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Investigating the Classical Pathway of the Complement System: Activation and Inhibition

Research output: Book/anthology/dissertation/reportPh.D. thesis

  • Alessandra Zarantonello
The complement system of innate immunity is a surveillance mechanism performing the functions of phagocytosis, inflammation and cell lysis of pathogens, but it also contributes to homeostasis of the organism by removal of apoptotic material and synaptic pruning in the central nervous system. Complement proceeds as a series of proteolytic reactions conveying structural rearrangements important for the function of each component. The classical pathway of complement is initiated by the heteropentameric C1 complex binding to a susceptible activator causing C1 autoactivation. The mechanism of this process has been investigated for more than 30 years, yet its molecular details remain elusive. The activated C1 complex in turn activates C4, generating the fragment C4b covalently attached to the activator surface, where binding of zymogen C2 results in C4b2, the next in the pathway reaction cascade. C4b2 is cleaved by the C1 complex to form the C4b2a proteolytic enzyme, with substrate C3. Multiple regulators assist factor I with cleaving C4b rendering it inactive. In humans, two C4 isotypes exist, C4A and C4B, and unbalanced expression towards the C4A isotype was recently linked to the neurodevelopmental disorder schizophrenia, but the underlying molecular mechanism is unknown. Possibly, the two C4 isotypes differ in their interactions with C1, C2 and the regulators. A detailed understanding of the activation mechanism of C1, C4 cleavage, and of the formation of the C4b2 complex offers a puzzle in the molecular framework for C4 biology. The classical pathway is also involved in the autoimmune diseases systemic lupus erythematosus and rheumatoid arthritis. Developing a strategy for its regulation could contribute to therapeutic treatment of autoimmune diseases.
The thesis presents the progress made in the reconstitution of the activated C1 complex for structural studies aimed at understanding its activation mechanism. Moreover, the 3.3 Å resolution crystal structure of C4b in complex with a nanobody inhibitor of the classical pathway is presented. The structure explains the inhibitory mechanism, which is based on steric hindrance of C2 binding to C4b. Functional characterization of the inhibitor in vitro and in vivo reveals its applicability in broad contexts. Other non-inhibitory nanobodies were applied as tools in structural studies of the C4b2 complex, and a negative stain electron microscopy reconstruction of the proconvertase is presented. Molecular dynamics simulations on C4b combined with the electron microscopy envelope of C4b2 and an atomic model of the C1 complex suggest a possible mode of activation of C4b2 in the classical pathway.
Original languageEnglish
Place of publicationAarhus
PublisherAarhus Universitet
Number of pages389
Publication statusPublished - 31 Jan 2020

Bibliographical note

Forsvaret 28/4 2020

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