A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint.

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 µM binding affinity for CtUGGTGT24in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 µM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9.

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.

Attenuation of Polysialic Acid Biosynthesis in Cells by the Small Molecule Inhibitor 8-Keto-sialic acid.

Sialic acids are key mediators of cell function, particularly with regard to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here, we describe a strategy to block the formation of α2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, cannot be elongated. 8-Keto-sialic acid is nontoxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.

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. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.

Discovery of ß-N-acetylglucosaminidases from screening metagenomic libraries and their use as thioglycoligase mutants.

ß-N-Acetylhexosaminidases (HexNAcases) are versatile biocatalysts that cleave terminal N-acetylhexosamine units from various glycoconjugates. Established strategies to generate glycoside-forming versions of the wild type enzymes rely on the mutation of their catalytic residues; however, successful examples of synthetically useful HexNAcase mutants are scarce. In order to expand the range of HexNAcases available as targets for enzyme engineering, we functionally screened a metagenomic library derived from a human gut microbiome. From a pool of hits, we characterized four of the more active candidates by sequence analysis and phylogenetic mapping, and found that they all belonged to CAZy family GH20. After detailed kinetic analysis and characterization of their substrate specificities, active site mutants were generated which resulted in the identification of two new thioglycoligases. BvHex E294A and AsHex E301A catalyzed glycosyl transfer to all three of the 3-, 4- and 6-thio-N-acetylglucosaminides (thio-GlcNAcs) that were tested. Both mutant enzymes also catalyzed glycosyl transfer to a cysteine-containing variant of the model peptide Tab1, with AsHex E301A also transferring GlcNAc onto a thiol-containing protein. This work illustrates how large scale functional screening of expressed gene libraries allows the relatively rapid development of useful new glycoside-forming mutants of HexNAcases, expanding the pool of biocatalysts for carbohydrate synthesis.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.

Prospecting for microbial α-N-acetylgalactosaminidases yields a new class of GH31 O-glycanase.

α-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type-determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that specifically cleaves the initial α-GalNAc from mucin-type O-glycans. This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces.

A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans.

We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a ß1,3-galactosyl transferase and UDP-Glc(NAc)-4-epimerase, both from Campylobacter jejuni, and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon α-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli.

Facile Formation of ß-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase.

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29.

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

Proximity Ligation-Based Fluorogenic Imaging Agents for Neuraminidases.

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase-imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus-specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti-neuraminidase antibodies.

Structural Dissection of Helianthamide Reveals the Basis of Its Potent Inhibition of Human Pancreatic α-Amylase.

Helianthamide is a potent inhibitor of human pancreatic α-amylase (HPA) ( KI = 0.01 nM) produced by the Caribbean sea anemone Stichodactyla helianthus. Helianthamide was previously shown to be structurally homologous to the ß-defensins and represents a new structural class of protein inhibitors of α-amylase. To understand the source of this potent inhibition, we performed site-directed mutagenesis studies on helianthamide fusion proteins. A novel YIYH inhibitory motif that interacts with conserved active site residues was originally proposed as being central to inhibitory activity based on the X-ray crystal structure of the porcine pancreatic α-amylase-helianthamide complex. However, variants in which these polar residues were replaced, individually, with alanine or phenylalanine bound only 5-46-fold more weakly than wild-type helianthamide, suggesting modest contributions from these interactions. In contrast, individual replacement of helianthamide's six cysteine residues with alanine resulted in much larger decreases in potency (a ≤1.3 × 104 increase in KI compared to that of the wild type). In a complementary approach, a series of small peptides based on helianthamide's sequence were synthesized and tested. Of these 19 synthetic peptides, only two showed any appreciable affinity for HPA, with inhibition constants of 141 and 396 µM, significantly higher than that of intact helianthamide. These results suggest that helianthamide's potent HPA inhibition does not rely so much on the accumulation of individual polar contacts but rather its ability to form an extensive hydrophobic interface with the enzyme and occlude the active site cleft.

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A.

Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.

Refolding the unfoldable: A systematic approach for renaturation of Bacillus circulans xylanase.

Xylanases are important polysaccharide-cleaving catalysts for the pulp and paper, animal feeds and biofuels industries. They have also proved to be valuable model systems for understanding enzymatic catalysis, with one of the best studied being the GH11 xylanase from Bacillus circulans (Bcx). However, proteins from this class are very recalcitrant to refolding in vitro. This both limits their high level expression in heterologous hosts, and prevents experimental approaches, such as peptide ligation or chemical modifications, to probe and engineer their stability and function. To solve this problem, a systematic screening approach was employed to identify suitable buffer conditions for renaturing Bcx in vitro. The fractional factorial screen employed identified starting conditions for refolding, which were then refined and developed into a generic protocol for renaturing preparative amounts of active Bcx in a 50-60% yield from inclusion bodies. The method is robust and proved equally proficient at refolding circularly permuted versions that carry cysteine mutations. This general approach should be applicable to related GH11 xylanases, as well as proteins adopting a similar ß-jellyroll fold, that are otherwise recalcitrant to refolding in vitro.

Rapid Discovery of Potent and Selective Glycosidase-Inhibiting De Novo Peptides.

Human pancreatic α-amylase (HPA) is responsible for degrading starch to malto-oligosaccharides, thence to glucose, and is therefore an attractive therapeutic target for the treatment of diabetes and obesity. Here we report the discovery of a unique lariat nonapeptide, by means of the RaPID (Random non-standard Peptides Integrated Discovery) system, composed of five amino acids in a head-to-side-chain thioether macrocycle and a further four amino acids in a 310 helical C terminus. This is a potent inhibitor of HPA (Ki = 7 nM) yet exhibits selectivity for the target over other glycosidases tested. Structural studies show that this nonapeptide forms a compact tertiary structure, and illustrate that a general inhibitory motif involving two phenolic groups is often accessed for tight binding of inhibitors to HPA. Furthermore, the work reported here demonstrates the potential of this methodology for the discovery of de novo peptide inhibitors against other glycosidases.

Proteolytic Cleavage Driven by Glycosylation.

Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc-transferase (OGT) (Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) Science 342, 1235-1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolytic activity. However, the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed.

5-Fluoro derivatives of 4-epi-isofagomine as D-galactosidase inhibitors and potential pharmacological chaperones for GM1-gangliosidosis as well as Fabry's disease.

Electrophilic fluorination of an exocyclic methoxymethylene enol ether derived from N-tert-butyloxycarbonyl-1,5-dideoxy-1,5-imino-3,4-O-isopropylidene-D-erythro-pent-2-ulose (11) provided the 5-fluoro derivative of the powerful ß-galactosidase inhibitor 4-epi-isofagomine (8). This structural alteration, in combination with N-alkylation, led to considerably improved α-galactosidase selectivity. New compounds may serve as leads en route to new pharmacological chaperones for Fabry's disease.

Enhancement of biological reactions on cell surfaces via macromolecular crowding.

The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors.

Fluorinated mechanism-based inhibitors: common themes and recent developments.

Mechanism-based inhibitors are relatively chemically inert compounds that become activated when processed by their target enzyme, leading to covalent enzyme inactivation. Fluorine substitution confers a number of properties that are beneficial to the chemistry of such inhibitors and to their potential use as pharmaceuticals, and indeed several fluorinated mechanism-based inhibitors have made it to clinical usage over the past 50 years. Well-known examples are the 5- fluorouracil metabolite, 5-fluoro-2'-deoxyuridine-5'-monophosphate, which is used in the treatment of cancer, and α- difluoromethylornithine for the treatment of African sleeping sickness. As the prevalence of fluorine in medicinal chemistry continues to rise, more and more medically relevant fluorinated mechanism-based inhibitors are being developed with a variety of interesting properties and uses.

A plate-based high-throughput activity assay for polysialyltransferase from Neisseria meningitidis.

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiter-plate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z' score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.