Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species

N-glycosyltransferase (NGT) is responsible for transferring hexose monosaccharides to the asparagine side chain of proteins and polypeptides in the consensus sequon (N-(X≠P)-T/S) with nucleotide-activated sugars as donor substrates. Here, we expressed and purified four different N-glycosyltransferases derived from diverse bacteria, including Actinobacillus pleuropneumoniae, Aggregatibacter aphrophilus, Kingella kingae and Bibersteinia trehalosi, and measured their catalytic activities of four synthesized peptides via in vitro glycosylation assays. RP-HPLC and mass spectrometry were used to identify and quantify the glycopeptide formation by distinct NGT isoforms. We then analyzed and compared the glycosylation efficiencies of different peptides for these four NGT isoforms, which showed distinct substrate selectivities. We sought to probe peptide specificities among various NGT isoforms, which could broaden the application of NGT-catalyzed N-glycosylation of a variety of therapeutic proteins.