Search

Sialic acids are commonly found on the terminal ends of biologically important carbohydrates, including intestinal mucin O-linked glycans. Pathogens such as Clostridium perfringens, the causative agent of necrotic enteritis in poultry and humans, have the ability to degrade host mucins and colonize the mucus layer, which involves removal of the terminal sialic acid by carbohydrate-active enzymes (CAZymes). Here, we present the structural and biochemical characterization of the GH33 catalytic domains of the three sialidases of C. perfringens and probe their substrate specificity. The catalytically active domains, which we refer to as NanH GH33 , NanJ GH33 , and NanI GH33 , displayed differential activity on various naturally occurring forms of sialic acid. We report the X-ray crystal structures of these domains in complex with relevant sialic acid variants revealing the molecular basis of how each catalytic domain accommodates different sialic acids. NanH GH33 displays a distinct preference for α-2,3-linked sialic acid, but can process α-2,6-linked sialic acid. NanJ GH33 and NanI GH33 both exhibit the ability to process α-2,3- and α-2,6-linked sialic acid without any significant apparent preference. All three enzymes were sensitive to generic and commercially available sialidase inhibitors, which impeded sialidase activity in cultures as well as the growth of C. perfringens on sialylated glycans. The knowledge gained in these studies can be applied to in vivo models for C. perfringens growth and metabolism of mucin O-glycans, with a view toward future mitigation of bacterial colonization and infection of intestinal tissues., Competing Interests: Conflict of interest A. B. Boraston is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for JBC and was not involved in the editorial review or the decision to publish this article. The other authors declare they have no conflicts of interest with the contents of this article., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe β-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. β-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. β-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N -glycans show that β-carbafucose is not incorporated into the antibody N -glycans at detectable levels. We expect that β-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes., Competing Interests: Competing interests statement:R.B. and D.J.V. are co-founders and shareholders of Carbaform Biosciences. Carbaform Biosciences has licensed SFU patents covering the materials and uses described within this manuscript.

Cardiolipin is a mitochondrial phospholipid that is also detected in serum inferring its extracellular release; however, this process has not been directly demonstrated for any of the brain cell types. Nevertheless, extracellular cardiolipin has been shown to modulate several neuroimmune functions of microglia and astrocytes, including upregulation of their endocytic activity. Low cardiolipin levels are associated with brain aging, and may thus hinder uptake of amyloid-β (Αβ) in Alzheimer's disease. We hypothesized that glial cells are one of the sources of extracellular cardiolipin in the brain parenchyma where this phospholipid interacts with neighboring cells to upregulate the endocytosis of Αβ. Liquid chromatography-mass spectrophotometry identified 31 different species of cardiolipin released from murine BV-2 microglial cells and revealed this process was accelerated by exposure to Aβ42. Extracellular cardiolipin upregulated internalization of fluorescently-labeled Aβ42 by primary murine astrocytes, human U118 MG astrocytic cells, and murine BV-2 microglia. Increased endocytic activity in the presence of extracellular cardiolipin was also demonstrated by studying uptake of Aβ42 and pHrodo™ Bioparticles™ by human induced pluripotent stem cells (iPSCs)-derived microglia, as well as iPSC-derived human brain organoids containing microglia, astrocytes, oligodendrocytes and neurons. Our observations indicate that Aβ42 augments the release of cardiolipin from microglia into the extracellular space, where it can act on microglia and astrocytes to enhance their endocytosis of Aβ42. Our observations suggest that the reduced glial uptake of Aβ due to the decreased levels of cardiolipin could be at least partially responsible for the extracellular accumulation of Aβ in aging and Alzheimer's disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2022. Published by Elsevier Inc.)

Terpene volatiles define the flavor of terpenic grape cultivars. However, grape terpene concentrations can vary 2- to 3-fold across seasons and vineyards, impacting vintage quality. The plant hormone methyl jasmonate (MeJA) stimulates grape terpene production but is expensive and can decrease berry weight and maturity. The synthetic jasmonate prohydrojasmon (PDJ) is cost-effective yet has not been evaluated on grape maturity and terpene production. Here, we performed in vitro (berry culture) and in vivo (vineyard) experiments using Gewürztraminer ( Vitis vinifera L.) to evaluate the time- and concentration-dependent sensitivity of maturity parameters and terpene content to MeJA and PDJ. In vitro berry weight was reduced by high MeJA and PDJ concentration across timings. Terpenes were most sensitive to low MeJA concentration at veraison (increased 24-fold) in vitro . Moderate PDJ concentration applied at veraison doubled (increased twofold) terpene concentration in vivo without impacting berry weight or maturity. In conclusion, PDJ may provide a solution to mitigate seasonal variability in terpene production in terpenic grape cultivars.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene-1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, a technique amenable to equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel by simple diffusion and subsequent solid-phase extraction (SPE)-based desalting, affords a single glycan species free of excess labeling reagents and buffer components. The described protocol also offers a simple, rapid method for the simultaneous removal of excess APTS and unlabeled glycan material from reaction mixtures. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG., Competing Interests: The authors have declared that no competing interests exist.

Terpenes play a formative role in grape and wine flavor, particularly for high-terpenic cultivars. Differences in terpene profiles influence grape varietal character and vintage quality. Little is known about the endogenous factors controlling terpene biosynthesis in grape. Through multiple experiments, six hormones (abscisic acid, ABA; ethylene, ETH; jasmonic acid, JA; methyl jasmonate, MeJA; indole-3-acetic acid, IAA; 1-naphthaleneacetic acid, NAA) that either promote or repress ripening were applied to Gewürztraminer clusters near veraison to gauge their effect on ripening and terpene biosynthesis. Jasmonates (JA, MeJA) increased terpene concentrations and the expression of terpene genes in grapes. Such increases were not associated to increases of other ripening-related metabolites such as sugars or anthocyanins. MeJA also affected the expression of several hormone related genes, increased IAA levels, and reduced sugar and anthocyanin concentration in grapes. This research provides novel insights into terpene regulation by ripening-related hormones and jasmonates in grapes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Vineyard exposure to wildfire smoke can taint grapes and wine. To understand the impact of this taint, it is imperative that the analytical methods used are accurate and precise. This study compared the variance across nine commercial and research laboratories following quantitative analysis of the same set of smoke-tainted wines. In parallel, correlations between the interlaboratory consensus values for smoke-taint markers and sensory analyses of the same smoke-tainted wines were evaluated. For free guaiacol, the mean accuracy was 94 ± 11% in model wine, while the free cresols and 4-methylguaiacol showed a negative bias and/or decreased precision relative to guaiacol. Similar trends were observed in smoke-tainted wines, with the cresols and glycosidically bound markers demonstrating high variance. Collectively, the interlaboratory results show that data from a single laboratory can be used quantitatively to understand smoke-taint. Results from different laboratories, however, should not be directly compared due to the high variance between study participants. Correlations between consensus compositional data and sensory evaluations suggest the risk of perceivable smoke-taint can be predicted from free cresol concentrations, overcoming limitations associated with the occurrence of some volatile phenols, guaiacol in particular, as natural constituents of some grape cultivars and of the oak used for barrel maturation.

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N -acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus ( At S) and Trichophyton rubrum ( Tr S). Unlike A. fumigatus Kdnase ( Af S), hydrolysis with the Neu5Ac substrates was negligible for Tr S and At S; thus, Tr S and At S are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by At S is similar to that by Af S but 30-fold higher by Tr S. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both At S and Tr S show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus , A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d- manno -oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of Af S in vesicles at the cell surface.

The immunomodulatory family of Siglecs recognizes sialic acid-containing glycans as " self ", which is exploited in cancer for immune evasion. The biochemical nature of Siglec ligands remains incompletely understood, with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs─CHST1, CHST2, or CHST4─significantly enhance the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, a pharmacological blockade of N - and O -glycan maturation reveals a cell-type-specific pattern of importance for either class of glycan. Production of a highly homogeneous Siglec-3 (CD33) fragment enabled a mass-spectrometry-based binding assay to determine ≥8-fold and ≥2-fold enhanced affinity for Neu5Acα2-3(6- O -sulfo)Galβ1-4GlcNAc and Neu5Acα2-3Galβ1-4(6- O -sulfo)GlcNAc, respectively, over Neu5Acα2-3Galβ1-4GlcNAc. CD33 shows significant additivity in affinity (≥28-fold) for the disulfated ligand, Neu5Acα2-3(6- O -sulfo)Galβ1-4(6- O -sulfo)GlcNAc. Moreover, joint overexpression of CHST1 with CHST2 in cells greatly enhanced the binding of CD33 and several other Siglecs. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a general mechanism for tuning Siglec ligands on cells, including in cancer.

We previously observed that sialylated bovine milk oligosaccharides (BMOs) decline in both absolute and relative abundances over the initial stages of bovine lactation, with initial evidence suggesting that this decline occurred due to increased concentrations of unique sulfated BMOs. Since both sulfated and sialylated BMOs have distinct bioactivites, a follow up study was launched in order to more clearly define relative changes in these classes of BMOs over the first week of lactation in dairy cattle. Capillary electrophoresis (CE) and several liquid chromatography mass spectrometry (LC-MS) methods, including a novel multiplexed tandem MS method, were used to profile the BMOs extracted from milk collected from the same 20 Holstein cows at milkings 1, 2, 3, 4, 8, and 14 post-partum. In addition to clearly validating that sulfated and sialylated BMOs exist in direct biosynthetic completion, our study has identified over 170 unique BMOs including 14 unique glucuronic acid-containing trisaccharides., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing β-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42-50 ng/g) increases in free 4-ethylphenol and p -cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-β-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae β-glucosidase activity appear to be influenced by the VP moiety.

Smoke taint in wine is thought to be caused by smoke-derived volatile phenols (VPs) that are absorbed into grape tissues, trapped as conjugates that are imperceptible by smell, and subsequently released into wines as their free odor-active forms via metabolism by yeasts during fermentation. Blocking VP uptake into grapes would, therefore, be an effective way for vineyards to protect ripening grape crops exposed to smoke. Here, we re-evaluated a biofilm that had previously shown promise in pilot studies in reducing levels of smoke-derived VPs. A suite of nine free and acid-labile VPs were quantitated in Pinot Noir grapes that had been exposed to smoke after being coated with the biofilm one, seven or 14 days earlier. In contrast with earlier studies, our results demonstrated that in all cases, the biofilm treatments led to increased concentrations of both free and total VPs in smoke-exposed grapes, with earlier applications elevating concentrations of some VPs more than the later time points. Tracking VP concentrations through the grape ripening process demonstrated that some (phenol, p / m -cresol, and guaiacol) were not entirely sequestered in grapes as acid-labile conjugates, suggesting the presence of VP storage forms beyond simple glycosides. Free VPs in grapes, though a minor portion of the total, most clearly correlated with concentrations present in the resulting wines. Finally, red table grapes, available year round, were observed to replicate the effects of the biofilm treatments and were capable of transforming most VPs into acid-labile conjugates in under 24 h, indicating that they might be an effective model for rapidly assessing smoke-taint prophylactic products in the laboratory.

Scope: The transgenerational impact of dietary fat remains unclear. Here, the role of maternal fat consumption as a modulator of gut microbial communities and infectious disease outcomes in their offspring is explored., Methods and Results: C57BL/6 mice are fed isocaloric high-fat diets throughout breeding, gestation and lactation. Diets contained either milk fat (MF), olive oil (OO) or corn oil (CO), with or without fish oil. The pups born to maternally exposed mice are weaned on to chow and raised into adulthood. At 8 weeks, the offsprings are either euthanized for colonic 16S rRNA analysis or challenged with the enteric pathogen, Citrobacter rodentium. Maternal CO exposure resulted in unique clustering of bacterial communities in offspring compared with MF and OO. Diets rich in CO reduced survival in offspring challenged with C. rodentium. The addition of fish oil did not improve mortality caused by CO and worsened disease outcomes when combined with OO. Unlike the unsaturated diets, MF is protective with and without fish oil., Conclusions: Overall, these data reveal that maternal intake of fatty acids do have transgenerational impacts on their offspring's bacteriome and enteric infection risk. Based on this study, saturated fats should be included in maternal diets., (© 2021 Wiley-VCH GmbH.)

Background: This study utilized a chicken model of chronic physiological stress mediated by corticosterone (CORT) administration to ascertain how various host metrics are altered upon challenge with Clostridium perfringens . Necrotic enteritis (NE) is a disease of the small intestine of chickens incited by C. perfringens , which can result in elevated morbidity and mortality. The objective of the current study was to investigate how physiological stress alters host responses and predisposes birds to subclinical NE., Results: Birds administered CORT exhibited higher densities of C. perfringens in their intestine, and this corresponded to altered production of intestinal mucus. Characterization of mucus showed that C. perfringens treatment altered the relative abundance of five glycans. Birds inoculated with C. perfringens did not exhibit evidence of acute morbidity. However, histopathologic changes were observed in the small intestine of infected birds. Birds administered CORT showed altered gene expression of tight junction proteins (i.e. CLDN3 and CLDN5 ) and toll-like receptors (i.e. TLR2 and TLR15 ) in the small intestine. Moreover, birds administered CORT exhibited increased expression of IL2 and G - CSF in the spleen, and IL1β , IL2 , IL18 , IFNγ , and IL6 in the thymus. Body weight gain was impaired only in birds that were administered CORT and challenged with C. perfringens ., Conclusion: CORT administration modulated a number of host functions, which corresponded to increased densities of C. perfringens in the small intestine and weight gain impairment in chickens. Importantly, results implicate physiological stress as an important predisposing factor to NE, which emphasizes the importance of managing stress to optimize chicken health., Competing Interests: Competing interestsThe authors declare no financial or competing interests., (© The Author(s) 2020.)

Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5- N -glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Quin et al.)

Stable, long-term interactions between fungi and algae or cyanobacteria, collectively known as lichens, have repeatedly evolved complex architectures with little resemblance to their component parts. Lacking any central scaffold, the shapes they assume are casts of secreted polymers that cement cells into place, determine the angle of phototropic exposure and regulate water relations. A growing body of evidence suggests that many lichen extracellular polymer matrices harbor unicellular, non-photosynthesizing organisms (UNPOs) not traditionally recognized as lichen symbionts. Understanding organismal input and uptake in this layer is key to interpreting the role UNPOs play in lichen biology. Here, we review both polysaccharide composition determined from whole, pulverized lichens and UNPOs reported from lichens to date. Most reported polysaccharides are thought to be structural cell wall components. The composition of the extracellular matrix is not definitively known. Several lines of evidence suggest some acidic polysaccharides have evaded detection in routine analysis of neutral sugars and may be involved in the extracellular matrix. UNPOs reported from lichens include diverse bacteria and yeasts for which secreted polysaccharides play important biological roles. We conclude by proposing testable hypotheses on the role that symbiont give-and-take in this layer could play in determining or modifying lichen symbiotic outcomes., (© FEMS 2020.)

Smoke-taint is a wine defect that may occur when ripening grape crops absorb volatile phenols (VPs), compounds associated with the negative sensory attributes of smoke-taint, due to exposure of grapes to wildfire smoke. This study examined potential methods to reduce the impact that smoke-exposure has on wine grapes. Specifically, agricultural sprays normally used to protect grapes from fungal pathogens and a spray used to prevent cracking in soft-fleshed fruits were assessed for their capacity to inhibit increases in VP concentrations in wine grapes following on-vine smoke-exposure. The results indicated that an artificial grape cuticle applied 1 week before exposure to simulated forest fire smoke (at 1-2 weeks after veraison) can significantly hinder an increase in VP concentrations in smoke-exposed grapes at commercial maturity. This reduction in VP concentrations may mitigate crop losses experienced globally by the wine industry due to exposure of grapes on-vine (at key phenological stages) to wildfire smoke.

Clostridium perfringens is a Gram-positive opportunistic pathogen that is the principal etiological agent of necrotic enteritis (NE) in poultry. The ability of C. perfringens to incite NE depends upon its ability to penetrate the protective mucus barrier within the small intestine, which is largely composed of heavily glycosylated proteins called mucins. Mucins are decorated by N- and O-linked glycans that serve both as a formidable gel-like barrier against invading pathogens and as a rich carbon source for mucolytic bacteria. The composition of avian O-linked glycans is markedly different from mucins in other vertebrates, being enriched in sulfated monosaccharides and N-acetyl-d-neuraminic acid (Neu5Ac, sialic acid). These modifications increase the overall negative charge of mucins and are believed to impede colonization by enteric pathogens. The mechanism by which C. perfringens penetrates the poultry intestinal mucus layer during NE is still unknown. However, the CAZome (i.e., the total collection of proteins encoded within a genome active on carbohydrates) of C. perfringens strain CP1 encodes several putative and known enzymes with activities consistent with the modification of mucin. To further investigate this relationship, O-glycans from Gallus gallus domesticus mucus were extracted from the small intestine and characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Chicken mucin monosaccharides included l-fucose (Fuc), d-mannose (Man), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), N-acetyl-d-glucosamine (GlcNAc), and Neu5Ac (sialic acid). Using these monosaccharides as sole carbon sources, we showed that C. perfringens CP1 grew on Neu5Ac, Man, Gal, and GlcNAc but not on Fuc and GalNAc. We also demonstrated C. perfringens grew on different native-state preparations of intestinal mucins and mucus including porcine mucins, chicken mucus, and chicken mucins. Finally, anaerobic incubation of chicken mucin O-glycans with C. perfringens and subsequent analysis of the glycans revealed that there was preferential removal of Neu5Ac. These observations are discussed in the context of the predicted metabolic potential of C. perfringens CP1 and the mucolytic enzymes encoded within its CAZome., (© 2019 Poultry Science Association Inc.)

Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions., (© 2018 Institute of Zoology, Chinese Academy of Sciences.)

Main Conclusion: The exposure of Vitis vinifera L. berries to forest fire smoke changes the concentration of phenylpropanoid metabolites in berries and the resulting wine. The exposure of Vitis vinifera L. berries (i.e., wine grapes) to forest fire smoke can lead to a wine defect known as smoke taint that is characterized by unpleasant "smoky" and "ashy" aromas and flavors. The intensity of smoke taint is associated with the concentration of organoleptic volatile phenols that are produced during the combustion-mediated oxidation of lignocellulosic biomass and subsequently concentrated in berries prior to fermentation. However, these same smoke-derived volatile phenols are also produced via metabolic pathways endogenous to berries. It follows then that an influx of exogenous volatile phenols (i.e., from forest fire smoke) could alter endogenous metabolism associated with volatile phenol synthesis, which occurs via the shikimic acid/phenylpropanoid pathways. The presence of ozone and karrikins in forest fire smoke, as well as changes to stomatal conductance that can occur from exposure to forest fire smoke also have the potential to influence phenylpropanoid metabolism. This study demonstrated changes in phenylpropanoid metabolites in Pinot noir berries and wine from three vineyards following the exposure of Vitis vinifera L. vines to simulated forest fire smoke. This included changes to metabolites associated with mouth feel and color in wine, both of which are important sensorial qualities to wine producers and consumers. The results reported are critical to understanding the chemical changes associated with smoke taint beyond volatile phenols, which in turn, may aid the development of preventative and remedial strategies.

The exposure of Vitis vinifera L. vines to smoke from wildland fires can alter the chemical composition of the berries, such that the resulting wine can possess a defect known as smoke-taint. This work constitutes a complete method for the analysis of simple volatile phenol glycosides (VP-glycosides) that can be elevated in berries and wine following smoke exposure. We synthesized 16 model VP-glycosides, four of which are not reported previously, to facilitate method development. Fragmentation analysis using high-resolution accurate-mass spectrometry demonstrated that the glycone and aglycone influenced the fragmentation pattern of VP-glycosides. Diagnostic fragmentation patterns for the synthesized VP-glycosides were applied to identify several VP-glycosides in smoke-exposed berries and wine. The fragmentation pattern of VP-disaccharides should facilitate the characterization of modified glycones. Putative non-VP glycosides elevated in smoke-exposed berries are demonstrated for the first time. In tandem with VP-glycosides, such compounds may contribute to the expression of smoke taint., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Cannabis sativa L. (cannabis) is utilised as a therapeutic and recreational drug. With the legalisation of cannabis in many countries and the anticipated regulation of potency that will accompany legalisation, analytical testing facilities will require a broadly applicable, quantitative, high throughput method to meet increased demand. Current analytical methods for the biologically active components of cannabis (phytocannabinoids) suffer from low throughput and/or an incomplete complement of relevant phytocannabinoids., Objective: To develop a rapid, quantitative and broadly applicable liquid chromatography-tandem mass spectrometry analytical method for 11 phytocannabinoids in cannabis with acidic and neutral character., Methodology: Bulk diffusion coefficients were calculated using the Taylor-Aris open tubular method, with four reference compounds used to validate the experimental set-up. Three columns were quantitatively evaluated using van Deemter plots and fit-to-purpose performance metrics. Low (1.2 μL 2 ) and standard (3.6 μL 2 ) extra-column variance ultra-high pressure liquid chromatography (UPLC) configurations were contrasted. Method performance was demonstrated with methanolic cannabis flower extracts., Results: Bulk diffusion coefficients and van Deemter plots for 11 phytocannabinoids are reported. The developed chromatographic method includes the challenging Δ 8 /Δ 9 -tetrahydrocannabinol isobars and, at 6.5 min, is faster than existing methods targeting similar panels of biologically active phytocannabinoids., Conclusions: The bulk diffusion coefficients and van Deemter curves informed the development of a rapid quantitative method and will facilitate potential expansion to include additional compounds, including synthetic cannabinoids. The developed method can be implemented with low or standard extra-column variance UPLC configurations., (Copyright © 2018 John Wiley & Sons, Ltd.)

Bovine milk oligosaccharides (BMOs), like their analogues in human milk, have important prebiotic functions. Environmental factors have previously been linked to variation in BMO structures, and thus to test the hypothesis that the bovine diet may lead to these changes in relative BMO abundances, a rapid capillary electrophoresis (CE)-based work flow was developed to profile the BMOs extracted from the milk of cows fed distinctly different diets. Over the first week of lactation, few significant differences were observed between the different diet groups, with the dominant changes being clearly linked to lactation period. CE analyses indicated the presence of ten unusually anionic BMOs, which were predicted to be phosphorylated and sulfated species. Nine unique sulfated BMOs were detected by high-resolution accurate mass spectrometry, none of which have been previously described in bovine milk. The biosynthesis of these was in direct competition with 3'-sialyllactose, the most abundant BMO in bovine milk.

Glycosidically bound, but not free, dietary sialic acids are used for the biosynthesis of new glycoconjugates in humans, making the quantitation of these two forms in infant food sources important, as in neonates the demand for sialic acid may exceed the de novo biosynthetic supply. Here, a rapid high-performance liquid chromatography-mass spectrometry method was developed to identify and quantitate glycosidically bound and free sialic acids in infant formulas. The sialic acid contents of eight commercially available infant formulas with varying protein source or manufacturer were investigated. The formula protein sources (whey vs casein) did not have a large impact on the ratios of free to bound sialic acids, nor did protein hydrolysis or sample form (solid vs liquid). Hydrolyzed bovine whey protein-based formulas were found to contain the highest amount of the most abundant human sialic acid, 5- N-acetylneuraminic acid (Neu5Ac). O-Acetylated Neu5Ac was quantified in all formulas tested and, for the first time, 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn) was detected in several infant formulas.

O-Linked glycosylation of serine and threonine residues of nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) residues is catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc is conserved within mammals and is implicated in a wide range of physiological processes. Herein, we describe metabolic precursor inhibitors of OGT suitable for use both in cells and in vivo in mice. These 5-thiosugar analogues of N-acetylglucosamine are assimilated through a convergent metabolic pathway, most likely involving N-acetylglucosamine-6-phosphate de-N-acetylase (NAGA), to generate a common OGT inhibitor within cells. We show that of these inhibitors, 2-deoxy-2-N-hexanamide-5-thio-d-glucopyranoside (5SGlcNHex) acts in vivo to induce dose- and time-dependent decreases in O-GlcNAc levels in various tissues. Decreased O-GlcNAc correlates, both in vitro within adipocytes and in vivo within mice, with lower levels of the transcription factor Sp1 and the satiety-inducing hormone leptin, thus revealing a link between decreased O-GlcNAc levels and nutrient sensing in peripheral tissues of mammals., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Smoke-taint is a wine defect linked to organoleptic volatile phenols (VPs) in Vitis vinifera L. berries that have been exposed to smoke from wildland fires. Herein, the levels of smoke-taint-associated VPs are reported in Cabernet Franc berries from veraison to commercial maturity and in wine after primary fermentation following on-vine exposure to simulated wildland fire smoke. VPs increased after smoke exposure were rapidly stored as acid-labile conjugates, and the levels of both free VPs and conjugated forms remained constant through ripening to commercial maturity. An increase in total VPs after primary fermentation suggested the existence of VP-conjugates other than the acid-labile VP-glycosides already reported. This conclusion was supported with base hydrolysis on the same samples. Relative to published results, the data suggested a multifactorial regional identity for smoke-taint and they inform efforts to produce a predictive model for perceptible smoke-taint in wine based on the chemical composition of smoke-exposed berries.

Accurate methods for quantitating volatile phenols (i.e., guaiacol, syringol, 4-ethylphenol, etc.) in smoke-exposed Vitis vinifera berries prior to fermentation are needed to predict the likelihood of perceptible smoke taint following vinification. Reported here is a complete, cross-validated analytical workflow to accurately quantitate free and glycosidically bound volatile phenols in smoke-exposed berries using liquid-liquid extraction, acid-mediated hydrolysis, and gas chromatography-tandem mass spectrometry. The reported workflow addresses critical gaps in existing methods for volatile phenols that impact quantitative accuracy, most notably the effect of injection port temperature and the variability in acid-mediated hydrolytic procedures currently used. Addressing these deficiencies will help the wine industry make accurate, informed decisions when producing wines from smoke-exposed berries.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene 1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, which utilizes equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel and subsequent solid-phase extraction (SPE), yields single glycan species free of excess labeling reagents and buffer components. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

Pyrrolidine-based iminocyclitols are a promising class of glycosidase inhibitors. Reported herein is a convenient epimerization strategy that provides direct access to a range of stereoisomeric iminocyclitol inhibitors of O-GlcNAcase (OGA), the enzyme responsible for catalyzing removal of O-GlcNAc from nucleocytoplasmic proteins. Structural details regarding the binding of these inhibitors to a bacterial homologue of OGA reveal the basis for potency. These compounds are orally available and permeate into rodent brain to increase O-GlcNAc, and should prove useful tools for studying the role of OGA in health and disease., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role played by N-glycosylation within the secretory pathway.

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.

Sialyl-Lewis X (sLe(X)) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe(X) biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe(X) in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe(X) presentation on HepG2 cells with an EC(50) in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe(X) levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe(X) formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.

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

The ability of gold (Au) nanoparticles (NPs) to generate heat efficiently by absorbing visible and near-infrared (NIR) light holds great promise as a means to trigger chemical and biochemical events near the NPs. Previous demonstrations show that pulsed laser irradiation can selectively elicit the release of a fluorescent dye covalently anchored to the NP surface through a heat-labile linker without measurably changing the temperature of the surroundings. This article reports that the authors demonstrate the biological efficacy of this approach to photodelivery by showing that the decorated Au NPs are rapidly internalized by cells, are stable under physiological conditions, are nontoxic, and exhibit nonlethal photorelease following exposure to pulsed laser radiation. These observations, further supported by the versatility of our delivery motif, reaffirm the potential for further development of nonlethal photothermal therapeutics and their future relevance to such fields as gene therapy and stem-cell differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The limited proteolysis of proteins by the proprotein convertases (PCs) is a common means of producing bioactive proteins or peptides. The PCs are associated with numerous human pathologies and their activity can be reduced through the use of specific inhibitors. Here, we demonstrate an alternative approach to inhibiting PCs by altering their N-glycosylation. Through site-directed mutagenesis, we show that the convertase PC1/3 contains two N-glycans, only one of which is critical for its prosegment cleavage. The exact structure of PC1/3 N-glycans does not significantly affect its zymogen activation within endocrine cells, but glycosylation of Asn(146) is critical. Processing of the PC1/3's substrate proopiomelanocortin (POMC) was used in a cell-based assay to screen a collection of 45 compounds structurally related to known glycosidase inhibitors. Two 5-thiomannose-containing disaccharide derivatives were discovered to block PC1/3 and POMC processing into the analgesic peptide β-endorphin. These compounds also reduced the zymogen activation of the convertase subtilisin kexin isozyme-1 (SKI-1), blocked the processing of its substrate the sterol regulatory element-binding protein SREBP-2 and altered its glycosylation. Thus, modification of PC glycosylation may also be a means of blocking their activity, an effect which, in the case of SKI-1, may be of possible therapeutic use since SREBP-2 regulates sterol levels including cholesterol biosynthesis and its metabolism.

The modification of N-glycans by α-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for α1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-α1,6-mannosidase activity consistent with specificity for N-linked glycans having their α1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (α/α)(6)-fold, recognize and hydrolyze the α1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-α1,6-mannosidases.

Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC(50). This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.

The galactofuran region of the mycobacterial cell wall consists of alternating 5- and 6-linked beta-d-galactofuranose (beta-D-Galf) residues, essential for viability. UDP-galactofuranose (UDP-Galf), the donor for Galf, is synthesised from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (UGM), which is not found in humans, rendering it a therapeutic target. The in vitro properties, i.e. enzymatic activity, antimycobacterial activity, cellular toxicity, activity in mycobacterial-infected macrophages and activity against non-replicating persistent mycobacteria, of (4-chlorophenyl)-[1-(4-chlorophenyl)-3-hydroxy-5-methyl-1H-pyrazol-4-yl]-methanone and 3-(4-iodophenyl)-2-[4-(3,4-dichlorophenyl)-thiazol-2-ylamino]-propionic acid were studied. The former compound, a pyrazole, was an inhibitor of UGM from Mycobacterium tuberculosis and Klebsiella pneumoniae and was effective against Mycobacterium smegmatis, Mycobacterium bovis BCG and M. tuberculosis but ineffective against other bacterial strains tested. This compound showed potency against mycobacteria in infected macrophages but exhibited moderate cellular toxicity and was ineffective against non-replicating persistent mycobacteria. This is the first report of a compound both with UGM inhibitory properties and broad antimycobacterial activities. The latter compound, an aminothiazole, was active against UGM from K. pneumoniae and M. tuberculosis but was ineffective against M. bovis BCG or M. tuberculosis as well as demonstrating higher cellular toxicity. These data validate the choice of UGM as a target for active antimycobacterial therapy and confirm the pyrazole compound as a viable lead candidate., (Copyright 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

A simple and reproducible capillary-zone electrophoresis (CZE) method was developed for the separation and quantitation of sulfonium-ion-containing compounds isolated from plants of the Salacia genus which are traditionally used in Ayurvedic medicine for the treatment of type-2 diabetes. The method sufficiently resolved four different compounds with confirmed glucosidase inhibitory activity, namely, salacinol, ponkoranol, kotalanol and de-O-sulfonated kotalanol. Separation could be achieved in less than 9 min, and calibration curves showed good linearity. Detection limits were determined to be in the low mug/mL range. This method was used to demonstrate that de-O-sulfonated kotalanol isolated from natural sources has identical ionic mobility to a synthetic standard. Furthermore, new extraction conditions were developed by which the zwitterionic compounds (salacinol, ponkoranol, and kotalanol) could be separated from de-O-sulfonated kotalanol in a single solid-phase extraction (SPE) procedure. The extraction gave reproducibly high recoveries and was used to process four commercial Salacia extracts for CZE analysis to reduce the complexity of resulting electropherograms and to facilitate the detection of the four inhibitors in question. De-O-sulfonated kotalanol was detected in two of four Salacia samples while ponkoranol was present in all four. A comparison of all samples tested demonstrated that they had remarkably similar patterns of peaks, suggesting that this CZE method may be useful in the chemical fingerprinting of Salacia-containing products.

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