Helicobacter pylori β1,3-N-acetylglucosaminyltransferase for versatile synthesis of type 1 and type 2 poly-LacNAcs on N-linked, O-linked and I-antigen glycans.
Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Galβ1-3GalNAcα/β) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the β1-4 or β1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans.
Glycan Array Synthesis Core D, Consortium for Functional Glycomics, The Scripps Research Institute, La Jolla, CA 92037, USA.
Wenjie
W
Peng
Jennifer
J
Pranskevich
Corwin
C
Nycholat
Michel
M
Gilbert
Warren
W
Wakarchuk
James C
JC
Paulson
Nahid
N
Razi
D000117:Acetylglucosamine / Q000737:chemistry; D000606:Amino Sugars / Q000096:biosynthesis* / Q000737:chemistry; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D016480:Helicobacter pylori / Q000201:enzymology*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D000606:Amino Sugars; D006028:Glycosphingolipids; C078965:I-antigen; D011134:Polysaccharides; C066929:polylactosamine; C000458:N-acetyllactosamine; D017351:N-Acetylglucosaminyltransferases; D000117:Acetylglucosamine