Defining the Structural Features and Functional Characteristics of Complement-Active IgM

Abstract

Immunoglobulin M (IgM) is an extremely important, yet surprisingly under-studied glycoprotein that is shared among all vertebrates on an evolutionary timeline spanning more than 500 million years. In addition to the genetically conserved subset of ‘natural’ IgM that conveys an innate degree of protection before birth, the adaptive immune system also produces ‘induced’ IgM as the initial response to all foreign pathogens. IgM is the most potent known activator of the classical complement cascade, making it an attractive therapeutic platform for the potential treatment of a wide array of disease states; while multiple IgG molecules must bind with coordination of their Fc regions together at the membrane of a pathogenic target, a single molecule of IgM is sufficient for the full-scale activation of complement, owing to the multivalency of its secreted pentameric and hexameric isoforms. Although the relationship between IgM and complement has been appreciated for decades, the biophysical details that underlie its activation mechanism still remain largely unknown. Many past attempts have been made to uncover the structural details surrounding the IgM-mediated activation of complement, but the same physical traits that give IgM its unique properties and relationship with the immune system have also earned it a notorious reputation for being technically difficult to handle and investigate experimentally. The large size, extensive glycosylation, and high degree of flexibility has rendered IgM largely refractive to many established structural techniques, and to date no fully intact IgM structures have been resolved; all structural characterizations and protein:protein interfaces have so far been inferred from combinations of fragmented structures, homology modeling, electron microscopy, X-ray scattering, and molecular dynamics simulations. While a number of current IgM drug candidates have shown promise for the treatment of several different cancers, the biophysical details of IgM-mediated complement activation still remain poorly understood. The objective of this dissertation is to establish a baseline set of structural features and functional characteristics that define the complement-active, antigen bound form of IgM – commonly referred to as the ‘staple’ conformation – and differentiate it further from other sub-active and inactive forms. Chapter 1 provides a historical review and outlines the contextual background that currently surrounds some of the major gaps in knowledge regarding the structure and function of IgM as it relates to the activation of complement. In Chapter 2, background work is presented on the development of a low-cost, automated LC-MS system designed to improve both the throughput and consistency of decoupled hydrogen/deuterium exchange mass spectrometry (HDX-MS) samples, which is the primary structural technique used to assess the defining features of the staple conformation. In Chapter 3, the functional aspects of complement-active IgM are explored through a combination of novel ligand binding and activity assay approaches, revealing a number of key variables that directly impact the binding kinetics and activation rates of the initiating molecules of the classical cascade pathway, C1q and C1. The mechanistic role of IgM binding valency/avidity was also investigated through a prebinding approach that, to our surprise, indicated there to be no hard stoichiometric cutoff for the number of surface bound arms of IgM required to bind/activate C1q/C1. The functional characteristics determined in Chapter 3 pair directly with the structural features found in Chapter 4 (via HDX-MS) in order to complete the defining structure/function relationship that underlies the active staple conformation of IgM. Strong evidence of a mechanistic hinge, compensatory rearrangement of domains within the Fc core, and the identity of previously unreported residues that may comprise a portion of the C1q binding site (in addition to the longstanding putative location) were discovered within the multivalent surface bound form of complement-active IgM. Additionally, the existence of an unexpectedly extensive network of allosterism was observed within solution bound IgM that could add much needed clarity to the litany of confusing reports found throughout the classical literature. Finally, in Chapter 5, a brief project conclusion is provided along with a discussion of future directions. Collectively, this work establishes key structural and functional qualities that define the complement-active form of IgM, along with a number of newly developed methods, improved protocols, and a foundation for IgM structural work upon which future research can be built.