Helicobacter Pylori Beta 1,3-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 beta 3-N-acetylglucosaminyltransferase (beta 3GlcNAcT) 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 (Gal beta 1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in beta 1-3 linkage to type-1 (Gal beta 1-3GlcNAc) or type-3/4 (Gal beta 1-3GalNAc alpha/beta) sequences. Thus, by alternating the use of the H. pylori beta 3GlcNAcT with galactosyltransferases that make the beta 1-4 or beta 1-3 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 beta 3GlcNAcT 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 beta 3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans.