Glycosyltransferases (GTs) are ubiquitous enzymes that catalyze the assembly of glycoconjugates found throughout all kingdoms of nature. A longstanding problem is the rational design of probes that can be used to manipulate GT activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analogue that inhibits, with high potency both in vitro and in cells, the human GT responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analogue. Treatment of cells with the precursor decreases O-GlcNAc in a targeted manner with a single digit micromolar EC50. This approach to inhibition of GTs should be applicable to other members of this increasingly interesting superfamily of enzymes and enable their manipulation in a biological setting.

Host Cell Factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the C-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above UDP-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.

Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive, but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-GlcNAc transferase, an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.

Photothermal release of DNA from gold nanoparticles either by thermolysis of the Au-S bonds used to anchor the oligonucleotides to the nanoparticle or by thermal denaturation has great therapeutic potential, however, both processes have limitations (a decreased particle stability for the former process and a prohibitively slow rate of release for the latter). Here we show that these two mechanisms are not mutually exclusive and can be controlled by adjusting laser power and ionic strength. We show this using two different double-stranded (ds)DNA-nanoparticle conjugates, in which either the anchored sense strand or the complementary antisense strand was labeled with a fluorescent marker. The amounts of release due to the two mechanisms were evaluated using fluorescence spectroscopy and capillary electrophoresis, which showed that irradiation of the decorated particles in 200 mM NaOAc containing 10 mM Mg(OAc)(2) with a pulsed 532 nm laser operating at 100 mW favors denaturation over Au-S cleavage to an extent of more than six-to-one. Due to the use of a pulsed laser, the process occurs on the order of minutes rather than hours, which is typical for continuous wave lasers. These findings encourage continued research toward developing photothermal gene therapeutics.

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon–derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role played by N-glycosylation within the secretory pathway.

The modification of N-glycans by ␣-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for ␣1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-␣1,6-mannosidase activity consistent with specificity for N-linked glycans having their ␣1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substratebound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (␣/␣) 6 -fold, recognize and hydrolyze the ␣1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-␣1,6-mannosidases.A feature of emerging importance to bacteria that colonize or infect humans is their capacity to process host glycans. Streptococcus pneumoniae is one notable human pathogen that relies on this ability for its full virulence (1). Among its known carbohydrate active virulence factors are NanA, StrH, BgaA, and EndoD. NanA Glycoside hydrolases, enzymes that break glycosidic bonds through a hydrolytic mechanism, are presently classified into 123-amino acid sequence based families (2). ␣-Mannosidases known to process N-glycans are found in families 38, 47, 76, 92, and 99. Very recent studies have shown the bacterial family 38 ␣-mannosidase from Streptococcus pyogenes (SpyGH38) to be a specific exo-␣1,3-mannosidase that is tolerant of the ␣1,6-branches in N-glycans (3). Analysis of family 92 glycoside hydrolases from the human gut symbiont Bacteroides thetaiotaomicron revealed an expanded repertoire of ␣-mannosidases (4). These enzymes displayed activity primarily toward ␣1,2-and ␣1,3-mannosidic linkages with some having low ␣1,6-mannosidase activity. In addition to the established ability of S. pneumoniae to exo-hydrolytically process the distal arms of complex glycans, which comprise sialic acid, galactose, and N-acetylglucosamine, consideration of additional putative carbohydrate-active enzymes found in this organism suggests it can partly degrade the mannose component of N-glycans using enzymes similar to those found in S. pyogenes and B. thetaiotaomicron. Through these observations it has become clear that some bacteria, possibly including S. pneumoniae, have the capacity to process the mannose component of N-glycans. A noteworthy gap, however, in the known bacterial N-glycan de...