O ‐linked N ‐acetylglucosamine ( O ‐GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O ‐GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O ‐GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O ‐GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate‐assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O ‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidene)amino‐ N ‐phenylcarbamate ( K i=5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala‐Cys(‐ S ‐GlcNAc)‐Ala, and has OGA activity against O ‐GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA.

• In wild type C. albicans yeast cells grown in standard lab conditions: • Chitin microfibrils are interspersed throughout the inner layer of the cell wall. • Cell wall proteins are embedded throughout the inner layer of the cell wall. • The outer fibrillar layer represents N-mannan outer chains. • The length of fibrils correlates with the length of the α(1,6)-N-mannan backbone. • Side chains extend from the α(1,6)-backbone at fixed angles every 10 mannose residues.

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is a Gram-positive human-exclusive pathogen, responsible for more than 500 000 deaths annually worldwide. Upon infection, GAS commonly triggers mild symptoms such as pharyngitis, pyoderma and fever. However, recurrent infections or prolonged exposure to GAS might lead to life-threatening conditions. Necrotizing fasciitis, streptococcal toxic shock syndrome and post-immune mediated diseases, such as poststreptococcal glomerulonephritis, acute rheumatic fever and rheumatic heart disease, contribute to very high mortality rates in non-industrialized countries. Though an initial reduction in GAS infections was observed in high-income countries, global outbreaks of GAS, causing rheumatic fever and acute poststreptococcal glomerulonephritis, have been reported over the last decade. At the same time, our understanding of GAS pathogenesis and transmission has vastly increased, with detailed insight into the various stages of infection, beginning with adhesion, colonization and evasion of the host immune system. Despite deeper knowledge of the impact of GAS on the human body, the development of a successful vaccine for prophylaxis of GAS remains outstanding. In this review, we discuss the challenges involved in identifying a universal GAS vaccine and describe several potential vaccine candidates that we believe warrant pursuit.

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.