Specific Glycosidases Research Articles

The History of the Glycosidase Inhibiting Hyacinthacine C-type Alkaloids: From Discovery to Synthesis.

Background:The inherent glycosidase inhibitory activity and potentially therapeutic value of the polyhydroxylated pyrrolizidine alkaloids containing a hydroxymethyl substituent at the C-3 position have been well documented. Belonging to this class, the naturally occurring hyacinthacine C-type alkaloids are of general interest among iminosugar researchers. Their selective micromolar α-glycosidase inhibitory ranges (10 – 100 µM) suggest that these azasugars are potential leads for treating type II diabetes. However, the structures of hyacinthacine C1, C3 and C4 are insecure with hyacinthacine C5 being recently corrected.Objective:This review presents the hyacinthacine C-type alkaloids: their first discovery to the most recent advancements on the structures, biological activities and total synthesis.Conclusion:The hyacinthacine C-type alkaloids are of exponentially increasing interest and will undoubtedly continue to be reported as synthetic targets. They represent a challenging but rewarding synthetic feat for the community of those interested in accessing biologically active iminosugars. Since 2009, ten total syntheses have been employed towards accessing similarly related products but only three have assessed the glycosidase inhibitory activity of the final products. This suggests the need for an accessible and universal glycosidase inhibitory assay so to accurately determine the structure-activity relationship of how the hyacinthacine C-type alkaloids inhibit specific glycosidases. Confirming the correct structures of the hyacinthacine C-type alkaloids as well as accessing various analogues continues to strengthen the foundation towards a marketable treatment for type II diabetes and other glycosidase related illnesses.

Phytochemical composition and biological activity of Armenian Muscari (Muscari armeniacum Leichtlin)

Введение. В настояшей статье рассматривается состав биологически активных соединений в надземных органах и луковицах мускари армянского (Muscari armeniacum Leichtlin) семейства Спаржевые (Asparagaceae Juss) и возможные фармакотерапевтические эффекты препаратов на его основе.

Multivalent Effect in Glycosidase Inhibition: The End of the Beginning.

Glycosidases are ubiquitous enzymes involved in a diversity of key biological processes such as energy uptake or cell wall degradation. The design of specific glycosidase inhibitors has been therefore the subject of intense research efforts in academia and pharmaceutical industry. However, until recently, the study of the impact of multivalency on glycosidase inhibition was almost completely neglected. The following account will review our ten year journey on the design of multivalent glycomimetics within our research group, from the discovery of the first strong multivalent effect in glycosidase inhibition to the high-resolution crystal structures of Jack bean α-mannosidase in complex with the multimeric inhibitor displaying the largest binding enhancements reported so far.

MUSCARI ARMENIACUM LEICHTLIN (GRAPE HYACINTH): PHYTOCHEMISTRY AND BIOLOGICAL ACTIVITIES REVIEW

This review focuses on the Muscari armeniacum Leichtlin (Asparagaceae Juss) biologically active substances composition presented in the Aireal and underground parts and finding their possible therapeutic effects. The systematic review is dedicated to the composition of biologically active substances, including recent advances in the biological activity investigation, phytochemical studies, and biotechnology methods of plant material producing. Various electronic search engines such as Google, Google Scholar, scientific literature, publishing sites, and electronic databases such as PubMed, Wiley, Springer, and Science Direct had been searched and data obtained. Other online academic libraries such as E-library and specific ethnopharmacological literature had been searched systematically for more exhaustive information on the crude herbal drug. The chemical composition of M. armeniacum biologically active substances is established; it contains anthocyanins (delphinidin and cyanidin derivatives), homoisoflavonoids, polyhydroxylated pyrrolizidine alkaloids (hyacinthacines A1, A2, A3, and B3), oligoglycosides (Muscarosides), and ribosome-inactivating proteins (musarmins). Recent physicochemical analytical procedures for components determination and hyacinthacines synthesis pathways are mentioned. Moreover, future prospects and trends in the research of this plant have been proposed. We have reviewed researches conducted on M. armeniacum especially in areas of its use in medicine, phytochemicals, biological activity, and developed analytical methods. M. armeniacum possesses antioxidant, antimutagenic activity, and specific glycosidase inhibitory activity; M. armeniacum can be used for the production of potential anticancer, antiviral, antidiabetic, and anti-obesity drugs. It should be noted that more pharmacognostic, pharmacological studies are needed for giving further information on the clinical practice and standardization procedures for the crude herbal drug.

MUSCARI ARMENIACUM LEICHTLIN (GRAPE HYACINTH): PHYTOCHEMISTRY AND BIOLOGICAL ACTIVITIES REVIEW

This review focuses on the Muscari armeniacum Leichtlin (Asparagaceae Juss) biologically active substances composition presented in the Aireal and underground parts and finding their possible therapeutic effects. The systematic review is dedicated to the composition of biologically active substances, including recent advances in the biological activity investigation, phytochemical studies, and biotechnology methods of plant material producing. Various electronic search engines such as Google, Google Scholar, scientific literature, publishing sites, and electronic databases such as PubMed, Wiley, Springer, and Science Direct had been searched and data obtained. Other online academic libraries such as E-library and specific ethnopharmacological literature had been searched systematically for more exhaustive information on the crude herbal drug. The chemical composition of M. armeniacum biologically active substances is established; it contains anthocyanins (delphinidin and cyanidin derivatives), homoisoflavonoids, polyhydroxylated pyrrolizidine alkaloids (hyacinthacines A1, A2, A3, and B3), oligoglycosides (Muscarosides), and ribosome-inactivating proteins (musarmins). Recent physicochemical analytical procedures for components determination and hyacinthacines synthesis pathways are mentioned. Moreover, future prospects and trends in the research of this plant have been proposed. We have reviewed researches conducted on M. armeniacum especially in areas of its use in medicine, phytochemicals, biological activity, and developed analytical methods. M. armeniacum possesses antioxidant, antimutagenic activity, and specific glycosidase inhibitory activity; M. armeniacum can be used for the production of potential anticancer, antiviral, antidiabetic, and anti-obesity drugs. It should be noted that more pharmacognostic, pharmacological studies are needed for giving further information on the clinical practice and standardization procedures for the crude herbal drug.

Sialidase down-regulation reduces non-HDL cholesterol, inhibits leukocyte transmigration, and attenuates atherosclerosis in ApoE knockout mice

Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1hypoApoe-/-). We found that the hypomorphic NEU1 expression in male Apoe-/- mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis. Transplantation of Apoe-/- bone marrow (BM) into Neu1hypoApoe-/- mice significantly increased atherosclerotic lesion development and had no effect on serum lipoprotein levels. Moreover, Neu1hypoApoe-/- mice exhibited a reduction in circulating monocyte and neutrophil levels and had reduced hyaluronic acid and P-selectin adhesion capability on monocytes/neutrophils and T cells. Consistent with these findings, administration of a sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, had a significant anti-atherogenic effect in the Apoe-/- mice. In summary, the reduction in NEU1 expression or function decreases atherosclerosis in mice via its significant effects on lipid metabolism and inflammatory processes. We conclude that NEU1 may represent a promising target for managing atherosclerosis.

Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids.

Understanding the detailed mechanisms of enzyme-catalyzed hydrolysis of the glycosidic bond is fundamentally important, not only to the design of tailored cost-efficient, stable and specific catalysts but also to the development of specific glycosidase inhibitors as therapeutics. Retaining glycosidases employ two key carboxylic acid residues, typically glutamic acids, in a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. One Glu functions as a nucleophile while the other acts as a general acid/base. A significant part of enzymatic proficiency is attributed to a "perfect match" of the electrostatics provided by these key residues, a hypothesis that has been remarkably difficult to prove in model systems or in enzymes themselves. We experimentally probe this synergy by preparing synthetic variants of a model glycosidase Bacillus circulans β-xylanase (Bcx) with the nucleophile Glu78 substituted by 4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity. These Bcx variants were semisynthesized by preparation of optically pure fluoroglutamic acid building blocks, incorporation into synthetic peptides, and ligation onto a truncated circular permutant of Bcx. By measuring the effect of altered electrostatics in the active site on enzyme kinetic constants, we show that lowering the nucleophile p Ka by two units shits the pH-dependent activity by one pH unit. Linear free energy correlations using substrates of varying leaving group ability indicate that by reducing nucleophilic catalysis the concerted mechanism of the enzyme is disrupted and shifted toward a dissociative pathway. Our study represents the first example of site-specific introduction of fluorinated glutamic acids into any protein. Furthermore, it provides unique insights into the synergy of nucleophilic and acid/base catalysis within an enzyme active site.

Immobilization of β-galactosidase and α-mannosidase onto magnetic nanoparticles: A strategy for increasing the potentiality of valuable glycomic tools for glycosylation analysis and biological role determination of glycoconjugates

Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae β-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70–90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40–50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.

Glycan profiling of proteins using lectin binding by Surface Plasmon Resonance

Glycan profiling of proteins was studied through their lectin binding activity by Surface Plasmon Resonance (SPR). To validate the method, we monitored specific lectin binding with sequential removal of sugar moieties from human transferrin using specific glycosidases. The results clearly indicated that glycans on the protein can be identified by their selective binding activity to various lectins. Using this method, we characterized Fc glycosylation profiles of therapeutic peptibodies and antibodies expressed in mammalian cells (CHO and HEK 293 6E cells), with E. coli expressed proteins as the negative controls. We observed that antibodies expressed in CHO cells did not contain any sialic acid, while antibodies expressed in 293 6E cells contained sialic acid. CHO cell expressed antibodies were also more heavily fucosylated than the ones expressed by 293 6E cells. We further applied this method to measure the fucose composition of glycan engineered mouse antibodies, as well as to determine mannose composition of human antibody variants with depletion or enrichment of high mannose. The glycan profiles generated using this method were comparable to results from 2-AB labeled glycan analysis of normal-phase separated glycans, and Fc gamma receptor binding activity of the glycan engineered antibodies were consistent with their glycan profiles. Hence, we demonstrated that SPR lectin binding analysis can be a quick alternative method to profile protein glycosylation.

Analysis of N- and O-Glycosylation of Lysosomal Glycoproteins.

The vast majority of lysosomal proteins are heavily glycosylated. The present protocol describes the method of analyzing N- and O-linked glycans in lysosomal proteins of interest. The method is based on using deglycosylating enzymes, endoglycosidases, and exoglycosidases. Endoglycosidases catalyze the cleavage of an internal bond in an oligosaccharide, while exoglycosidases remove terminal carbohydrates from glycans. Different types of carbohydrate residues or chains can be removed by specific glycosidases. Removing oligosaccharides with glycosidases increases the electrophoretic mobility of a protein. This increase in mobility depends on the size and number of removed carbohydrate chains. Therefore, the treatment of lysosomal proteins with specific glycosidases followed by a western blot analysis of a protein of interest provides a way to determine which types of glycans are present in the protein by comparing the gel mobility before and after treatment.

A universal fluorometric assay strategy for glycosidases based on functional carbon quantum dots: β-galactosidase activity detection in vitro and in living cells.

The development of highly sensitive assays for glycosidases is of critical significance to understand their functions, facilely detect associated diseases and screen potential new drugs. In this work, we develop a universal assay strategy for glycosidase enzymes and inhibitor screening based on functional carbon quantum dots through a combined host-guest recognition and specific static quenching-induced signal transduction mechanism. This detection strategy is established in terms of the following facts: (1) β-cyclodextrin as a perfect host can selectively associate with p-nitrophenol due to its hydrophobic character and right size match of the cavity, which renders specific binding between β-cyclodextrin and p-nitrophenol via a host-guest recognition. (2) The formation of an inclusion complex between β-cyclodextrin modified carbon quantum dots (β-CD-CQDs) and p-nitrophenol results in fluorescence quenching with a high quenching efficiency due to the static quenching mechanism. Glycoconjugates of p-nitrophenol as the substrates could be rapidly hydrolyzed to corresponding glycose and p-nitrophenol in the presence of specific glycosidase, and the resulting p-nitrophenol induces the following host-guest interaction and static quenching leading to a change in the fluorescence signal. The activity of different glycosidase enzymes could be evaluated in the same way as long as the glycosyl unit of glycosylated substrates was changed. Here we take β-galactosidase as an example to demonstrate the applicability of the proposed detection strategy because it can act as a molecular target for primary ovarian cancers. A highly sensitive assay for β-galactosidase activity in terms of linear correlation of the fluorescence change with the β-galactosidase level was established with a low detection limit of 0.6 U L-1. Its function of inhibitor screening was also assessed by using d-galactal as the inhibitor for β-galactosidase, and the positive results indicated its feasibility to screen potential inhibitors. It is also illustrated that the nanoprobe possesses excellent biocompatibility, and can sensitively monitor the intracellular β-galactosidase level in ovarian cancer cells. This work provides a general detection method for glycosidase activity, demonstrates its applicability of monitoring the enzyme level in living cells, and broadens fluorogenic probes in fluorescence-guided diagnostics.

ChemInform Abstract: Are Enzymes Sensitive to the Multivalent Effect? Emerging Evidence with Glycosidases

AbstractReview: [49 refs.

Inhibitor versus chaperone behaviour of d-fagomine, DAB and LAB sp2-iminosugar conjugates against glycosidases: A structure–activity relationship study in Gaucher fibroblasts

A library of sp2-iminosugar conjugates derived from the piperidine iminosugar d-fagomine and the enantiomeric pyrrolidine iminosugars DAB and LAB has been generated in only two steps involving direct coupling of the fully unprotected polyhydroxylated heterocycles with isothiocyanates, to give monocyclic thiourea adducts, and further intramolecular nucleophilic displacement of the δ-located primary hydroxyl group by the thiocarbonyl sulphur atom, affording bicyclic isothioureas. These transformations led to a dramatic shift in the inhibitory selectivity from α- to β-glucosidases, with inhibition potencies that depended strongly on the nature of the aglycone-type moiety in the conjugates. Some of the new derivatives behaved as potent inhibitors of human β-glucocerebrosidase (GCase), the lysosomal enzyme whose dysfunction is responsible for Gaucher disease. Moreover, GCase inhibition was 10-fold weaker at pH 5 as compared to pH 7, which is generally considered as a good property for pharmacological chaperones. Surprisingly, most of the compounds strongly inhibited GCase in wild type fibroblasts at rather low concentrations, showing an unfavourable chaperone/inhibitor balance on disease-associated GCase mutants in cellulo. A structure–activity relationship analysis points to the need for keeping a contiguous triol system in the glycone moiety of the conjugates to elicit a chaperone effect. In any case, the results reported here represent a proof of concept of the utmost importance of implementing diversity-oriented strategies for the identification and optimization of potent and specific glycosidase inhibitors and chaperones.

Glycoprotein labeling with click chemistry (GLCC) and carbohydrate detection

Molecular labeling and detection techniques are essential to research in life science. Here, a method for glycoprotein labeling/carbohydrate detection through glycan replacement, termed glycoprotein labeling with click chemistry (GLCC), is described. In this method, a glycoprotein is first treated with specific glycosidases to remove certain sugar residues, a procedure that creates acceptor sites for a specific glycosyltransferase. A ‘clickable’ monosaccharide is then installed onto these sites by the glycosyltransferase. This modified glycoprotein is then conjugated to a reporter molecule using a click chemistry reaction. For glycoproteins that already contain vacant glycosylation sites, deglycosylation is not needed before the labeling step. As a demonstration, labeling on fetal bovine fetuin, mouse immunoglobulin IgG and bacterial expressed human TNFα and TNFβ are shown. Compared to traditional ways of protein labeling, labeling at glycosylation sites with GLCC is considerably more specific and less likely to have adverse effects, and, when utilized as a method for carbohydrate detection, this method is also highly specific and sensitive.

Glycoprotein Labeling with Click Chemistry (GLCC)

Protein labeling is an essential technique to research in life science. Here, a method for glycoprotein labeling through glycan replacement, termed as glycoprotein labeling with click chemistry (GLCC), is described. In this method, a glycoprotein is first treated with specific glycosidases or chemicals to remove certain sugar residues, which creates acceptor sites for a specific glycosyltransferase. A clickable monosaccharide is then installed to these sites by the glycosyltransferase. The modified glycoprotein is then conjugated to a reporter molecule using a click chemistry reaction. For labeling proteins or glycoproteins that already contain vacant glycosylation sites, such as proteins purified from E.coli, deglycosylation is not needed before the labeling step. As demonstrations, labeling on bovine fetuin, mouse immunoglobulin IgG and bacterial expressed human TNFα and TNFβ are shown. Compared to traditional way of protein labeling, labeling with GLCC is through glycosylation sites, therefore is more specific, likely has less adverse effect on target proteins.

Synthesis and Inhibition Study of Bicyclic Iminosugar‐Based Alkaloids, Scaffolds, and Libraries towards Glucosidase

AbstractA small library of bicyclic iminosugar‐based alkaloids and scaffolds possessing a polyhydroxylated pyrrolidine and a varied ring skeleton have been synthesized. Through rapid diversification of the scaffold via an amide coupling with random carboxylic acids, structurally diverse bicyclic iminosugar‐based libraries were prepared with substituent diversity, core diversity, and configurational diversity. This discovery process allowed us to efficiently sieve out potent and specific glycosidase inhibitors, and a bicyclic, conformationally restricted iminosugar was demonstrated to be more potent than the monocyclic ones in this study. The most potent and selective inhibitor discovered was found to have a Ki value of 71 nM against α‐glucosidase.

Polymeric iminosugars improve the activity of carbohydrate-processing enzymes.

Multivalent iminosugars have recently emerged as powerful tools to inhibit the activities of specific glycosidases. In this work, biocompatible dextrans were coated with iminosugars to form linear and ramified polymers with unprecedently high valencies (from 20 to 900) to probe the evolution of the multivalent inhibition as a function of ligand valency. This study led to the discovery that polyvalent iminosugars can also significantly enhance, not only inhibit, the enzymatic activity of specific glycoside-hydrolase, as observed on two galactosidases, a fucosidase, and a bacterial mannoside phosphorylase for which an impressive 70-fold activation was even reached. The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase. The possibility of using these polymers as "artificial enzyme effectors" may therefore open up new perspectives in therapeutics and biocatalysis.

A defined α-helix in the bifunctional O-glycosylated natriuretic peptide TcNPa from the venom of Tropidechis carinatus.

Natriuretic peptides (NP) play important roles in human cardiac physiology through their guanylyl cyclase receptors NPR-A and NPR-B. Described herein is a bifunctional O-glycosylated natriuretic peptide, TcNPa, from Tropidechis carinatus venom and it unusually targets both NPR-A and NPR-B. Characterization using specific glycosidases and ETD-MS identified the glycan as galactosyl-β(1-3)-N-acetylgalactosamine (Gal-GalNAc) and was α-linked to the C-terminal threonine residue. TcNPa contains the characteristic NP 17-membered disulfide ring with conserved phenylalanine and arginine residues. Both glycosylated and nonglycosylated forms were synthesized by Fmoc solid-phase peptide synthesis and NMR analysis identified an α-helix within the disulfide ring containing the putative pharmacophore for NPR-A. Surprisingly, both forms activated NPR-A and NPR-B and were relatively resistant towards proteolytic degradation in plasma. This work will underpin the future development of bifunctional NP peptide mimetics.

ChemInform Abstract: Multivalent Inhibitors for Carbohydrate‐Processing Enzymes: Beyond the “Lock‐and‐Key” Concept

AbstractReview: [41 refs.

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.