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Bacillus anthracis is the causative agent of the infectious disease anthrax. The disease is caused after taking up the spores by either inhalation, ingestion or through skin lesion. They then germinate inside the body and start secreting the tripartite anthrax toxin. This toxin can hijack the two anthrax toxin receptors, CMG2 (ANTXR2) or TEM8 (ANTXR1) to enter host cells. Once inside the host cell, the toxin will start to interfere with essential cellular pathways, ultimately leading to severe damage and death. The two anthrax toxin receptors have been well described in the context of anthrax toxin pathogenesis. Their normal, physiological function however has been studied far less. Mutations in either one of the two proteins leads to a severe genetic disease, underlining the importance CMG2 and TEM8 normally have. My thesis therefore aimed at better understanding the physiological function of the two receptors. In a first part, we chose to study the impact of N-glycosylation on the folding, trafficking and, finally, the function of CMG2 and TEM8. N-glycosylation is a very common posttranslational modification and has been shown to play an important role in various cellular processes such as protein folding, stability, cell adhesion, endocytosis, cell-cell recognition and trafficking. We show that both receptors are glycosylated on the predicted sites, with varying impacts on different functions of the proteins. TEM8 crucially relies on the presence of the sugar sidechains, as a non-glycosylated mutant will fail to fold, will be retained in the ER and is non-functional. CMG2 is less sensitive to defects in glycosylation, even a non-glycosylated mutant will be able to reach its target destination and function correctly. However, in the simultaneous presence of mutations seen in Hyaline Fibromatis Syndrome, N-glycosylation seems to function as a buffer, stabilizing the mutated protein. N-glycosylation helps to increase the folding capacity of both receptors and therefore impacts on their physiological function. In a second part our objective was to find new interaction partners of CMG2 and study their effect on toxin endocytosis as a suitable functional output. We identified 4 new proteins that regulate anthrax toxin entry via CMG2: RNF41 and Cbl, both ubiquitin E3 ligases, MARK2, a serine-threonine kinase and USP8, a deubiquitinating enzyme. In the absence of any of these four proteins, toxin entry into cells was strongly delayed or blocked. Of these four new regulators, Cbl and MARK2 are conserved for toxin entry via TEM8. We found that both RNF41 and Cbl can ubiquitinate CMG2, but regulate different steps of toxin entry. We also established a flow cytometry-based endocytosis assay for CMG2 and TEM8, enabling us to look at endocytosis and internalization kinetics in the absence of the pathogenic ligand anthrax toxin. Both receptors continuously internalize from the plasma membrane in an endocytic process, which is regulated by ligand-binding and the multimerization of the receptors. Interestingly, CMG2 and TEM8 show a striking difference in their endocytosis rate, suggesting an important functional role of this behavior as well.

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