Terminal N-acetylglucosamine Residues Research Articles

Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa−/− mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

N-glycans of complex glycosylated biopharmaceuticals and their impact on protein clearance

N-glycosylation is a common post-translational modification of biopharmaceutical products. Certain types of N-glycans have been shown to influence important properties of monoclonal antibody products including pharmacokinetics and effector functions. Complex biopharmaceuticals e.g. Fc fusion proteins may contain several N- and O-glycosylation sites. Domain specific characterization of two Fc fusion proteins showed an Fc N-glycosylation pattern comparable to IgG molecules. The receptor N-glycosylation was found to contain some larger and more complex N-glycans compared to the Fc part. Analyses of samples from non-clinical studies of the two studied fusion proteins indicate that their N-glycans impact pharmacokinetic properties. Interestingly, besides the type of N-glycan this influence on the pharmacokinetics depends also on the glycosylation site and thus the accessibility on the protein. The same type of N-glycan can influence the clearance of fusion proteins when located at the receptor part, but not if located at the Fc part. In this study, it is shown that N-glycans with terminal galactose or N-acetylglucosamine residues have a negative impact on serum half-life when located at the receptor part. Terminal sialylation of galactose residues prevents this faster clearance even when only one sialic acid is present. O-acetylation, a modification of sialic acids does not impact pharmacokinetics. Thus, type and accessibility of N-glycan moieties of fusion proteins both play an important role in pharmacokinetics. Finally, detailed site specific analysis is critical in the development of biopharmaceuticals.

Unique Binding Specificities of Proteins toward Isomeric Asparagine-Linked Glycans

The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine (Asn)-linked N-glycans, we synthesized a library of isomeric multiantennary N-glycans that vary in terminal non-reducing sialic acid, galactose, and N-acetylglucosamine residues, as well as core fucose. We identified specific recognition of N-glycans by several plant lectins, human galectins, influenza viruses, and Siglecs, and explored the influence of sialic acid linkages and branching of the N-glycans. These results show the unique recognition of complex-type N-glycans by a wide variety of glycan-binding proteins and their abilities to distinguish isomeric structures, which provides new insights into the biological roles of these proteins and the uses of lectins in biological applications to identify glycans.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Transgenic Production of an Anti HIV Antibody in the Barley Endosperm.

Barley is an attractive vehicle for producing recombinant protein, since it is a readily transformable diploid crop species in which doubled haploids can be routinely generated. High amounts of protein are naturally accumulated in the grain, but optimal endosperm-specific promoters have yet to be perfected. Here, the oat GLOBULIN1 promoter was combined with the legumin B4 (LeB4) signal peptide and the endoplasmic reticulum (ER) retention signal (SE)KDEL. Transgenic barley grain accumulated up to 1.2 g/kg dry weight of recombinant protein (GFP), deposited in small roundish compartments assumed to be ER-derived protein bodies. The molecular farming potential of the system was tested by generating doubled haploid transgenic lines engineered to synthesize the anti-HIV-1 monoclonal antibody 2G12 with up to 160 μg recombinant protein per g grain. The recombinant protein was deposited at the periphery of protein bodies in the form of a mixture of various N-glycans (notably those lacking terminal N-acetylglucosamine residues), consistent with their vacuolar localization. Inspection of protein-A purified antibodies using surface plasmon resonance spectroscopy showed that their equilibrium and kinetic rate constants were comparable to those associated with recombinant 2G12 synthesized in Chinese hamster ovary cells.

Glycosylation patterns of proteins in trophoblast and decidua of the cat placenta

Glycosylation patterns of proteins in trophoblast and decidua of the cat placenta

Intermediate Filament Proteins Expression and Carbohydrate Moieties in Trophoblast and Decidual Cells of Mature Cat Placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Enzyme-Mediated Methodology for the Site-Specific Radiolabeling of Antibodies Based on Catalyst-Free Click Chemistry

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal (89)Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with (89)Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing (89)Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to highly selective tumor uptake (67.5 ± 5.0%ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.

Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-acetylglucosamine (GlcNAc) residues on live cell surface.

On the basis of the crystal structure of bovine β4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289L-β4Gal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn(2+) concentrations required to selectively modify GlcNAc residues on the surface of live cells, we have engineered a Mg(2+)-dependent enzyme. Previously, we found that the mutation of the metal-binding residue Met-344 to His-344 in bovine β4Gal-T1 enzyme altered its metal-ion specificity in such a way that the M344H-β4Gal-T1 enzyme exhibits better catalytic activity with Mg(2+) than with Mn(2+). Here, we find that, when these two mutations are combined, the double mutant, Y289L-M344H-β4Gal-T1, transfers GalNAc and its analogue sugars to the acceptor GlcNAc in the presence of Mg(2+). Using this mutant enzyme, we have detected free GlcNAc residues on the surface glycans of live HeLa cells and platelets. The specific transfer of a synthetic sugar with a chemical handle to the terminal GlcNAc residues on the surface of live cells provides a novel tool for selective modification, detection, and isolation of GlcNAc-ending glycans present on the cellular surface.

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.

Rapid Analysis of Listeria monocytogenes Cell Wall Teichoic Acid Carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of Gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria.

Comparative analysis reveals selective recognition of glycans by the dendritic cell receptors DC-SIGN and Langerin

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) and Langerin are homologous C-type lectins expressed as cell-surface receptors on different populations of dendritic cells (DCs). DC-SIGN interacts with glycan structures on HIV-1, facilitating virus survival, transmission and infection, whereas Langerin, which is characteristic of Langerhans cells (LCs), promotes HIV-1 uptake and degradation. Here we describe a comprehensive comparison of the glycan specificities of both proteins by probing a synthetic carbohydrate microarray comprising 275 sugar compounds using the bacterially produced and fluorescence-labeled, monomeric carbohydrate-recognition domains (CRDs) of DC-SIGN and Langerin. In this side-by-side study DC-SIGN was found to preferentially bind internal mannose residues of high-mannose-type saccharides and the fucose-containing blood-type antigens H, A, B, Le(a), Le(b) Le(x), Le(y), sialyl-Le(a) as well as sulfatated derivatives of Le(a) and Le(x). In contrast, Langerin appeared to recognize a different spectrum of compounds, especially those containing terminal mannose, terminal N-acetylglucosamine and 6-sulfogalactose residues, but also the blood-type antigens H, A and B. Of the Lewis antigens, only Le(b), Le(y), sialyl-Le(a) and the sialyl-Le(x) derivative with 6'-sulfatation at the galactose (sialyl-6SGal Le(x)) were weakly bound by Langerin. Notably, Ca(2+)-independent glycan-binding activity of Langerin could not be detected either by probing the glycan array or by isothermal titration calorimetry of the CRD with mannose and mannobiose. The precise knowledge of carbohydrate specificity of DC-SIGN and Langerin receptors resulting from our study may aid the future design of microbicides that specifically affect the DC-SIGN/HIV-1 interaction while not compromising the protective function of Langerin.

Expression of β-1,4-galactosyltransferase and suppression of β- N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of β- N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of β-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

β-N-Acetylhexosaminidases HEXO1 and HEXO3 Are Responsible for the Formation of Paucimannosidic N-Glycans in Arabidopsis thaliana

Most plant glycoproteins contain substantial amounts of paucimannosidic N-glycans instead of their direct biosynthetic precursors, complex N-glycans with terminal N-acetylglucosamine residues. We now demonstrate that two β-N-acetylhexosaminidases (HEXO1 and HEXO3) residing in different subcellular compartments jointly account for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. Total N-glycan analysis of hexo knock-out plants revealed that HEXO1 and HEXO3 contribute equally to the production of paucimannosidic N-glycans in roots, whereas N-glycan processing in leaves depends more heavily on HEXO3 than on HEXO1. Because hexo1 hexo3 double mutants do not display any obvious phenotype even upon exposure to different forms of abiotic or biotic stress, it should be feasible to improve the quality of glycoprotein therapeutics produced in plants by down-regulation of endogenous β-N-acetylhexosaminidase activities.

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis

Novel recombinant human lysosomal β-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases.

Neural-specific α3-fucosylation of N-linked glycans in the Drosophila embryo requires Fucosyltransferase A and influences developmental signaling associated with O-glycosylation

Addition of fucose (Fuc) to glycoprotein N-linked glycans or in O-linkage directly to Ser/Thr residues modulates specific cell-cell interactions and cell signaling events. Vertebrates and invertebrates add Fuc in α6-linkage to the reducing terminal N-acetylglucosamine residue of N-glycans. In Drosophila and other invertebrates, Fuc can also be added in α3-linkage to the same residue. These difucosylated N-glycans are recognized by anti-horseradish peroxidase (anti-HRP) antisera, providing a well-established marker for insect neural tissue. To understand the mechanisms and consequences of tissue-specific glycan expression, we identified a single α3-fucosyltransferase (FucTA) that produces the anti-HRP epitope in Drosophila embryos. FucTA transcripts are temporally and spatially restricted to cells that express the anti-HRP epitope and are missing in a mutant that lacks neural α3-fucosylation. Transgenic expression of FucTA, but not of any other candidate α3-fucosyltransferase, rescues the anti-HRP epitope in the embryonic nervous system of this mutant. Mass spectrometric characterization of the N-glycans of Drosophila embryos overexpressing FucTA confirms that this enzyme is indeed responsible for the biosynthesis of difucosylated glycans in vivo. Whereas ectopic expression of FucTA in the larval wing disc produces mild wing notching, the heterochronic, pan-neural expression of FucTA in early differentiating neurons generates neurogenic and cell migration phenotypes; this latter effect is associated with reduced GDP-Fuc levels in the embryo and indicates that the diversion of fucosylation resources towards fucosylation of N-glycans has an impact on developmental signaling associated with O-fucosylation.

Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system.

Plant glycosidases acting on protein-linked oligosaccharides

Glycosidases have been used as invaluable tools in glycobiology research for decades, and their role in glycoprotein maturation has been amply studied. The molecular biological coverage of this large group of enzymes has only recently reached an appreciable level. In this review, we present an overview of plant glycosidases, whose DNA/protein sequence has been identified and for which recombinant enzymes have been characterized. The physiological role in the maturation of glycoproteins is discussed as well as the biotechnological prospects arising from knowing the enzymes responsible for the removal of terminal N-acetylglucosamine residues. The current knowledge on plant fucosidases and of the first bits of information on glycosidases acting on arabinogalactan proteins is presented.

Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

The disaccharide peracetylated GlcNAcbeta1-3Galbeta-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3) GlcNAc; sLe(X)) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe(X) assembly, and (iii) generation of several glycans related to sLe(X). In this report, we show that GlcNAcbeta1-3Galbeta-O-naphthalenemethanol binds to the acceptor site of human beta1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcbeta1-2Manbeta1-6Man, which is present on N-linked glycans. The 4'-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe(X) formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N(3), -NH(2), or -OCH(3) instead of the hydroxyl groups at C-3'- and C-4'-positions of the terminal N-acetylglucosamine residue also blocked sLe(X) formation in cells. The reduction of sLe(X) by the 4'-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.