In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation.
Campylobacter jejuni has a general N-linked glycosylation pathway (encoded by the pgl gene cluster), which culminates in the transfer of a heptasaccharide: GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac [where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] from a membrane-anchored undecaprenylpyrophosphate (Und-PP)-linked donor to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Herein we report, the cloning, overexpression, and purification of four of the glycosyltransferases (PglA, PglH, PglI, and PglJ) responsible for the biosynthesis of the Und-PP-linked heptasaccharide. Starting with chemically synthesized Und-PP-linked Bac and various combinations of enzymes, we have deduced the precise functions of these glycosyltransferases. PglA and PglJ add the first two GalNAc residues on to the isoprenoid-linked Bac carrier, respectively. Elongation of the trisaccharide with PglH results in a hexasaccharide revealing the polymerase activity of PglH. The final branching glucose is then added by PglI, which prefers native lipids for optimal activity. The sequential activities of the glycosyl transferases in the pathway can be reconstituted in vitro. This pathway represents an ideal venue for investigating the integrated functions of a series of enzymatic processes that occur at a membrane interface.
Glover
Kerney Jebrell
KJ
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Weerapana
Eranthie
E
Imperiali
Barbara
B
D020816:Amino Acid Motifs / Q000235:genetics; D016123:Campylobacter jejuni / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006595:Hexosamines / Q000378:metabolism*; D008954:Models, Biological; D009841:Oligonucleotides; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*
D006595:Hexosamines; D009841:Oligonucleotides; D011106:Polyisoprenyl Phosphates; D011135:Polysaccharides, Bacterial; C088139:undecaprenyl pyrophosphate; C013920:4-deoxyneosamine C; D016695:Glycosyltransferases