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Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.

The late-onset form of Tay-Sachs disease displays when the activity levels of human β-hexosaminidase A (HexA) fall below 10% of normal, due to mutations that destabilise the native folded form of the enzyme and impair its trafficking to the lysosome. Competitive inhibitors of HexA can rescue disease-causative mutant HexA, bearing potential as pharmacological chaperones, but often also inhibit the enzyme O-glucosaminidase (GlcNAcase; OGA), a serious drawback for translation into the clinic. We have designed sp

Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked

We report the rational design and synthesis of C2-modified DGJ analogues to improve the selective inhibition of human GALA over other glycosidases. We prepare these analogues using a concise route from non-carbohydrate materials and demonstrate the most selective inhibitor 7c (∼100-fold) can act in Fabry patient cells to drive reductions in levels of the disease-relevant glycolipid Gb3.

All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi. The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major, the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Galpα1,3Galfβ and Galpα1,3Galfβ1,3Manpα glycotopes as diagnostic biomarkers for L. major-caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica-caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies.

Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): Galα (NGP3B), Galα(1,3)Galα (NGP17B), Galα(1,3)Galβ (NGP9B), Galα(1,6)[Galα(1,2)]Galβ (NGP11B), and Galα(1,3)Galβ(1,4)Glcβ (NGP1B) that are differentially recognized in sera from individuals withLeishmania majorinfection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing α-galactopyranosyl (α-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-α-Gal response in CL diagnosis.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

A route for the synthesis of glycuronamides via the intermediacy of 6-S-tolyl-substituted glycosides and uronic acid thioesters, is reported. The route, which is compatible with a variety of carbohydrate residues and protecting groups, was used to synthesize the repeating unit of the E. coli K54 capsular polysaccharide.

The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer’s disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.

Glucocerebrosidase (GCase) is a lysosomal glycoside hydrolase that cleaves the glycolipid glucosylceramide (GlcCer). Deficiencies of this enzyme lead to accumulation of GlcCer and the development of the lysosomal storage disease known as Gaucher's disease. Recently, loss-of-function mutations in the GBA1 gene that encodes GCase have been linked to Parkinson's disease. Currently pursued therapeutic strategies to increase GCase involve enzyme replacement therapy, chemical chaperone therapy, and GCase activators. A challenge associated with advancing such strategies is to efficiently monitor GCase activity within the lysosomes of live cells. In this chapter, we review the design and use of the fluorescent-quenched probe GBA1-FQ2 to quantitatively measure GCase activity in lysosomes of live cells.

Glucocerebrosidase (GCase) is a lysosomal glycoside hydrolase that cleaves the glycolipid glucosylceramide (GlcCer). Deficiencies of this enzyme lead to accumulation of GlcCer and the development of the lysosomal storage disease known as Gaucher's disease. Recently, loss-of-function mutations in the GBA1 gene that encodes GCase have been linked to Parkinson's disease. Currently pursued therapeutic strategies to increase GCase involve enzyme replacement therapy, chemical chaperone therapy, and GCase activators. A challenge associated with advancing such strategies is to efficiently monitor GCase activity within the lysosomes of live cells. In this chapter, we review the design and use of the fluorescent-quenched probe GBA1-FQ2 to quantitatively measure GCase activity in lysosomes of live cells.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease in humans. Linear and branched O-glycans with non-reducing, terminal α-galactosyl (α-Gal) glycotopes located on cell surface glycosylphosphatidylinositol (GPI)-anchored mucins of the infective trypomastigote form of the parasite are foreign to humans and elicit high levels of anti-α-Gal antibodies in Chagas disease patients (Ch anti-α-Gal antibodies). These antibodies have the capability to lyse the parasite in a complement-dependent or -independent manner. Ch anti-α-Gal antibodies have a considerably higher reactivity to the parasitic surface α-Gal glycotopes than the normal human serum (NHS) anti-α-Gal antibodies, which are present in every healthy human being. A series of ten mercaptopropyl saccharides with α-Gal moieties at the non-reducing end, all connected to another galactose unit, and five non-α-Gal-containing glycan controls were synthesized, and conjugated to maleimide-derivatized bovine serum albumin. This produced neoglycoproteins (NGPs), which were assembled into glycoarrays for the interrogation with sera of chronic Chagas disease patients and healthy individuals using chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA). This study identified the terminal Galα(1,3)Galβ disaccharide as an immunodominant T. cruzi glycotope and biomarker, which shows a considerable binding differential between Ch and NHS anti-α-Gal antibodies. Therefore, this glycotope is suitable for the diagnosis of Chagas disease, and could also be potentially used for follow-up studies for the effectiveness of chemotherapy in Chagas disease patients.

Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules, such as proteins and lipids, and alter their properties and functions, so glycan characterization is essential for understanding the effects they have on cellular systems. However, the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycan standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest that specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS. Graphical abstract Glycan isomers, such as fructose and glucose, show distinct separations in positive and negative ion mode.

An enantioselective de novo synthesis of a thioglycoside derivative of the 6-O-methyl-D-glycero-L-gluco-heptopyranose residue found in the Campylobacter jejuni NCTC11168 (HS:2) capsular polysaccharide is reported. The compound is obtained from a furfural-derived chiral diol in 11 steps. Notably, compared to the only previous synthesis of this molecule, this approach significantly reduces the number of purification steps required to obtain the target.

β-Configured peracetylated sugars are often used as easily accessible glycosyl donors that are typically activated with common Lewis acids such as boron trifluoride or trimethylsilyltrifluoromethane sulfonate. Often these glycosylations occur with unsatisfactory yields due to incomplete reactions or extensive byproduct formation, primarily as a result of loss of an additional acetyl group generating partially unprotected glycosides. Here we report a simple glycosylation–reacetylation protocol for the generation of predominantly β-configured peracetylated allyl glucoside, -galactoside, -lactoside, and -maltoside with substantially improved reaction yields.

A two-step route for introducing methyl phosphoramidate moieties onto carbohydrates is reported. The approach uses methyl pivolyl H-phosphonate as the phosphorylating reagent to produce an isolable carbohydrate H-phosphonate intermediate that is then oxidized by a Todd–Atherton reaction. The stability of the product methyl phosphoramidates was subsequently evaluated using various deprotection strategies.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81-176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity.

A synthetic glycoarray containing non-reducing α-galactopyranosyl moieties related to mucin O-glycans of the parasite Trypanosoma cruzi was evaluated by a chemiluminescent enzyme-linked immunosorbent assay with sera from patients with chronic Chagas disease. Our data revealed the disaccharide Galα(1,3)Galβ as the immunodominant glycotope, which may eventually be employed as a diagnostic antigen for Chagas disease.