Structure and Interactions of Human IgG-Fc

Abstract: This thesis involves structure and interaction studies of the Fc fragment of human IgG. For this purpose, hIgG-Fc of different subclasses were cloned and expressed in the eukaryotic host Pichia pastoris, where relevant protein modification at the post-translational level can be obtained.Sometimes, changes in pH, temperature and salt concentration or addition of moderate amounts of denaturants to a protein solution are associated with the protein forming non-natively folded states, such as the molten globule or the A state. IgG and some parts thereof are capable of forming another, so called alternatively folded state, usually induced by acidification in the presence of anions. This state is in many aspects related to the molten globule and the A state but with distinguishing properties related mainly to chemical stability and formation of oligomeric structures. The first part of this thesis describes two different alternatively folded states of hIgG-Fc of subclass 4. One of them was induced by decreasing the pH of the protein solution. Observed structural changes were highly dependent on the concentration of sodium chloride. The alternatively folded protein showed drastic changes in its secondary structure compared to the native protein and significant tertiary structure was lost. Moreover, it displayed an apparently increased chemical stability and had surface exposed hydrophobic patches resulting in the formation of higher order assemblies. In addition, it was shown for the first time that thermal induction of an alternatively folded state is also possible, with similar, but not identical, properties as the acid-induced state. Heat incubation for 20 hours at neutral pH and at a physiological salt concentration further resulted in the formation of protein aggregates. The dye Congo red had affinity for these aggregates, and when viewed under polarized light, it showed green birefringence. They also displayed binding of Thioflavin T and had a typical fibril appearance in the transmission electron microscope. Hence, the formed aggregates share key properties with structures constituting amyloid.The second part of this thesis is focused on interactions of the Fc-fragment with respect to both Fcγ-receptors on monocytes and the IgG autoantibody rheumatoid factor. Immune complexes and their binding to Fcγ-receptors are of pathogenic interest to rheumatoid arthritis. A surface mimic presenting full IgG molecules was designed as an in vitro immune complex model. Utilizing self-assembled monolayers composed of alkanethiolates with different chemical functionalities, the lateral IgG density could be tuned, enabling control of monocyte interaction with the surface. Importantly, the IgG molecules were homogeneously oriented to expose the Fc-fragment. The protein repellent properties of these  surfaces ensured that only differences in IgG concentration determined variations in cellular adhesion. In a separate study the specificities of IgG rheumatoid factor with respect to the different subclasses of hIgG-Fc were investigated, using sera from patients with early rheumatoid arthritis. Strikingly high IgG-RF reactivity against hIgG2-Fc was observed, together with raised levels against hIgG1-Fc and hIgG4-Fc. No reactivity against hIgG3-Fc was found.

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