistry
Background:
- Immunoglobulin G (IgG) has a modular structure crucial for antigen recognition and effector functions like complement-dependent cytotoxicity.
- IgG self-assembles into hexameric rings on antigen-presenting membranes, initiating complement pathway activation via C1q binding.
Purpose of the Study:
- To investigate the initial molecular interactions in the complement pathway.
- To quantitatively visualize the binding dynamics between mouse IgG and the C1 complex (C1q, C1r, C1s) using high-speed atomic force microscopy.
Main Methods:
- High-speed atomic force microscopy (HS-AFM) for quantitative visualization.
- Study of interactions between mouse IgG2b and C1 complex.
- Analysis of interactions between a CH1-deleted IgG2a variant and C1/C1q.
Main Results:
- C1q within the C1 complex exhibits restricted internal motion.
- The C1 complex demonstrates enhanced binding affinity to on-membrane IgG2b assemblages compared to C1q alone.
- A 1:1 interaction was observed between C1/C1q and a CH1-deleted IgG2a variant, involving a cryptic secondary binding site on the CL domain.
Conclusions:
- The restricted motion of C1q in the C1 complex and increased binding affinity to IgG assemblages are attributed to reduced conformational entropy loss.
- A novel, antigen-independent binding interaction mediated by the CL domain of IgG was identified.
- Findings provide a basis for developing novel therapeutic antibodies targeting the complement pathway.
Abstract:
Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.
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