N-linked protein glycosylation is a frequent post-translational modification that can be found in all three domains of life. In a canonical, highly conserved pathway, an oligosaccharide is transferred by a membranebound oligosaccharyltransferase from a lipid donor to asparagines in the sequon N-X-S/T of secreted polypeptides. The δ-proteobacterium Actinobacillus pleuropneumoniae encodes an unusual pathway for N-linked protein glycosylation. This pathway takes place in the cytoplasm and is mediated by a soluble Nglycosyltransferase (NGT) that uses nucleotideactivated monosaccharides to glycosylate asparagine residues. To characterize the process of cytoplasmic N-glycosylation in more detail, we studied the glycosylation in A. pleuropneumoniae and functionally transferred the glycosylation system to Escherichia coli. Nlinked glucose specific human sera were used for the analysis of the glycosylation process. We identified autotransporter adhesins as the preferred protein substrate of NGT in vivo and in-depth analysis of the modified sites in E. coli revealed a surprisingly relaxed peptide substrate specificity. While N-X-S/T is the preferred acceptor sequon, we detected glycosylation of alternative sequons, including modification of glutamine and serine residues. We also demonstrate the use of NGT to glycosylate heterologous proteins. Therefore, our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria.

Molecular analysis of an alternative N-glycosylation machinery by functional transfer from Actinobacillus pleuropneumoniae to Escherichia coli / Naegeli A; Neupert C; Fan YY; Lin CW; Poljak K; Papini AM; Schwarz F; Aebi M.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 289(4):(2014), pp. 2170-2179. [10.1074/jbc.M113.524462]

Molecular analysis of an alternative N-glycosylation machinery by functional transfer from Actinobacillus pleuropneumoniae to Escherichia coli.

PAPINI, ANNA MARIA;
2014

Abstract

N-linked protein glycosylation is a frequent post-translational modification that can be found in all three domains of life. In a canonical, highly conserved pathway, an oligosaccharide is transferred by a membranebound oligosaccharyltransferase from a lipid donor to asparagines in the sequon N-X-S/T of secreted polypeptides. The δ-proteobacterium Actinobacillus pleuropneumoniae encodes an unusual pathway for N-linked protein glycosylation. This pathway takes place in the cytoplasm and is mediated by a soluble Nglycosyltransferase (NGT) that uses nucleotideactivated monosaccharides to glycosylate asparagine residues. To characterize the process of cytoplasmic N-glycosylation in more detail, we studied the glycosylation in A. pleuropneumoniae and functionally transferred the glycosylation system to Escherichia coli. Nlinked glucose specific human sera were used for the analysis of the glycosylation process. We identified autotransporter adhesins as the preferred protein substrate of NGT in vivo and in-depth analysis of the modified sites in E. coli revealed a surprisingly relaxed peptide substrate specificity. While N-X-S/T is the preferred acceptor sequon, we detected glycosylation of alternative sequons, including modification of glutamine and serine residues. We also demonstrate the use of NGT to glycosylate heterologous proteins. Therefore, our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria.
2014
289(4)
2170
2179
Naegeli A; Neupert C; Fan YY; Lin CW; Poljak K; Papini AM; Schwarz F; Aebi M.
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/850102
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