Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation
Résumé
Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound β-domain and an α-domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several α-domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. Here we describe the effect of glycosylation on the extracellular α-domain of the E. coli autotransporter Ag43α, which induces frizzy colony morphology and cell settling. We identify 16 glycosylation sites and suggest two possible glycosylation motifs for Ser and Thr. To our knowledge this is the first bacterial glycosylation sequence analysis. 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 data 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 over the membrane. Our work demonstrates that glycosylation may play significant roles in structural and functional properties of bacterial autotransporters at many different levels.
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