Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain.
The mechanism of protein secretion mediated by the beta-domain of the Neisseria gonorrhoeae IgA protease, a paradigm of a family of secreted polypeptides of Gram-negative bacteria called autotransporters, has been examined using a single-chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the beta-module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N-terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S-S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N-passenger domain can be translocated through the beta-domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re-evaluation of the prevailing model for secretion of the N-domain of autotransporters.
Veiga
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Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Campus de Cantoblanco, 28049-Madrid, Spain.
de Lorenzo
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Fernández
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LA
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D066298:In Vitro Techniques; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000502:physiology*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase