Glycosyltransferases (GTs) catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. GTs are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new GTs are both difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with analysis by mass spectrometry to screen putative GTs. A total of 14,280 combinations of GT, acceptor and donor in four buffer conditions were screened and led to the identification and characterization of four new GTs. This work is significant because it provides a label-free method for the rapid functional annotation of putative enzymes.

Background: WaaL mediates the ligation of O-antigen onto lipid A-core. Results: This ligation was reconstituted in vitro using synthetic donor substrates and donor mimics bearing structural variations. All of them were accepted as substrates by WaaL. Conclusion: WaaL exhibits relaxed donor substrate specificity. Significance: This work, together with other previously published studies, lays important foundations for dissecting the mechanism of WaaL enzymes.

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Poly(ethylene glycol) (PEG) conjugation (i.e. PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids, however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with various sized carbohydrates, up to a molecular weight (MW) of 10,000 Da, thus, serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with various sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of a similar MW to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (504 to ~10,000 Da). Hb was selected based on the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb’s biophysical properties was investigated in vitro.

Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an α1,2-fucosyltransferase using a radioactivity based assay, thin layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from pH 6.5 to pH 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-β1,3-GalNAc-α-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe, on a milligram scale.