Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni.
The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.
Glover
Kerney Jebrell
KJ
Department of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Weerapana
Eranthie
E
Numao
Shin
S
Imperiali
Barbara
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006020:Glycopeptides / Q000096:biosynthesis*; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006602:Hexosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis*; D016415:Sequence Alignment; D013379:Substrate Specificity; D015854:Up-Regulation
D001426:Bacterial Proteins; D006020:Glycopeptides; D008565:Membrane Proteins; D010455:Peptides; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase