A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins.

Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins.

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses.

Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA's receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus-cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells.

Vinyl Halide-Modified Unsaturated Cyclitols are Mechanism-Based Glycosidase Inhibitors.

Suitably configured allyl ethers of unsaturated cyclitols act as substrates of β-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of β-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages.

Mammalian sialyltransferases allow efficient Escherichia coli-based production of mucin-type O-glycoproteins but can also transfer Kdo.

The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans.

Development of an active site titration reagent for α-amylases.

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining β-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available.