Recent progress in the relationship between carbohydrate cross-reactive determinants (CCDs) and allergic response highlights the importance of carbohydrate moieties in the innate immune system. Previous research pointed out that the protein allergen in Ginkgo biloba seeds is glycosylated, and the oligosaccharides conjugated to these proteins might also contribute to the allergy. The aim of this study was to analyze carbohydrate moieties, especially N-linked glycans, of glycoproteins from Ginkgo seeds originating from different places for detailed structures, to enable further research on the role played by N-glycans in Ginkgo-caused allergy. Results of monosaccharide composition and immunoblotting assays indicated the existence of N-glycans. Detailed structural elucidation of the N-glycans was further carried out by means of hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 14 out of 16 structures detected by UPLC were confirmed by MALDI-TOF-MS and tandem mass spectrometry, among which complex-type N-glycans bearing Lewis A determinants and high-mannose-type N-glycans were identified from Ginkgo seeds for the first time. Precise quantification of N-glycans was performed by use of an external standard, and both the absolute amount of each N-glycan and the percentage of different types of N-glycan showed significant diversity among the samples without any pattern of geographic variation.Keywords: Ginkgo seeds; N-glycome; quantification; structure analysis;

AbstractNovel sialic acid scaffolds have great significance in the development of influenza neuraminidase inhibitors. Here the enzymatic synthesis of a wide range of 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) analogues via aldol addition of pyruvate to d-mannose, d-glucose, d-galactose, 2-deoxy-d-glucose, d-arabinose, l-arabinose and l-rhamnose using a previously unstudied N-acetylneuraminic acid (Neu5Ac) aldolase derived from the bacterium Dyadobacter fermentas is exemplified. Several of the synthesized KDN analogues showed comparable or better inhibitory activity than unstudied Neu5Ac against the mutated influenza neuraminidases (A/California/04/2009 and A/Anhui/1/2005), which both show resistance to Neu5Ac-based neuraminidase inhibitors, demonstrating that these compounds are promising templates for the development of anti-influenza drugs.

•Mannoside phosphorylases are frequently found in bacteria and play an important role in carbohydrate processing.•A bacterial phosphorylase catalyzes the reversible phosphorolysis of novel β-1,3-oligomannose structures.•Discovery of an enzyme specifically targets β-1,3-mannoside linkages.•Functional extension of glycoside hydrolase family 130BackgroundMannoside phosphorylases are frequently found in bacteria and play an important role in carbohydrate processing. These enzymes catalyze the reversible conversion of β-1,2- or β-1,4-mannosides to mannose and mannose-1-phosphate in the presence of inorganic phosphate.MethodsThe biochemical parameters of this recombinantly expressed novel mannose phosphorylase were obtained. Furthermore purified reaction products were subjected to ESI- and MALDI-TOF mass spectrometry and detailed NMR analysis to verify this novel type of β-1,3-mannose linkage.ResultsWe describe the first example of a phosphorylase specifically targeting β-1,3-mannoside linkages. In addition to mannose, this phosphorylase originating from the bacterium Zobellia galactanivorans could add β-1,3-linked mannose to various other monosaccharides and anomerically modified 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars).ConclusionsAn unique bacterial phosphorylase specifically targeting β-1,3-mannoside linkages was discovered.General significanceFunctional extension of glycoside hydrolase family 130.Download high-res image (130KB)Download full-size image

•N-glycome derived from human and bovine milk were isolated for detailed structure analysis.•Bifidus growth promoting activity was found from both human and bovine milk derived N-glycome.•The consumption profiles of two types of N-glycome by B. infantis were different.•The intact N-glycan was decomposed by exo-glycosidases produced by the B. infantis.The ability of B. longum subsp. infantis ATCC15697 in utilizing milk N-glycans to shape an ideal gut microbiota in infants has not been extensively studied. Here we compared N-glycan structures from human and bovine milk using UHPLC and MALDI-TOF-MS/MS. The structural differences between the two were apparent. When being tested as bifidus growth promoting factors, milk N-glycans from both human and bovine showed bifidogenic activity, with a stronger effect from the former. Sequential comparison of glycoprofiles revealed an apparent difference in the bifidobacterial consumption of N-glycans from human and bovine milk, and a general preference for smaller structures. In summary, this work indicates that the consumption of the milk N-glycans by B. infantis ATCC15697 is depending on the glycan composition, and regulating the N-glycome composition of bovine milk through adding exogenous N-glycans favored by B. infantis ATCC15697 might provide improved effect on regulating the microbial composition of the infant gut.

N-Acetyl glucosamine (GlcNAc) is one of the most abundant biomolecules on Earth and is cheaply available from chitin, a major component of crustaceans. The key step in the conversion of GlcNAc to high-value products is the de-N-acetylation to glucosamine, in itself a valuable dietary supplement that is produced at over 29 000 tons scale per annum by chemical hydrolysis, a process that requires harsh reaction conditions and leads to side products requiring separation. Here, we report for the first time the isolation and characterisation of an enzyme, a deacetylase from Cyclobacterium marinum that is able to catalyse the highly selective quantitative hydrolysis of GlcNAc to glucosamine under mild reaction conditions. This enzyme is small (38 kDa), is easily obtainable by heterologous expression in E. coli, has high turnover rates (kcat = 61 s−1), tolerates high substrate concentrations (over 100 g L−1) and can be repeatedly re-used as an immobilised catalyst. When coupled with chitinase, the high selectivity of the enzyme for GlcNAc over other biomolecules allowed one-pot extraction of glucosamine from crude solid mushroom fractions containing chitin, thus allowing for alternative production of glucosamine from non-animal sources, of benefit to consumers with crustacean allergies and vegan diets. We suggest that the deacetylase fills an important gap in the sustainable exploitation of GlcNAc and chitin.

•Human milk N-glycome showed higher anti-pathogenic activity than bovine milk.•Anti-pathogenic activity of both dramatically decreased after defucosylation.•Functional difference almost vanished after defucosylation.Health differences between breast- and formula-fed infants have long been apparent despite great efforts in improving the function of baby formula by adjusting the levels of various milk nutritional components. However, the N-glycome, a type of oligosaccharide decorating a diverse range of proteins, has not been extensively studied in milk regarding its biological function. In this study, the anti-pathogenic function of the enzymatically released human and bovine milk N-glycome against 5 food-borne pathogens was investigated. The human milk N-glycome showed significantly higher activity than bovine milk. After enzymatic defucosylation of human and bovine N-glycan pool, UHPLC peak shifts were observed in both suggesting heavy fucosylation of samples. Furthermore, the anti-pathogenic activity of the defulosylated N-glycome decreased significantly, and the significance of functional difference between the two almost disappeared. This result indicates the essential role of fucosylation for the anti-pathogenic function of the milk N-glycome, especially in human milk.

•An extraction platform for the automated release of N-glycans from glycoproteins.•Designed and fabricated with a low-cost 3D manufacturing technology.•The usability of the platform was tested with fetuin as a model glycoprotein.The development of techniques for the rapid analysis of N-glycans is a key step in enabling the roles of glycoproteins in biological processes to be studied. Analysis is usually performed through the liberation of the carbohydrate moieties from proteins, followed by fluorescent labeling and identification using either standardized HPLC or mass spectrometry techniques. A simple and robust automated process for the release and isolation of N-glycans would greatly improve analytical throughput and reproducibility, and is thus highly desirable. Inspired by the increasing number of reported projects involving open source labware, which allows the design and construction of otherwise inaccessible laboratory equipment using low-cost 3D printers, we used this technique to fabricate a platform for the automated isolation of N-glycans. As a proof of concept, we demonstrated the successful recovery of glycan samples from the glycoprotein model fetuin using our self-made 3D-printed equipment.

Anomeric sugar kinases perform fundamental roles in the metabolism of carbohydrates. Under- or overexpression of these enzymes, or mutations causing functional impairments can give rise to diseases such as galactosaemia and so the study of this class of kinase is of critical importance. In addition, anomeric sugar kinases which are naturally promiscuous, or have been artificially made so, may find application in the synthesis of libraries of drug candidates (for example, antibiotics), and natural or unnatural oligosaccharides and glycoconjugates. In this review, we provide an overview of the biological functions of these enzymes, the tools which have been developed to investigate them, and the current frontiers in their study.

Herein we describe a UPLC-FLD-based method for the quantification of the sialic acid content of red meat, using a synthetic neuraminic acid derivative as an internal standard. X-Gal-α-2,6-N-propionylneuraminic acid was synthesized via a chemoenzymatic pathway and its hydrolytic stability was characterized. Known quantities of this compound were incubated with samples of red meat under sialic acid-releasing conditions. The released sialic acids were derivatized, analyzed by UPLC-FLD, and the Neu5Ac/Neu5Gc content of the meat sample was determined by comparison with the internal standard. A number of red meats were analyzed by this method with the following results (Neu5Ac μg/g tissue, Neu5Gc μg/g tissue ± s.d.): pork (68 ± 3, 15.2 ± 0.7), beef (69 ± 8, 36 ± 5), lamb (46 ± 2, 33 ± 1), rabbit (59 ± 2, 0.4 ± 0.4), and hare (50 ± 4, 1 ± 1). We envisage that this methodology will find application in investigating the health effects of dietary Neu5Gc.

Two putative α-mannosidase genes isolated from the rather unexplored soil bacterium Solitalea canadensis were cloned and biochemically characterised. Both recombinant enzymes were highly selective in releasing α-linked mannose but no other sugars. The α-mannosidases were designated Sca2/3Man2693 and Sca6Man4191, and showed the following biochemical properties: the temperature optimum for both enzymes was 37 °C, and their pH optima lay at 5.0 and 5.5, respectively. The activity of Sca2/3Man2693 was found to be dependent on Ca2+ ions, whereas Cu2+ and Zn2+ ions almost completely inhibited both α-mannosidases. Specificity screens with various substrates revealed that Sca2/3Man2693 could release both α1-2- and α1-3-linked mannose, whereas Sca6Man4191 only released α1-6-linked mannose. The combined enzymatic action of both recombinant α-mannosidases allowed the sequential degradation of high-mannose-type N-glycans. The facile expression and purification procedures in combination with strict substrate specificities make α-mannosidases from S. canadensis promising candidates for bioanalytical applications.

We present a generic method for screening small molecule kinases for their acceptor specificity. The release of the reaction byproduct adenosine diphosphate (ADP) triggers a concentration-dependent formation of amylose from sucrose, by using the combined enzymatic action of sucrose synthase and glycogen synthase. Kinase activities could be quantified photometrically after the formation of a dark-blue amylose–polyiodide complex. We demonstrate that this method can be used to profile both known and novel nucleotide- and sugar-kinases for their substrate specificity. Using a facile and widely available methodology, the amylose–polyiodide small-molecule kinase assay presented herein has the potential to perform substrate screenings of small molecule kinases in a high-throughput manner.

The quantification of potentially allergenic carbohydrate motifs of plant and insect glycoproteins is increasingly important in biotechnological and agricultural applications as a result of the use of insect cell-based expression systems and transgenic plants. The need to analyze N-glycan moieties in a highly parallel manner inspired us to develop a quick N-glycan analysis method based on a recently discovered bacterial protein N-glycanase (PNGase H+). In contrast to the traditionally used PNGase A, which is isolated from almond seeds and only releases N-glycans from proteolytically derived glycopeptides, the herein implemented PNGase H+ allows for the release of N-glycans directly from the glycoprotein samples. Because PNGase H+ is highly active under acidic conditions, the consecutive fluorescence labeling step using 2-aminobenzamide (2AB) can be directly performed in the same mixture used for the enzymatic deglycosylation step. All sample handling and incubation steps can be performed in less than 4 h and are compatible with microwell-plate sampling, without the need for tedious centrifugation, precipitation, or sample-transfer steps. The versatility of this methodology was evaluated by analyzing glycoproteins derived from various plant sources using ultra-performance liquid chromatography (UPLC) analysis and further demonstrated through the activity analysis of four PNGase H+ mutant variants.

•Four putative neuraminidases from a human gut bacterium were cloned and recombinantly expressed in active form.•Functional characterisation revealed that the enzymes had similar temperature optima, but distinct metal requirements and pH optima.•The enzymes showed different substrate specificities towards C5-modified sialic acid analogues.•Simple expression and purification procedure make these enzymes potentially valuable for bioanalytical and industrial applicationsSince the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu2+ and Zn2+, and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.

UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn2+ ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.

We herein report the use of 1,3-di(2-pyridyl)-1,3-propanedione (DPPD) as a fluorogenic labeling reagent for sugars. Reaction of DPPD with the anomeric carbon affords a fluorescent 2-pyridylfuran (2-PF) moiety that permits the sensitive HPLC-based detection of monosaccharides. 2-PF-labeled monosaccharides can be easily separated and analyzed from mixtures thereof, and the reported protocol compares favorably with established labeling reagents such as 2-aminobenzoic acid (2-AA) and 1-phenyl-3-methyl-5-pyrazolone (PMP), ultimately allowing subfemtomole detection of the galactose-derived product. Furthermore, we demonstrate the application of DPPD in the labeling of monosaccharides in complex biological matrices such as blood and milk samples. We envisage that DPPD will prove to be an excellent choice of labeling reagent in monosaccharide and carbohydrate analysis.