Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound β-domain and a passenger domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several passenger domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. In the present study we describe the effect of glycosylation on the extracellular passenger domain of the Escherichia coli autotransporter Ag43α, which induces frizzy colony morphology and cell settling. We identify 16 glycosylation sites and suggest two possible glycosylation motifs for serine and threonine residues. Glycosylation stabilizes against thermal and chemical denaturation and increases refolding kinetics. Unexpectedly, glycosylation also reduces the stabilizing effect of Ca2+ ions, removes the ability of Ca2+ to promote cell adhesion, reduces the ability of Ag43α-containing cells to form bacterial amyloid and increases the susceptibility of the resulting amyloid to proteolysis. In addition, our results indicate that Ag43α folds without a stable intermediate, unlike pertactin, indicating that autotransporters may arrive at the native state by a variety of different mechanisms despite a common overall structure. A small but significant fraction of Ag43α can survive intact in the periplasm if expressed without the β-domain, suggesting that it is able to adopt a protease-resistant structure prior to translocation across the membrane. The present study demonstrates that glycosylation may play significant roles in structural and functional properties of bacterial autotransporters at many different levels.

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