Garrett E. Whitworth

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimers disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.

Structural snapshots of the reaction coordinate for O-GlcNAc transferase

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.

Active Site Plasticity within the Glycoside Hydrolase NagZ Underlies a Dynamic Mechanism of Substrate Distortion.

NagZ is a glycoside hydrolase that participates in peptidoglycan (PG) recycling by removing β-N-acetylglucosamine from PG fragments that are excised from the bacterial cell wall during growth. Notably, the products formed by NagZ, 1,6-anhydroMurNAc-peptides, activate β-lactam resistance in many Gram-negative bacteria, making this enzyme of interest as a potential therapeutic target. Crystal structure determinations of NagZ from Salmonella typhimurium and Bacillus subtilis in complex with natural substrate, trapped as a glycosyl-enzyme intermediate, and bound to product, define the reaction coordinate of the NagZ family of enzymes. The structures, combined with kinetic studies, reveal an uncommon degree of structural plasticity within the active site of a glycoside hydrolase, and unveil how NagZ drives substrate distortion using a highly mobile loop that contains a conserved histidine that has been proposed as the general acid/base.

Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans.

The modification of N-glycans by α-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 α1,6-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-α1,6-mannosidase activity consistent with specificity for N-linked glycans having their α1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (α/α)6-fold, recognize and hydrolyze the α1,6-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-α1,6-mannosidases.

Differential recognition and hydrolysis of host carbohydrate-antigens by Streptococcus pneumoniae family 98 glycoside hydrolases

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-β-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-β-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine (Dol-P-GlcNAc), which contrasts with the polyprenyl-diphosphate intermediates that feature in both eukaryotes and bacteria. Intriguingly, AglK exhibits high sequence homology to dolichyl-phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked Dol-P-glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide the first biochemical evidence revealing that despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.

Mammalian Notch is modified by d-Xyl-α1-3-d-Xyl-α1-3-d-Glc-β1-O-Ser: Implementation of a method to study O-glucosylation

Notch is a key cell surface protein receptor that is a vital component of intercellular signaling occurring during development. The O-glucosylation of the extracellular Notch epidermal growth factor-like (EGF) repeats has recently been found to play an important role in the proper functioning of Notch in Drosophila. Previous efforts to identify the fine structure of the O-glucose-containing glycan of mammalian Notch have been hindered by limitations associated with approaches used to date. Here, we report the development of an alternative strategy that can be used to study this modification from a range of different tissues. To implement this approach, we have generated standards of the D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc trisaccharide, isomers of this structure, as well as the d-Xyl-alpha1-3-d-Glc disaccharide found previously on secreted EGF-containing proteins of the blood coagulation cascade. Following derivatization with 8-aminopyrene-1,3,6-trisulfonate (APTS), we use these standards in capillary electrophoretic analyses of O-glycans released from Notch1 EGF repeats in conjunction with exo-alpha-xylosidase digestion. These studies collectively reveal that the O-glucose-containing glycan decorating mammalian Notch is the D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc trisaccharide; an assignment in accord with previous predictions. Given the demonstrated importance of this modification in the function of Notch in Drosophila, we expect that the identification of this glycan decorating mammalian Notch1 should aid studies into the functional role of O-glycosylation of mammalian Notch isoforms. Wider application of this approach should facilitate identification of other EGF-containing proteins bearing this O-glycan and aid in their study.

Molecular Basis of 1,6-Anhydro Bond Cleavage and Phosphoryl Transfer by Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase

Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the γ phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-l-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide.

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE*

Background: The active site of O-GlcNAcase (OGA) is larger than needed to process O-GlcNAc from proteins. Results: GlcNGc is assimilated to form UDP-GlcNGc and to generate O-GlcNGc, which can be removed by OGA. Conclusion: OGA has a conserved active site that enables processing of O-GlcNGc. Significance: Substrate tolerance of OGA occurs to process metabolic variants of O-GlcNAc. The O-GlcNAc modification involves the attachment of single β-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865–28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.

Chemoenzymatic Assembly of Bacterial Glycoconjugates for Site-Specific Orthogonal Labeling

The cell surfaces of bacteria are replete with diverse glycoconjugates that play pivotal roles in determining how bacteria interact with the environment and the hosts that they colonize. Studies to advance our understanding of these interactions rely on the availability of chemically defined glycoconjugates that can be selectively modified under orthogonal reaction conditions to serve as discrete ligands to probe biological interactions, in displayed arrays and as imaging agents. Herein, enzymes in the N-linked protein glycosylation (Pgl) pathway of Campylobacter jejuni are evaluated for their tolerance for azide-modified UDP-sugar substrates, including derivatives of 2,4-diacetamidobacillosamine and N-acetylgalactosamine. In vitro analyses reveal that chemoenzymatic approaches are useful for the preparation of undecaprenol diphosphate-linked glycans and glycopeptides with site-specific introduction of azide functionality for orthogonal labeling at three specific sites in the heptasaccharide glycan. The uniquely modified glycoconjugates represent valuable tools for investigating the roles of C. jejuni cell surface glycoconjugates in host pathogen interactions.