| Abstract
| - The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a generalN-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunitof the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharidefrom a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conservedglycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using alibrary of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the aminoacids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency,whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to beconsistent with the trends observed from their peptidyl counterparts, though less dramatic because ofadditional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed theselectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward theglycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.
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