Identification of the effect of N-glycan modification and its sialylation on proteolytic stability and glucose-stabilizing activity of glucagon-like peptide 1 by site-directed enzymatic glycosylation.

In this study, an approach to prepare long-acting glucagon-like peptide 1 (GLP-1) by site-directed enzymatic glycosylation with homogeneous biantennary complex-type N-glycan has been developed. All the N-glycan-modified GLP-1 analogues preserved an unchanged secondary structure. The glycosylated GLP-1 analogues with sialyl complex-type N-glycan modified at Asn26 and Asn34 exhibited a 36.7- and 24.0-fold in vitro half-life respectively when incubated with dipeptidyl peptidase-IV (DPP-IV), and 25.0- and 13.9-fold respectively when incubated with mouse serum. Compared to native GLP-1, both glycosylated GLP-1 analogues modified at Asn34 by asialyl and sialyl N-glycan demonstrated lower maximum blood glucose levels, as well as more rapid and more persistent glucose-stabilizing capability in type 2 diabetic db/db mice. Our results indicated that the selection of an appropriate position (to avoid hindering the peptide-receptor binding) is crucial for N-glycan modification and its sialylation to improve the therapeutic properties of the modified peptides. The information learned would facilitate future design of therapeutic glycopeptides/glycoproteins with N-glycan to achieve enhanced pharmacological properties.

Chemoenzymatic modular assembly of O-GalNAc glycans for functional glycomics.

O-GalNAc glycans (or mucin O-glycans) play pivotal roles in diverse biological and pathological processes, including tumor growth and progression. Structurally defined O-GalNAc glycans are essential for functional studies but synthetic challenges and their inherent structural diversity and complexity have limited access to these compounds. Herein, we report an efficient and robust chemoenzymatic modular assembly (CEMA) strategy to construct structurally diverse O-GalNAc glycans. The key to this strategy is the convergent assembly of O-GalNAc cores 1-4 and 6 from three chemical building blocks, followed by enzymatic diversification of the cores by 13 well-tailored enzyme modules. A total of 83 O-GalNAc glycans presenting various natural glycan epitopes are obtained and used to generate a unique synthetic mucin O-glycan microarray. Binding specificities of glycan-binding proteins (GBPs) including plant lectins and selected anti-glycan antibodies towards these O-GalNAc glycans are revealed by this microarray, promoting their applicability in functional O-glycomics. Serum samples from colorectal cancer patients and healthy controls are assayed using the array reveal higher bindings towards less common cores 3, 4, and 6 than abundant cores 1 and 2, providing insights into O-GalNAc glycan structure-activity relationships.

Exogenous spatial attention shortens perceived depth.

Although spatial attention has been found to alter the subjective appearance of visual stimuli in several perceptual dimensions, no research has explored whether exogenous spatial attention can affect depth perception, which is a fundamental dimension in perception that allows us to effectively interact with the environment. Here, we used an experimental paradigm adapted from Gobell and Carrasco (Psychological Science, 16[8], 644-651, 2005) to investigate this question. A peripheral cue preceding two line stimuli was used to direct exogenous attention to either location of the two lines. The two lines were separated by a certain relative disparity, and participants were asked to judge the perceived depth of two lines while attention was manipulated. We found that a farther stereoscopic depth at the attended location was perceived to be equally distant as a nearer depth at the unattended location. No such effect was found in a control experiment that employed a postcue paradigm, suggesting that our findings could not be attributed to response bias. Therefore, our study shows that exogenous spatial attention shortens perceived depth. The apparent change in stereoscopic depth may be regulated by a mechanism involving direct neural enhancement on those tuned to disparity, or be modulated by an attentional effect on apparent contrast. This finding shows that attention can change not only visual appearance but also the perceived spatial relation between an object and an observer.

Microarray analyses of closely related glycoforms reveal different accessibilities of glycan determinants on N-glycan branches.

Glycans mediate a wide variety of biological roles via recognition by glycan-binding proteins (GBPs). Comprehensive knowledge of such interaction is thus fundamental to glycobiology. While the primary binding feature of GBPs can be easily uncovered by using a simple glycan microarray harboring limited numbers of glycan motifs, their fine specificities are harder to interpret. In this study, we prepared 98 closely related N-glycoforms that contain 5 common glycan epitopes which allowed the determination of the fine binding specificities of several plant lectins and anti-glycan antibodies. These N-glycoforms differ from each other at the monosaccharide level and were presented in an identical format to ensure comparability. With the analysis platform we used, it was found that most tested GBPs have preferences toward only one branch of the complex N-glycans, and their binding toward the epitope-presenting branch can be significantly affected by structures on the other branch. Fine specificities described here are valuable for a comprehensive understanding and applications of GBPs.

Biochemical characterization of Helicobacter pylori α1-3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides.

Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid truncated version of Helicobacter pylori α1-3-fucosyltransferase (Hp3FT) is a good candidate. Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of fucosides. While N-acetyllactosamine (LacNAc) and lactose were both suitable acceptors, the former is preferred. At a low guanosine 5'-diphospho-ß-L-fucose (GDP-Fuc) to acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics, diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, lacto-N-neofucopentaose (LNnFP) V, lacto-N-neodifucohexaose (LNnDFH) II, difuco- and trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH), were synthesized enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of multiple glycosyltransferase-catalyzed reactions.

Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins.

Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.

Decoding glycan protein interactions by a new class of asymmetric N-glycans.

N-Glycans are normally involved in crucial physiological and disease processes by interactions with glycan-binding proteins. So far structurally defined N-glycans have been good candidates for glycan binding study. Herein, a class of homogeneous asymmetric N-glycans was synthesized by coupling glycan-oxazoline and N-glycans using EndoM N175Q catalyzed quick glycan extension. Branch-biased binding and spacial inhibition caused by the bulky group on the other branch of N-glycan were observed in glycan protein interactions involving lectins and these glycans by glycan microarray study. These new compounds are valuable for functional glycomic studies to better understand new functions of glycans and glycan-binding proteins.

H. pylori α1-3/4-fucosyltransferase (Hp3/4FT)-catalyzed one-pot multienzyme (OPME) synthesis of Lewis antigens and human milk fucosides.

Helicobacter pylori α1-3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg L-1 culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of l-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, and x, O-sulfated Lewis x, and sialyl Lewis x and human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, and lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, the synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactosamine (LacNAc) component in lacto-N-neotetraose (LNnT).

An enzymatic strategy to asymmetrically branched N-glycans.

An enzymatic strategy was developed to generate asymmetrically branched N-glycans from natural sources by using a panel of glycosidases and glycosyltransferases. Briefly, LacZ ß-galactosidase was employed to selectively trim symmetrically branched N-glycans isolated from bovine fetuin. The yielding structures were then converted to asymmetrically branched core structures by robust glycosyltransferase for further extension.

Cation exchange assisted binding-elution strategy for enzymatic synthesis of human milk oligosaccharides (HMOs).

A cation exchange assisted binding-elution (BE) strategy for enzymatic synthesis of human milk oligosaccharides (HMOs) was developed. An amino linker was used to provide the cation ion under acidic condition which can be readily bound to cation exchange resin and then eluted off by saturated ammonium bicarbonate. Ammonium bicarbonate in the collections was easily removed by vacuum evaporation. This strategy circumvented the incompatible issue between glycosyltransferases and solid support or large polymers, and no purification was needed for intermediate products. With current approach, polyLacNAc backbones of HMOs and fucosylated HMOs were synthesized smoothly.

Betaine Improves Polymer-Grade D-Lactic Acid Production by Sporolactobacillus inulinus Using Ammonia as Green Neutralizer.

The traditional CaCO3-based fermentation process generates huge amount of insoluble CaSO4 waste. To solve this problem, we have developed an efficient and green D-lactic acid fermentation process by using ammonia as neutralizer. The 106.7 g/L of D-lactic acid production and 0.89 g per g of consumed sugar were obtained by Sporolactobacillus inulinus CASD with a high optical purity of 99.7% by adding 100 mg/L betaine in the simple batch fermentation process. The addition of betaine was experimentally proven to protect cell at high concentration of ammonium ion, increase the D-lactate dehydrogenase specific activity and thus promote the production of D-lactic acid.

Identification of the binding roles of terminal and internal glycan epitopes using enzymatically synthesized N-glycans containing tandem epitopes.

Glycans play diverse roles in a wide range of biological processes. Research on glycan-binding events is essential for learning their biological and pathological functions. However, the functions of terminal and internal glycan epitopes exhibited during binding with glycan-binding proteins (GBPs) and/or viruses need to be further identified. Therefore, a focused library of 36 biantennary asparagine (Asn)-linked glycans with some presenting tandem glycan epitopes was synthesized via a combined Core Isolation/Enzymatic Extension (CIEE) and one-pot multienzyme (OPME) synthetic strategy. These N-glycans include those containing a terminal sialyl N-acetyllactosamine (LacNAc), sialyl Lewis x (sLex) and Siaα2-8-Siaα2-3/6-R structures with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) sialic acid form, LacNAc, Lewis x (Lex), α-Gal, and Galα1-3-Lex; and tandem epitopes including α-Gal, Lex, Galα1-3-Lex, LacNAc, and sialyl LacNAc, presented with an internal sialyl LacNAc or 1-2 repeats of an internal LacNAc or Lex component. They were synthesized in milligram-scale, purified to over 98% purity, and used to prepare a glycan microarray. Binding studies using selected plant lectins, antibodies, and viruses demonstrated, for the first time, that when interpreting the binding between glycans and GBPs/viruses, not only the structure of the terminal glycan epitopes, but also the internal epitopes and/or modifications of terminal epitopes needs to be taken into account.

Site-Directed Glycosylation of Peptide/Protein with Homogeneous O-Linked Eukaryotic N-Glycans.

Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine α-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans.

"Happy goat says": The effect of a food selection inhibitory control training game of children's response inhibition on eating behavior.

Recent studies suggest that when inhibitory control is lacking, people are more inclined to indulge in high-calorie food, but inhibitory control can be trained. In this study, a daily-life training game was used to train children and investigate whether strengthening or weakening inhibitory control influences food intake in opposite directions. The baseline of response inhibition was measured by the go/no-go task, and the baseline of food intake was measured by a bogus food taste task. Then, participants performed a food selection training game named "Happy goat says" with three within-subject conditions: the first type of instruction was always paired without a go signal (inhibition manipulation); the second type of instruction was always presented with a go signal (impulsivity manipulation); and the third type of instruction was presented either with a go or no-go signal, both in 50% of the time (control manipulation). Following these manipulations, they went through the go/no-go task and bogus food taste task. In the pre-training food taste task, commission errors were positively correlated with body mass index. Relative to a control group playing Lego blocks (n = 20), the trained group showed a performance improvement on the go/no-go task. The intake of food in the inhibition manipulation was significantly less in the post-training food taste task. These findings demonstrate that children can gain control over the consumption of high-calorie food after a daily-life response inhibition training game.

[Active sites of N-acetylhexosamine 1-kinase from Bifidobacterium longum].

OBJECTIVE: To study the active sites of N-acetylhexosamine 1-kinase (NahK) from Bifidobacterium longum JCM12 17. METHODS: We obtained expression strains of 10 single-mutants at 4 sites of NahK by site-directed mutagenesis, and expressed and purified both wild-type (WT) and mutant enzymes. Then, their optimum pH and optimum concentration of Mg²âº were determined by DNS assay and NADH-coupled microplate photometric assay, and their kinetic parameters were measured. RESULTS: Four mutants (D208A, D208N, D208E and I24A) lost most part of the catalytic activity. The optimum pH of mutants H31A, H31V, F247A and I24V switched from pH 7.5 (for WT) to pH 7.0, and the optimum concentration of Mg²âº of mutants H31A and F247A increased to 10 mmol/L from 5 mmol/L (for WT). The kinetic parameters of WT and mutants indicate that mutant F247Y had higher enzymatic activity toward GlcNAc, GalNAc and ATP than WT. CONCLUSION: The key amino acids that affect the catalytic activity of NahK were determined by site-directed mutagenesis, and together with the mutant that has higher catalytic efficiency, this has laid a foundation for further modification and evolution of NahK.

A General Chemoenzymatic Strategy for the Synthesis of Glycosphingolipids.

A concise, prototypical, and stereoselective strategy for the synthesis of therapeutically and immunologically significant glycosphingolipids has been developed. This strategy provides a universal platform for glycosphingolipid synthesis by block coupling of enzymatically prepared free oligosaccharideglycans to lipids using glycosyl N-phenyltrifluoroacetimidates as efficient activated intermediates. As demonstrated here, two different types of glycosphingolipids were obtained in excellent yields using the method.

Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7.

Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galß1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

Crystallization and X-ray diffraction of an ectoine hydroxylase from Bacillus pseudofirmus OF4.

OBJECTIVE: To study crystallization and X-Ray diffraction of ectoine hydroxylase from Bacillus pseudofirmus OF4. METHODS: We cloned the gene BpectD from B. pseudofirmus OF4 with a His6 tag and overexpressed it in E. coli BL21 (DE3). Then, we purified protein BpEctD by Ni2 -chelating affinity and size-exclusion chromatography. After that, crystals were grown by the sitting-drop vapour-diffusion method at 289 K and diffracted at 100K using an in-house X-ray source. RESULTS: Protein BpEctD was expressed and purified successfully. We obtained well diffracting crystals of about 360 µm x 240 µm x 60 µm in size using a solution consisting of 0.2 mol/L magnesium chloride hexahydrate, 0.1 mol/L bis-tris pH6. 5, 25% (W/V) polyethylene glycol 3,350 at a protein concentration of 6. 5 mg/mL, and collected X-ray diffraction data to 2.40 Å resolution in the anorthic space group P1, with unit-cell parameters a = 45.18Å, b = 58.87Å, c = 68.81 Å, α = 77.48 degrees, ß = 86.03 degrees, γ = 66.97 degrees. The asymmetric unit contains two molecules of BpEctD with a Mattews coefficient of about 2.44 Å3/Da and a solvent content of 49.53%. CONCLUSION: According to the X-ray diffraction data, the three-dimensional structure of BpEctD from B. pseudofirmus OF4 soon will be analyzed, and it will provide insights into the biochemical properties of ectoine hydroxylase.

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.

Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry.

Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.