Structural and Functional Characterization of Pentameric and Hexameric IgM Antibodies in Complement System Activation

Abstract

Immunoglobulin M (IgM) antibodies are critical components of the immune system, characterized by their pentameric or hexameric structure and exceptional binding avidity. Understanding the structural and functional differences between these forms is essential for advancing therapeutic antibody development and furthering our understanding of immune responses. This dissertation comprises interconnected studies that advance our understanding of IgM biology through methodological innovation and biophysical characterization.Chapter 1 will introduce and provide relevant background information on the topics discussed herein including IgM biology and function, hydrogen deuterium exchange mass spectrometry (HDX-MS) as a biophysical tool to study complex systems, complement cascade activation and its role in the immune system, and the myriad other techniques employed in the studies conducted. Chapter 2 addresses a fundamental methodological challenge in hydrogen-deuterium exchange mass spectrometry: hydrogen scrambling during analysis. We systematically mapped activation thresholds for scrambling, revealing that it is a global process with peptide-specific thresholds, providing critical parameters for optimizing site-specific HDX-MS experiments. Chapter 3 presents comprehensive structural characterization of pentameric and hexameric IgM using HDX-MS, electron microscopy, and small-angle X-ray scattering. These studies reveal distinct conformational properties between the two forms, with hexameric IgM showing greater flexibility in the Fc region and more extensive conformational changes upon binding to antigen-displaying nanoparticles. Chapter 4 presents functional characterization demonstrating that hexameric IgM activates complement slightly more efficiently than pentameric IgM. Integration of structural and functional data reveals that the optimal Fc presentation in hexameric IgM facilitates more effective C1q binding and complement activation. Chapter 5 presents preliminary data on C1 complex interactions and conformational dynamics and discusses future directions for understanding the complete molecular mechanism of IgM-mediated complement activation, with implications for therapeutic antibody development.