ALBERT

Description: In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in k cat . It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in k cat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield.

Description: After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N -glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N -glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.

Description: Neisseria meningitidis serogroups A, B, C, Y, W135 and X are responsible for most cases of meningococcal meningitis. Neisseria meningitidis serogroup X has recently emerged as a contributor to outbreaks of disease in Africa, but there is currently no vaccine against serogroup X. Understanding of the biosynthesis of the serogroup X capsular polysaccharide would provide useful tools for vaccine production. The serogroup X polysaccharide is a homopolymer of (α1-〉4)-linked N -acetylglucosamine (GlcNAc)-1-phosphate. It has been shown that the gene cluster xcb ABC encodes synthesis of this polysaccharide. The xcbA gene product has significant homology with sac B, which is responsible for synthesis of the Neisseria serogroup A capsular polysaccharide, an (α1-〉6)- N -acetylmannosamine-1-phosphate homopolymer. The xcbA protein also shares homology with the catalytic domain of human N -acetylglucosamine-1-phosphoryltransferase, a key enzyme in the mannose-6-phosphate receptor pathway. In this study, we show that xcbA in the appropriate background is sufficient for the synthesis of N. meningitidis serogroup X polysaccharide. By ELISA we detected polysaccharide in fractions of Escherichia coli expressing the xcbA gene. We isolated polysaccharide from an E. coli strain expressing XcbA and demonstrated that this polysaccharide has a 13 C-NMR spectrum identical to that of polysaccharide isolated from N. meningitidis Group X. We also demonstrate that the purified XcbA protein is an N -acetylglucosamine-1-phosphotransferase that transfers N -acetylglucosamine-1-phosphate from UDP-GlcNAc to the 4-hydroxyl of an N -acetylglucosamine-1-phosphate oligosaccharide. Oligosaccharides fluorescently labeled at the aglycon are extended by XcbA only after the 4-phosphate occupying the non-reducing GlcNAc has been removed. The minimum size of fluorescent acceptors is a trisaccharide.

Description: The invasion of host cells by the intracellular protozoan Trypanosoma cruzi requires interactions with host cell molecules, and the replication of the parasite requires escape from a parasitophorous vacuole into the host cell cytosol. Galectin-3, a member of β-galactosidase-binding lectin family, has numerous extracellular and intracellular functions. In this study, we investigated the role of galectin-3 during the invasion and intracellular trafficking of T. cruzi extracellular amastigotes (EAs). Endogenous galectin-3 from mouse peritoneal macrophages accumulated around the pathogen during cell invasion by EAs. In addition, galectin-3 accumulated around parasites after their escape from the parasitophorous vacuole. Thus, galectin-3 behaved as a novel marker of phagolysosome lysis during the infection of host cells by T. cruzi .

Description: Neisseria meningitidis ( Nm ) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is Nm X for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of Nm X called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the Nm X capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with Nm X-specific antibodies. Moreover, the chemical identity of in vitro produced Nm X polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the Nm X CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.

Description: Glycoside hydrolase family 42 (GH42) includes β-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different β- d -galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite –1 to be key specificity determinants. Bga42A thus prefers the β1-3-galactosidic linkage from human milk and other β1-3- and β1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galβ1-4Galβ1-4Glc) as well as 4-galactobiose (Galβ1-4Gal) and 4-galactotriose (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside-rich gut niche and emphasize the importance and diversity of β-galactoside metabolism in probiotic bifidobacteria.

Description: Human milk oligosaccharides (HMOs) have been paid much attention due to their beneficial effects observed in vitro, e.g., prebiotic, anti-infective and anti-inflammatory properties. However, in vivo investigations with regard to HMO metabolism and functions are rare. The few data available indicate that HMOs are absorbed to a low extent and excreted via urine without noteworthy modifications, whereas the major proportion reaches infant's colon undigested. Via intrinsic 13 C-labeling of HMOs during their biosynthesis in the mammary gland of 10 lactating women, we were able to follow the fate of 13 C-labeled oligosaccharides (OSs) from their secretion in milk to the excretion in the urine of their breastfed infants. To a certain extent, we could therefore discriminate between original HMOs and non-labeled OSs derived from degradation of HMOs or endogenous glycoconjugates. By means of our novel, rapid, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based approach, we found a homogeneous time pattern of isotopomer enrichment in milk among all subjects and between single OS species. In contrast, the time curves from infants' urine varied strongly between individuals and OS species, though the overall MALDI-TOF MS profile resembled those of the mothers' milk. Our data suggest that neutral HMOs might be processed and/or utilized differentially after or upon absorption from the gut, as deduced from their structure-dependent variation in the extent of tracer enrichment and in the retention times in infant's organism. This sheds new light on the role of HMOs within infant's body, beyond the intestine and its microbiota alone.

Description: Our previous studies showed that mouse β4-galactosyltransferase 5 (β4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the β4GalT5 gene. We isolated a stable clone named E5 whose β4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous β4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the β4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by β4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Description: O-glycans are suitable targets as novel and useful tumor markers. The structures of O-glycans in human sera from four healthy controls were precisely analyzed to obtain the reference O-glycan database. O-glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine and identified using two-dimensional mapping, enzymatic digestion and mass spectrometry (MS) together with methanolysis and the use of newly synthesized sulfated oligosaccharides as standards. O-glycans, present at more than 0.01% of the total O-glycans, were analyzed, and 18 kinds of acidic and 2 kinds of neutral glycans were identified. NeuAcα2-3Galβ1-3 N -acetylgalactosamine (GalNAc) (61–64%), NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAc (15–26%) and Galβ1-3GalNAc (6–14%) were major components while other sialylated glycans, Galβ1-3(NeuAcα2-6)GalNAc, Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAc and NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAc were relatively minor components, accounting for ~1–2%. Very minor glycans accounting for ~0.01–0.1% of the total include (i) the neutral glycan, Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc, (ii) sialylated glycans, having sialyl Tn antigen, agalacto and trisialylated structures, (iii) fucosylated glycans forming blood type H antigen, blood type A antigen, blood type B antigen, Lewis X antigen and sialyl Lewis X antigen and (iv) sulfated glycans, having 6-sulfo and 3'-sulfo structures. Two kinds of clinically applied tumor markers namely sialyl Tn antigen and sialyl Lewis X antigen in healthy controls sera were revealed to be present at ~0.1–0.2% of the total. However, other markers such as CA19-9 and DU-PAN-2 were not found, suggesting the relative amounts of these glycans to be 〈0.01%. These detailed O-glycan profiles will help to find novel carbohydrate tumor markers.

Description: The repeat unit structure of the K2 capsule from an extensively antibiotic-resistant Acinetobacter baumannii global clone 2 (GC2) strain was determined. The oligosaccharide contains three simple sugars, d -glucopyranose, d -galatopyranose and N -acetyl- d -galactosamine, and the complex sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy- l - glycero - l - manno -non-2-ulosonic acid (Pse5Ac7Ac or pseudaminic acid), which has not previously been reported in any A. baumannii capsule. The strain was found to carry all the genes required for the synthesis of the sugars and construction of the K2 structure. The linkages catalyzed by the initiating transferase, three glycosyltransferases and the Wzy polymerase were also predicted. Examination of publicly available A. baumannii genome sequences revealed that the same gene cluster, KL2, often occurs in extensively antibiotic-resistant GC2 isolates and in further strain types. The gene module responsible for the synthesis of pseudaminic acid was also detected in four other K loci. A related module including genes for an acylated relative of pseudaminic acid was also found in two new KL types. A polymerase chain reaction scheme was developed to detect all modules containing genes for sugars based on pseudaminic acid and to specifically detect KL2.

Description: The cell wall peptidoglycan (PG) of Burkholderia cenocepacia , an opportunistic pathogen, has not yet been characterized. However, the B. cenocepacia genome contains homologs of genes encoding PG biosynthetic functions in other bacteria. PG biosynthesis involves the formation of the undecaprenyl-pyrophosphate-linked N-acetyl glucosamine- N -acetyl muramic acid-pentapeptide, known as lipid II, which is built on the cytosolic face of the cell membrane. Lipid II is then translocated across the membrane and its glycopeptide moiety becomes incorporated into the growing cell wall mesh; this translocation step is critical to PG synthesis. We have investigated candidate flippase homologs of the MurJ family in B. cenocepacia . Our results show that BCAL2764, herein referred to as murJ Bc , is indispensable for viability. Viable B. cenocepacia could only be obtained through a conditional mutagenesis strategy by placing murJ Bc under the control of a rhamnose-inducible promoter. Under rhamnose depletion, the conditional strain stopped growing and individual cells displayed morphological abnormalities consistent with a defect in PG synthesis. Bacterial cells unable to express MurJ Bc underwent cell lysis, while partial MurJ Bc depletion sensitized the mutant to the action of β-lactam antibiotics. Depletion of MurJ Bc caused accumulation of PG precursors consistent with the notion that this protein plays a role in lipid II flipping to the periplasmic compartment. Reciprocal complementation experiments of conditional murJ mutants in B. cenocepacia and Escherichia coli with plasmids expressing MurJ from each strain indicated that MurJ Bc and MurJ Ec are functional homologs. Together, our results are consistent with the notion that MurJ Bc is a PG lipid II flippase in B. cenocepacia .

Description: Roe deer ( Capreolus capreolus ) are seasonal breeders and cyclic structural changes of roe bucks' testis come along with a totally arrested (winter) and a highly activated spermatogenesis (summer). For this reason, roe buck represents an interesting model to study general mechanisms of initiation and termination of spermatogenesis. We investigated if polysialic acid (polySia)—a linear homopolymer of α2,8-linked sialic acids, which could act as a negative regulator of cell–cell adhesion—might be involved in the activation and/or inactivation of spermatogenesis. To address this point, testis samples of adult male roe deer were collected at different time point of the year. Intriguingly, we observed that polySia attached to the neural cell adhesion molecule was enhanced during the onset of spermatogenesis in April. In addition, polySia was highly expressed in December. Predominantly, polySia was detectable between Sertoli cells and spermatogonia in the basal regions of testicular tubules and in the adluminal part of Sertoli cells. Interestingly, similar polySia distributions were observed during early testis development of other mammalians when gonocytes (pre-spermatogonia) and Sertoli cells represent the only cell populations in tubuli seminiferi . Thus, polySia is expressed during key steps of the "on/off mechanisms" of spermatogenesis and might represent one mediator of the interaction and communication between Sertoli cells and germ cell precursors.

Description: O -Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2- O and/or 3- O positions of the xylopyranosyl (Xyl p ) units, but the distribution of different O -acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O -acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7 , irx9-1 , irx10 , irx14 and gux1gux2 . The relative contents of the Xyl p structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xyl p units constituted 44 and 6% of the AcGX backbone, respectively; while (4- O -methyl)-glucopyranosyluronic acid (1 -〉 2)-linked Xyl p units, most of which also carry 3 -O -acetylation, represented 13%. The DA was decreased in irx7 , irx9-1 and irx14 due to the decrease in monoacetylation (2- O and 3- O ), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2 , but monoacetylation was nonetheless elevated in gux1gux2 . This indicates that acetylation occurs after addition of Glc p A to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xyl p unit.

Description: Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell–cell interactions. To date, the majority of data gathered on glycan expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.

Description: Hypertrophy is central to several heart diseases; however, not much is known about the role of glycosphingolipids (GSLs) in this phenotype. Since GSLs have been accorded several physiological functions, we sought to determine whether these compounds affect cardiac hypertrophy. By using a rat cardiomyoblast cell line, H9c2 cells and cultured primary neonatal rat cardiomyocytes, we have determined the effects of GSLs on hypertrophy. Our study comprises (a) measurement of [ 3 H]-leucine incorporation into protein, (b) measurement of cell size and morphology by immunofluorescence microscopy and (c) real-time quantitative mRNA expression assay for atrial natriuretic peptide and brain natriuretic peptide. Phenylephrine (PE), a well-established agonist of cardiac hypertrophy, served as a positive control in these studies. Subsequently, mechanistic studies were performed to explore the involvement of various signaling transduction pathways that may contribute to hypertrophy in these cardiomyocytes. We observed that lactosylceramide specifically exerted a concentration- (50–100 µM) and time (48 h)-dependent increase in hypertrophy in cardiomyocytes but not a library of other structurally related GSLs. Further, in cardiomyocytes, LacCer generated reactive oxygen species, stimulated the phosphorylation of p44 mitogen activated protein kinase and protein kinase-C, and enhanced c-jun and c-fos expression, ultimately leading to hypertrophy. In summary, we report here that LacCer specifically induces hypertrophy in cardiomyocytes via an "oxygen-sensitive signal transduction pathway."

Description: Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The final products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efficacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively.

Description: Heparin-like saccharides play an essential role in binding to the fibroblast growth factor (FGF)-1 and to their membrane receptors fibroblast growth factor receptor forming a ternary complex that is responsible of the internalization of the signal, via the dimerization of the intracellular regions of the receptor. In this study, we report the binding affinities between five synthetic hexasaccharides with human FGF-1 obtained by surface plasmon resonance experiments, and compare with the induced mitogenic activity previously obtained. These five oligosaccharides differ in sulfation pattern and in sequence. We have previously demonstrated that all the five hexasaccharides have similar 3D structure of the backbone. Consequently, the differences in binding affinity should have their origin in the substitution pattern. Subsequently, the different capacity for induction of mitogenic activity can be, at least partially, explained from these binding affinities. Interestingly, one of the oligosaccharides lacking axially symmetry ( 3 ) was biologically inactive, whereas the other ( 2 ) was the most active. The difference between both compounds is the order of the FGF-binding motifs along the chain relative to the carbohydrate polarity. We can conclude that the directionality of the GAG chain is essential for the binding and subsequent activation. The relative biological activity of the compounds with regular substitution pattern can be inferred from their values of IC 50. Remarkably, the sulfate in position 6 of d -glucosamine was essential for the mitogenic activity but not for the interaction with FGF-1.

Description: Galectin-3 has been reported to regulate the functions of a number of immune cell types. We previously reported that galectin-3 is translocated to immunological synapses in T cells upon T-cell receptor engagement, where it associates with ALG-2-interacting protein X (Alix). Alix is known to coordinate with the endosomal sorting complex required for transport (ESCRT) to promote human immunodeficiency virus (HIV)-1 virion release. We hypothesized that galectin-3 plays a role in HIV-1 viral budding. Cotransfection of cells of the Jurkat T line with galectin-3 and HIV-1 plasmids resulted in increased HIV-1 budding, and suppression of galectin-3 expression by RNAi in Hut78 and primary CD4+ T cells led to reduced HIV-1 budding. We used immunofluorescence microscopy to observe the partial colocalization of galectin-3, Alix and Gag in HIV-1-infected cells. Results from co-immunoprecipitation experiments indicate that galectin-3 expression promotes Alix-Gag p6 association, whereas the results of Alix knockdown suggest that galectin-3 promotes HIV-1 budding through Alix. HIV-1 particles released from galectin-3-expressing cells acquire the galectin-3 protein in an Alix-dependent manner, with proteins primarily residing inside the virions. We also found that the galectin-3 N-terminal domain interacts with the proline-rich region of Alix. Collectively, these results suggest that endogenous galectin-3 facilitates HIV-1 budding by promoting the Alix-Gag p6 association.

Description: Besides regulating multiple physiological processes such as cellular growth, migration and differentiation, glycosaminoglycans (GAGs) are also largely explored in the global market as clinical or nutraceutical agents. Generally, these physiological and biomedical events are ultimately triggered by the quality and nature of molecular interactions between GAGs and functional proteins. Nuclear magnetic resonance (NMR) spectroscopy participates significantly in the investigations of most carbohydrate–protein interactions. The present review covers in a very systematic and comprehensive way the major NMR-based studies of GAG–protein interactions undertaken within the last 3 years. The main objective herein is, besides revisiting the results from these recent works, to present for the current glycomic era an overview about the underlying biological inferences drawn from these results.

Description: The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and K D values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (UA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton–proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between 1 C 4 chair and 2 S o skew-boat structure of the internal iduronic acid residue is preserved.

Description: Previous studies indicate that snake venom contains glycan-binding proteins (GBPs), although the binding specificity and biological activities of many of these GBPs is unclear. Here we report our studies on the glycan binding specificity and activities of galatrox, a Bothrops atrox snake venom-derived GBP. Glycan microarray analysis indicates that galatrox binds most strongly to glycans expressing N -acetyllactosamine (LacNAc), with a significant preference for Galβ1-4GlcNAcβ over Galβ1-3GlcNAcβ compounds. Galatrox also bound immobilized laminin, a LacNAc-dense extracellular matrix component, suggesting that this GBP can bind LacNAc-bearing glycoproteins. As several endogenous mammalian GBPs utilize a similar binding LacNAc binding preference to regulate neutrophil and monocyte activity, we hypothesized that galatrox may mediate B. atrox toxicity through regulation of leukocyte activity. Indeed, galatrox bound neutrophils and promoted leukocyte chemotaxis in a carbohydrate-dependent manner. Similarly, galatrox administration into the mouse peritoneal cavity induced significant neutrophil migration and the release of pro-inflammatory cytokines IL-1α and IL-6. Exposure of bone marrow-derived macrophages to galatrox induced generation of pro-inflammatory mediators IL-6, TNF-α, and keratinocyte-derived chemokine. This signaling by galatrox was mediated via its carbohydrate recognition domain by activation of the TLR4-mediated MyD88-dependent signaling pathway. These results indicate that galatrox has pro-inflammatory activity through its interaction with LacNAc-bearing glycans on neutrophils, macrophages and extracellular matrix proteins and induce the release of pro-inflammatory mediators.

Description: To fulfill their potential, leukocytes must be able to exit the vasculature and reach the site of inflammation within the tissue. This process of leukocyte extravasation is a tightly regulated sequence of events that is governed by a host of cell adhesion molecules, cytokines, chemokines and lipid mediators. Of major importance to this process and the function of many of the proteins and lipids involved is the posttranslational modification of these moieties by glycosylation. The glycosylation process is coordinated by multiple enzymes that add and remove saccharides to/from glycan structures on proteins and lipids, resulting in a unique molecular signature that affords specificity to the molecules involved in leukocyte recruitment. This review will discuss how glycosylation impacts the function of these key molecules involved in the recruitment of leukocytes during inflammation and the function of specific lectins (carbohydrate-binding proteins) that have a role in leukocyte trafficking.

Description: Sulfated polysaccharides of brown algae (fucoidans) attract great attention due to their high and strongly diversified biological activity. This review summarizes recent data on the structural variability of these polysaccharides and reports their anti- and proangiogenic properties. Recent publications have revealed that fucoidans isolated from different algal species may differ considerably in the structures of their backbones and branches, in both monosaccharide composition and sulfate content. It was found that the degree of sulfation significantly influences the biological properties of fucoidans. Additionally, fucoidan action in angiogenesis is highly dependent on molecular weight: antiangiogenic activity is connected with the high-molecular weight of polysaccharide molecules, whereas the low-molecular-weight fractions may act as proangiogenic agents. The influence of other fine structural details of fucoidans on angiogenesis remains to be established.

Description: Abnormal glycosylation is a typical hallmark of the transition from healthy to neoplastic tissues. Although the importance of glycans and glycan-binding proteins in cancer-related processes such as tumor cell adhesion, migration, metastasis and immune escape has been largely appreciated, our awareness of the impact of lectin-glycan recognition in tumor vascularization is relatively new. Regulated glycosylation can influence vascular biology by controlling trafficking, endocytosis and signaling of endothelial cell (EC) receptors including vascular endothelial growth factor receptors, platelet EC adhesion molecule, Notch and integrins. In addition, glycans may control angiogenesis by regulating migration of endothelial tip cells and influencing EC survival and vascular permeability. Recent evidence indicated that changes in the EC surface glycome may also serve "on-and-off" switches that control galectin binding to signaling receptors by displaying or masking-specific glycan epitopes. These glycosylation-dependent lectin-receptor interactions can link tumor hypoxia to EC signaling and control tumor sensitivity to anti-angiogenic treatment.

Description: Ocular neovascularization can affect almost all the tissues of the eye: the cornea, the iris, the retina, and the choroid. Pathological neovascularization is the underlying cause of vision loss in common ocular conditions such as diabetic retinopathy, retinopathy of prematurity and age-related macular neovascularization. Glycosylation is the most common covalent posttranslational modification of proteins in mammalian cells. A growing body of evidence demonstrates that glycosylation influences the process of angiogenesis and impacts activation, proliferation, and migration of endothelial cells as well as the interaction of angiogenic endothelial cells with other cell types necessary to form blood vessels. Recent studies have provided evidence that members of the galectin class of β-galactoside-binding proteins modulate angiogenesis by novel carbohydrate-based recognition systems involving interactions between glycans of angiogenic cell surface receptors and galectins. This review discusses the significance of glycosylation and the role of galectins in the pathogenesis of ocular neovascularization.

Description: This study is focused on the analysis and mutagenesis of β-mannosidase from Bacteroides thetaiotaomicron with the aim of broadening its substrate specificity to 2-acetamido-2-deoxy-β- d -mannopyranosyl (β-ManNAc) derivatives. Various conformations ( 4 C 1 , 4 H 5 and 1 S 5 ) of native and modified ligands were docked to the binding site of the protein to determine the most suitable conformation of sugars for further hydrolysis. Key amino acid residues were mutated in silico focusing on stabilizing the acetamido group of β-ManNAc as well as forming the oxazoline intermediate needed for hydrolysis. The results of large set of 5 ns molecular dynamic simulations showed that the majority of the active site residues are involved in substrate interaction and do not exhibit a higher flexibility except for Asn178. Mutations of Asn178 to alanine and Asp199 to serine could lead to a stabilization of the acetamido group in the binding site. So far, in vitro mutagenesis and the screen of a large variety of biological sources were unable to extend β-mannosidase's activity to include β-ManNAc derivatives.

Description: The vascular endothelial glycocalyx contains several anionic sugars, one of which is a sialic acid attached to both N - and O -glycans. Platelet endothelial cell adhesion molecule (PECAM), a member of the Ig superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis and angiogenesis, has recently been shown to recognize α2,6-sialic acid. In endothelial cells that lack α2,6-sialic acid because of sialyltransferase ST6Gal I deficiency, impairment of the homophilic PECAM interaction and PECAM-dependent cell survival signaling is observed. In this review, we will introduce part of the biological role of PECAM, and discuss how the lectin activity of PECAM is related to angiogenesis.

Description: Glycosaminoglycans (GAGs) interact with many proteins to regulate processes such as hemostasis, cell adhesion, growth and differentiation and viral infection. Yet, majority of these interactions remain poorly understood at a molecular level. A major reason for this state is the phenomenal structural diversity of GAGs, which has precluded analysis of specificity of their interactions. We had earlier presented a computational protocol for predicting "high-specificity" GAG sequences based on combinatorial virtual library screening (CVLS) technology. In this work, we expand the robustness of this technology through rigorous studies of parameters affecting GAG recognition of proteins, especially antithrombin and thrombin. The CVLS approach involves automated construction of a virtual library of all possible oligosaccharide sequences (di- to octasaccharide) followed by a two-step selection strategy consisting of "affinity" (GOLD score) and "specificity" (consistency of binding) filters. We find that "specificity" features are optimally evaluated using 100 genetic algorithm experiments, 100,000 evolutions and variable docking radius from 10 Å (disaccharide) to 14 Å (hexasaccharide). The results highlight critical interactions in H/HS oligosaccharides that govern specificity. Application of CVLS technology to the antithrombin–heparin system indicates that the minimal "specificity" element is the GlcA p (1 -〉 4)GlcN p 2S3S disaccharide of heparin. The CVLS technology affords a simple, intuitive framework for the design of longer GAG sequences that can exhibit high "specificity" without resorting to exhaustive screening of millions of theoretical sequences.

Description: Vascular endothelial cells, in addition to many other mammalian cell types, express proteins that are highly modified with mucin-type O-glycosylation, a specific type of glycosylation that begins with the addition of an N -acetylgalactosamine moiety to serine or threonine residues within the peptide backbone. Recently, it has become evident that O-glycosylation governs the separation of blood and lymphatic vessels throughout life and plays a critical role in maintaining vascular integrity in specific tissues such as the brain and lymph node. This mini-review seeks to highlight some of these recent advances regarding in vivo functions of mucin-type O -glycans.

Description: The N-terminal domain of the Epa1p adhesin from Candida glabrata (N-Epa1p) is a calcium-dependent lectin, which confers the opportunistic yeast the ability to adhere to human epithelial cells. This lectin domain is able to interact with galactosides and, more precisely, with glycan molecules containing the Galβ-1,3-GalNAc disaccharide, also known as the T-antigen. Based on the crystallographic structure of the N-Epa1p domain and the role of the variable loop CBL2 in glycan binding, saturation mutagenesis on some residues of the CBL2 loop was used to increase the binding affinity of N-Epa1p for fibronectin, which was selected as a model of a human glycoprotein. Two adhesin mutants, E227A and Y228W, with improved binding features were obtained. More importantly, a glycan array screening revealed that single-point mutations in the CBL2 could produce significant changes in the carbohydrate specificity of the protein. In particular, lectin molecules were generated with a high affinity for sulfated glycans, which may find an application as molecular probes for the identification of 6-sulfogalactose containing glycans and glycoconjugates.

Description: Hyaluronic acid (HA) is an ubiquitous nonsulfated glycosaminoglycan of the extracellular matrix in all mammalian connective tissues. Along with the age growth, HA will lose its N -acetyl groups in vivo; however, the significance of this physiological process remains largely unknown. Herein, three highly N -deacetylated HAs (dHAs), dHA-5 kDa (Mw: 5 kDa, DD: 100%), dHA-16 kDa (Mw: 16 kDa, DD: 94%) and dHA-110 kDa (Mw: 110 kDa, DD: 72%), were generated after hydrazinolysis. Their capability in the activation of antigen-presenting cells (APCs) was compared with that of their respective HAs. Our results demonstrated that both HAs and dHAs could activate the nuclear factor-kappa B (NF-B) transcription factor in APCs and induced cytokine production through the Toll-like receptor (TLR)/MyD88 pathway. Notably, the capacity of dHAs in cytokine induction was much lower than that of HAs. In addition, the TLR-2 pathway was much involved following the appearance of zwitterionic motifs in dHAs. Thus, our findings highlight that N-deacetylation renders HA divergences in immune response, which might be implicated in age-induced functional change in endogenous glycosaminoglycans due to the structural modification in vivo.

Description: Under normal conditions, platelets do not interact with blood vessel walls; however, upon activation, platelets firmly attach to endothelial cells. Communication between platelets and endothelial cells during the normal or activated state takes place at multiple levels. Cross-talk may occur over a distance via transient interactions or through receptor-mediated cell–cell adhesion. Platelets may release or transfer substances that affect endothelial cell function and vice versa . Excessive dialogue between platelets and the endothelium exists in several disease states as a causative factor and/or as a consequence of the disease process. Glycans are covalent assemblies of sugars that exist in either free form or in covalent complexes with proteins or lipids. Among other functions, glycans confer stability to the proteins to which they are attached, play key roles in signal transduction and control cell development and differentiation. Glycans not only influence the structure and function of hemostatic molecules but are also increasingly recognized as key molecules regulating platelet-endothelial interactions. The present review outlines the current knowledge regarding glycan-mediated interactions between platelets and endothelial cells and their role in physiopathological processes.

Description: Trehalose dimycolate, an unusual glycolipid in the outer membrane of Mycobacterium tuberculosis , stimulates macrophages by binding to the macrophage receptor mincle. This stimulation plays an important role both in infection by mycobacteria and in the use of derivatives of mycobacteria as adjuvants to enhance the immune response. The mechanism of trehalose dimycolate binding to the C-type carbohydrate-recognition domain in human mincle has been investigated using a series of synthetic analogs of trehalose dimycolate and site-directed mutagenesis of the human protein. The results support a mechanism of binding acylated trehalose derivatives to human mincle that is very similar to the mechanism of binding to bovine mincle, in which one glucose residue in the trehalose headgroup of the glycolipid is ligated to the principle Ca 2+ -binding site in the carbohydrate-recognition domain, with specificity for the disaccharide resulting from interactions with the second glucose residue. Acyl chains attached to the 6-OH groups of trehalose enhance affinity, with the affinity dependent on the length of the acyl chains and the presence of a hydrophobic groove adjacent to the sugar-binding sites. The results indicate that the available crystal structure of the carbohydrate-recognition domain of human mincle is unlikely to be in a fully active conformation. Instead, the ligand-binding conformation probably resembles closely the structure observed for bovine mincle in complex with trehalose. These studies provide a basis for targeting human mincle as a means of inhibiting interactions with mycobacteria and as an approach to harnessing the ability of mincle to stimulate the immune response.

Description: Although mucin O-glycosylation of sputum from individuals suffering from cystic fibrosis (CF) is known to be altered relative to their unaffected counterparts, protein N-glycosylation of CF sputum remains structurally and functionally under-characterized. We report the first N -glycome of soluble proteins in sputum derived from five CF patients, two pathogen-free and two pathogen-infected/colonized non-CF individuals suffering from other pulmonary conditions. N -Glycans were profiled using porous graphitized carbon-liquid chromatography-negative ion-tandem mass spectrometry following enzymatic release from sputum proteins. The composition, topology and linkage isomers of 68 N -glycans were characterized and relatively quantified. Recurring structural features in all sputum N -glycomes were terminal α2,6-sialylation, α1,6-core fucosylation, β1,4-bisecting GlcNAcylation and compositions indicating paucimannosylation. Despite covering different genotypes, age, gender and microbial flora, the sputum N -glycomes showed little interpatient and longitudinal variation within CF patients. Comparative N -glycome analysis between inter-patient group revealed that lung infection/colonization, in general, extensively enriches the CF sputum N-glycosylation phenotype with paucimannose with simultaneous over-sialylation/fucosylation and under-bisecting GlcNAcylation of complex/hybrid N -glycans. In contrast, the sputum from CF patients had only slightly increased abundance of paucimannose N -glycans relative to pathogen-infected/colonized non-CF individuals. Similar to mucin O-glycosylation, protein N-glycosylation appears to be regulated primarily as a secondary effect of bacterial infection and inflammation rather than the CF pathogenesis in sputum. This study provides new structural N- glycan information to help understand the complex cellular and molecular environment of the CF affected respiratory tract.

Description: N -acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

Description: The extent of the role of N- linked glycans ( N- glycans) in shielding influenza A hemagglutinin (HA) against host antibodies has proved controversial, with different authors making widely different assumptions. One common assumption is that N -glycans physically shield surface residues that are near to glycosylation sites, thereby preventing antibodies from binding to them. However, it is unclear, from existing experimental evidence, whether antibodies that bind close to N- glycans are a rare or commonplace feature of human herd immune responses to influenza AHA. The aim of this paper is to present a computational analysis of mutations in the vicinity of N- glycans that will facilitate a better understanding of their protective role. We identify, from an analysis of over 6000 influenza A H3N2 sequences, a set of residues adjacent to N - glycosylation sites that are highly likely to be involved in antigenic escape from host antibodies. Fifteen of these residues occur within 10 Å of an N - glycosylation site. Hence, we conclude that it is relatively common for antibodies to bind in close proximity to N- glycans on the surface ofHA, with any shielding effect largely attributable to the inability of host antibodies to bind across an N- glycan attachment site, rather than to the physical masking of neighboring residues.

Description: Intestinal T-cell receptor-bearing intraepithelial lymphocytes ( IELs) play a multifaceted role in maintaining mucosal homeostasis. In order to investigate the relationship between O-glycosylation and inflammation, we carried out an in-depth mass spectrometric comparison of the intestinal O-glycosylation profile of mice lacking IELs (TCR –/– ) and of their wild-type (WT) littermates. A total of 69 nonsulfated and 59 sulfated compositional types of O -glycans were identified in the small intestine and colon of TCR –/– and WT mice. Our results demonstrated structural differences in intestinal glycosylation in TCR –/– mice compared with WT littermates. TCR –/– colons contained a lower proportion of core-2 structures and an increased proportion of core-1 structures whereas TCR –/– small intestines had a decreased percentage of core-3 structures. The glycan antennae in TCR –/– colon and small intestine showed altered structural diversity compared with WT mice. There were significant differences in the sialylated species between the TCR –/– and WT mice with the sialylated Tn antigen found exclusively in the TCR –/– small intestine, whereas the sulfation pattern remained mostly unchanged. These findings provide novel molecular insights underpinning the role of IELs in maintaining gut homeostasis.

Description: Recombinant human interferon β-1a (IFN-β-1a) is extensively used as the first-line treatment of relapsing forms of multiple sclerosis. Its glycosylation is recognized as having a complex impact on a wide range of molecule characteristics and functions. The present study reports the enrichment of IFN-β-1a glycoforms and their physicochemical and biological characterization by means of electrospray ionization-mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays (antiproliferative, antiviral, immunomodulatory and reporter gene assay). The glycoforms were fractionated by means of cation-exchange chromatography using recombinant IFN-β-1a derived from Chinese Hamster Ovary cell culture as starting material. The obtained fractions contained bi- and higher-antennarity glycans as described in the European Pharmacopoeia monograph ( Nr. 1639E, Interferon beta 1a concentrated solution ). The in vitro bioassay responses revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms have biological activity and play a role in modulating the overall IFN-β biological activity with higher-antennarity glycoforms being able to better sustain IFN-β-1a bioactivity over time. These data indicate the role of IFN-β-1a glycosylation in vivo and shed new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.

Description: In this study, we selected 181 nematode glycogenes that are orthologous to human glycogenes and examined their RNAi phenotypes. The results are deposited in the Caenorhabditis elegans Glycogene Database (CGGDB) at AIST, Tsukuba, Japan. The most prominent RNAi phenotypes observed are disruptions of cell cycle progression in germline mitosis/meiosis and in early embryonic cell mitosis. Along with the previously reported roles of chondroitin proteoglycans, glycosphingolipids and GPI-anchored proteins in cell cycle progression, we show for the first time that the inhibition of the functions of N -glycan synthesis genes (cytoplasmic alg genes) resulted in abnormal germline formation, ER stress and small body size phenotypes. The results provide additional information on the roles of glycoconjugates in the cell cycle progression mechanisms of germline and embryonic cells.

Description: Hybrid quantum mechanics/molecular mechanics calculations were used to study the catalytic mechanism of the retaining human α -(1,3)-galactosyltransferase (GTB WT ) and its E303C mutant (GTB E303C ). Both backside (via covalent glycosyl-enzyme intermediate, CGEI) and frontside S N i-like mechanisms (via oxocarbenium-ion intermediate, OCII) were investigated. The calculations suggest that both mechanisms are feasible in the enzymatic catalysis. The nucleophilic attack of the acceptor substrate to the anomeric carbon of OCII is the rate-determining step with an overall reaction barrier ( E = 19.5 kcal mol –1 ) in agreement with an experimental rate constant ( k cat = 5.1 s –1 ). A calculated α-secondary kinetic isotope effect (α-KIE) of 1.27 (GTB WT ) and 1.26 (GTB E303C ) predicts dissociative character of the transition state in agreement with experimentally measured α-KIE of other retaining glycosyltransferases. Remarkably, stable CGEI in GTB E303C compared with its counterpart in GTB WT may explain why the CGEI has been detected by mass spectrometry only in GTB E303C ( Soya N, Fang Y, Palcic MM, Klassen JS. 2011 . Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases. Glycobiology , 21: 547–552).

Description: Invasive aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Diagnosis of this infection frequently employs detection of the circulating galactomannan in the patient serum using enzyme immunoassay (EIA), a highly sensitive and specific system. Although there are many structural studies of the galactomannan of Aspergillus fumigatus , some inconsistencies are present in these results. In this study, to clarify the relationship between the growth conditions and structure of the galactomannans, we cultured A. fumigatus using two distinct yeast/fungal cultivation media, i.e. the yeast extract–peptone–dextrose (YPD) medium and yeast nitrogen base (YNB) medium. Galactomannans prepared from the resulting culture supernatants were structurally characterized by 1 H and 13 C nuclear magnetic resonance, methylation analysis, acetolysis and α-mannosidase degradation. These assays revealed that the galactomannan from the YPD cultivation had short β-1,5-linked galactofuranose (Gal f ) oligosaccharide chains in both the O- and N-linked carbohydrate moieties, while the galactomannan from the YNB cultivation incorporated long Gal f oligosaccharide chains. The galactomannans derived from the two culture conditions significantly differed in reactivity based on the EIA diagnostic system. We also demonstrated the presence of a novel Gal f -containing branched oligosaccharide in the O-linked moiety.

Description: Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.

Description: Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted proteoglycans (PGs) displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.

Description: N -Acetylneuraminate lyase synthase (NeuB; E.C. 2.5.1.56) is a key enzyme in pathogenic microorganisms for producing N -acetylneuraminic acid through the irreversible condensation of N -acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N -acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 ( Il NeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40°C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-β-synthase domain (cystathionine-β-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I–X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (〉700 residues) NeuB, such as that existing in domain architectures 3 and 4.

Description: Most currently available glycan structure databases use their own proprietary structure representation schema and contain numerous annotation errors. These cause problems when glycan databases are used for the annotation or mining of data generated in the laboratory. Due to the complexity of glycan structures, curating these databases is often a tedious and labor-intensive process. However, rigorously validating glycan structures can be made easier with a curation workflow that incorporates a structure-matching algorithm that compares candidate glycans to a canonical tree that embodies structural features consistent with established mechanisms for the biosynthesis of a particular class of glycans. To this end, we have implemented Qrator, a web-based application that uses a combination of external literature and database references, user annotations and canonical trees to assist and guide researchers in making informed decisions while curating glycans. Using this application, we have started the curation of large numbers of N -glycans, O -glycans and glycosphingolipids. Our curation workflow allows creating and extending canonical trees for these classes of glycans, which have subsequently been used to improve the curation workflow.

Description: Galectin-8 (gal-8) is a " tandem-repeat " - type galectin, containing two carbohydrate recognition domains connected by a linker peptide. gal-8 is expressed both in the cytoplasm and nucleus in vascular endothelial cells (ECs) from normal and tumor-associated blood vessels, and in lymphatic endothelial cells. Herein, we describe a novel role for gal-8 in the regulation of vascular and lymphatic angiogenesis and provide evidence of its critical implications in tumor biology. Functional assays revealed central roles for gal-8 in the control of capillary-tube formation, EC migration and in vivo angiogenesis. So far, two endothelial ligands have been described for gal-8, namely podoplanin in lymphatic vessels and CD166 (ALCAM, activated leukocyte cell adhesion molecule) in vascular ECs. Other related gal-8 functions are also summarized here, including cell adhesion and migration, which collectively demonstrate the multi-functionality of this complex lectin. Thus, gal-8 is an important component of the angiogenesis network, and an essential molecule in the extracellular matrix by providing molecular anchoring to this surrounding matrix. The implications of gal-8 in tumor angiogenesis remain to be further explored, but it is exciting to speculate that modulating gal-8-glycan interactions could be used to block lymphatic-vascular connections vital for metastasis.

Description: Galectin-3 is a member of the family of β-galactoside-binding lectins characterized by evolutionarily conserved sequences defined by structural similarities in their carbohydrate-recognition domains. Galectin-3 is a unique, chimeric protein consisting of three distinct structural motifs: (i) a short NH 2 terminal domain containing a serine phosphorylation site; (ii) a repetitive proline-rich collagen-α-like sequence cleavable by matrix metalloproteases; and (iii) a globular COOH-terminal domain containing a carbohydrate-binding motif and an NWGR anti-death motif. It is ubiquitously expressed and has diverse biological functions depending on its subcellular localization. Galectin-3 is mainly found in the cytoplasm, also seen in the nucleus and can be secreted by non-classical, secretory pathways. In general, secreted galectin-3 mediates cell migration, cell adhesion and cell–cell interactions through the binding with high affinity to galactose-containing glycoproteins on the cell surface. Cytoplasmic galectin-3 exhibits anti-apoptotic activity and regulates several signal transduction pathways, whereas nuclear galectin-3 has been associated with pre-mRNA splicing and gene expression. Its unique chimeric structure enables it to interact with a plethora of ligands and modulate diverse functions such as cell growth, adhesion, migration, invasion, angiogenesis, immune function, apoptosis and endocytosis emphasizing its significance in the process of tumor progression. In this review, we have focused on the role of galectin-3 in tumor metastasis with special emphasis on angiogenesis.

Description: Prostate cancer is the second most common cause of cancer and the sixth leading cause of cancer death among men worldwide. While localized prostate cancer can be cured, advanced and metastatic prostate cancer remains a significant therapeutic challenge. Malignant transformation is associated with important modifications of the cellular glycosylation profile, and it is postulated that these changes have a considerable relevance for tumor biology. Metastasis is a multiphasic process that encompasses angiogenesis, the spread of tumor cells and their growth at distant sites from the primary tumor location. Recognition of glycoconjugates by galectins, among other lectins, plays a fundamental role in the metastatic spread, tumor immune escape and the neovascularization process. Particularly in prostate cancer, both carbohydrates and galectins have been implicated in many cellular processes such as proliferation, apoptosis, migration and invasion. However, a limited number of studies assessed their potential implications in the induction of metastasis in prostate cancer patients or in animal models. Moreover, the role of galectin–glycan interactions in vivo still remains poorly understood; concerted effort should thus be made in order to shed some light on this question. This review summarizes current evidence on both the expression and role of glycans and galectins in prostate cancer, particularly turning our attention to the angiogenic and metastatic processes.

Description: Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.

Description: Radiation therapy is a main stay in treating solid tumors and plays a significant role in definitive and adjuvant therapy. Unfortunately, local control remains a challenge, in which the success of radiotherapy is largely dictated by tumor hypoxia, DNA damage repair and the antitumor immune response. Extensive efforts have therefore been devoted to targeting the factors that attenuate tumor radiosensitivity, although with limited success. Mounting evidence suggests that tumor and endothelial cells may utilize galectin-1 (Gal-1) for protection against radiation through several mechanisms. Targeting Gal-1 in combination with radiotherapy provides an exciting approach to address several radiation-prohibitive mechanisms.

Description: Crystal structure determination of the lectin domain of MSMEG_3662 from Mycobacterium smegmatis and its complexes with mannose and methyl-α-mannose, the first effort of its kind on a mycobacterial lectin, reveals a structure very similar to β-prism II fold lectins from plant sources, but with extensive unprecedented domain swapping in dimer formation. The two subunits in a dimer often show small differences in structure, but the two domains, not always related by 2-fold symmetry, have the same structure. Each domain carries three sugar-binding sites, similar to those in plant lectins, one on each Greek key motif. The occurrence of β-prism II fold lectins in bacteria, with characteristics similar to those from plants, indicates that this family of lectins is of ancient origin and had evolved into a mature system before bacteria and plants diverged. In plants, the number of binding sites per domain varies between one and three, whereas the number is two in the recently reported lectin domains from Pseudomonas putida and Pseudomonas aeruginosa . An analysis of the sequences of the lectins and the lectin domains shows that the level of sequence similarity among the three Greek keys in each domain has a correlation with the number of binding sites in it. Furthermore, sequence conservation among the lectins from different species is the highest for that Greek key which carries a binding site in all of them. Thus, it would appear that carbohydrate binding influences the course of the evolution of the lectin.

Description: The growth of new blood vessels out of the pre-existing vasculature, i.e., angiogenesis, is executed by endothelial cells that normally form the inner lining of blood vessels. During angiogenesis, these cells exert different activities which require interactions with other cells and with the extracellular environment. It has become evident that this frequently involves galectins since the members of this protein family facilitate interactions between cells and/or glycoproteins via carbohydrate binding. In addition, they can regulate intracellular processes like signaling and splicing via non-carbohydrate interactions. We have previously reported on the role of galectin-1 and -9 in endothelial cell function. Here, we review the current knowledge of these two galectins in endothelial cell biology and angiogenesis. In addition, we describe the contribution of both galectins to other processes that involve the endothelium, including inflammation and coagulation. Finally, we discuss the challenges for future research in order to better understand how this "sweet" couple exerts its multifunctional activities within the vasculature.

Description: Aberrant protein glycosylation is known to be associated with the development of cancers. The aberrant glycans are produced by the combined actions of changed glycosylation enzymes, substrates and transporters in glycosylation synthesis pathways in cancer cells. To identify glycosylation enzymes associated with aggressive prostate cancer (PCa), we analyzed the difference in the expression of glycosyltransferase genes between aggressive and non-aggressive PCa. Three candidate genes encoding glycosyltransferases that were elevated in aggressive PCa were subsequently selected. The expression of the three candidates was then further evaluated in androgen-dependent (LNCaP) and androgen-independent (PC3) PCa cell lines. We found that the protein expression of one of the glycosyltransferases, α (1,6) fucosyltransferase (FUT8), was only detected in PC3 cells, but not in LNCaP cells. We further showed that FUT8 protein expression was elevated in metastatic PCa tissues compared to normal prostate tissues. In addition, using tissue microarrays, we found that FUT8 overexpression was statistically associated with PCa with a high Gleason score. Using PC3 and LNCaP cells as models, we found that FUT8 overexpression in LNCaP cells increased PCa cell migration, while loss of FUT8 in PC3 cells decreased cell motility. Our results suggest that FUT8 may be associated with aggressive PCa and thus is potentially useful for its prognosis.

Description: Tri- N -acetylchitotriosyl moranoline, (GlcNAc) 3 -M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-β-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem . 288:6072–6082). The findings prompted us to examine the interaction of di- N -acetylchitobiosyl moranoline, (GlcNAc) 2 -M, with a family GH19 chitinase from moss, Bryum coronatum ( Bc Chi19A). Thermal unfolding experiments using Bc Chi19A and the catalytic acid-deficient mutant ( Bc Chi19A-E61A) revealed that the transition temperature ( T m ) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc) 2 -M, while the chitin dimer, (GlcNAc) 2 , elevated T m only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc) 3 and (GlcNAc) 2 -M with Bc Chi19A-E61A were determined to be –5.2 and –6.6 kcal/mol, respectively, while (GlcNAc) 2 was found to interact with Bc Chi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using 15 N-labeled Bc Chi19A and Bc Chi19A-E61A revealed that both (GlcNAc) 2 and (GlcNAc) 2 -M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc) 2 -M is markedly stronger than that of (GlcNAc) 2 for both enzymes. However, (GlcNAc) 2 -M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by Bc Chi19A (IC 50 = 130–620 μM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc) 2 -M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc) 2 -M did not fit into the catalytic cleft (subsite –1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the Bc Chi19A-catalyzed hydrolysis.

Description: Despite advances in diagnosis and treatment, the overall outcomes for patients with brain tumors remain unpredictable. New prognostic markers are still needed to identify high-risk patients for whom the standard treatment has poor outcomes and would thus be well suited for more aggressive therapies. Neovascularization has long been implicated as a salient feature of glioma progression. In fact, high-grade gliomas are among the most vascular of all solid tumors, and vascular proliferation is a pathological hallmark of glioblastomas. Galectins are known to play important roles in cancer biology, including cancer cell migration, tumor immune escape or tumor angiogenesis. Moreover, galectins were reported to be involved in glioma progression. Given the key role of angiogenesis in brain tumors, the expression of galectins in tumor-associated endothelial cells (EC) and the implication of galectins in angiogenesis, the present review will focus on the expression of galectins in ECs of normal brain and brain tumors.

Description: Galectins (Gals), a family of mammalian lectins, play diverse roles under physiological and pathological conditions. Here, we analyzed the tandem-repeat Gal-8 synthesis, secretion and effects on the endothelium physiology. Gal-8M and Gal-8L isoforms were secreted under basal conditions by human microvascular endothelial cells (HMEC-1). However, expression and secretion of the Gal-8M isoform, but not Gal-8L, were increased in response to bacterial lipopolysaccharide (LPS) stimulus and returned to control values after LPS removal. Similarly, cell surface Gal-8 exposure was increased after stimulation with LPS. To evaluate Gal-8 effects on the endothelium physiology, HMEC-1 cells were incubated in the presence of recombinant Gal-8M. Pretreated HMEC-1 cells became proadhesive to human normal platelets, indicating that Gal-8 actually activates endothelial cells. This effect was specific for lectin activity as it was prevented by the simultaneous addition of lactose, but not by sucrose. Endothelial cells also increased their exposition of von Willebrand factor after Gal-8 treatment, which constitutes another feature of cell activation that could be, in turn, responsible for the observed platelet adhesion. Several pro-inflammatory molecules were abundantly produced by Gal-8 stimulated endothelial cells: CXCL1 (GRO-α), GM-CSF, IL-6 and CCL5 (RANTES), and in a lower degree CCL2 (MCP-1), CXCL3 (GRO-) and CXCL8 (IL-8). In agreement, Gal-8M induced nuclear factor kappa B phosphorylation. Altogether, these results not only confirm the pro-inflammatory role we have already proposed for Gal-8 in other cellular systems but also suggest that this lectin is orchestrating the interaction between leukocytes, platelets and endothelial cells.

Description: N-Linked glycosylation (N-glycosylation) of proteins has long been associated with oncogenesis, but not until recently have the molecular mechanisms underlying this relationship begun to be unraveled. Here, we review studies describing how dysregulation of the N-glycosylation-regulating gene, DPAGT1 , drives oral cancer. DPAGT1 encodes the first and rate-limiting enzyme in the assembly of the lipid-linked oligosaccharide precursor in the endoplasmic reticulum and thus mediates N-glycosylation of many cancer-related proteins. DPAGT1 controls N-glycosylation of E-cadherin, the major epithelial cell–cell adhesion receptor and a tumor suppressor, thereby affecting intercellular adhesion and cytoskeletal dynamics. DPAGT1 also regulates and is regulated by Wnt/β-catenin signaling, impacting the balance between proliferation and adhesion in homeostatic tissues. Thus, aberrant induction of DPAGT1 promotes a positive feedback network with Wnt/β-catenin that represses E-cadherin-based adhesion and drives tumorigenic phenotypes. Further, modification of receptor tyrosine kinases (RTKs) with N -glycans is known to control their surface presentation via the galectin lattice, and thus increased DPAGT1 expression likely contributes to abnormal activation of RTKs in oral cancer. Collectively, these studies suggest that dysregulation of the DPAGT1 /Wnt/E-cadherin network underlies the etiology and pathogenesis of oral cancer.

Description: The C-type lectin E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells during the inflammatory process. In numerous studies, the S128R mutation of the E-selectin was associated with cardiovascular and autoimmune diseases. There is evidence that the S128R E-selectin mutation leads to a loss in ligand specificity, thus increasing leukocyte recruitment. Apart from the natural tetrasaccharide ligand sialyl Lewis x (sLe x ), it has previously been proposed that non-fucosylated carbohydrates also bind to S128R E-selectin. To evaluate the therapeutic potential of the antagonism of the E-selectin mutant, ligand specificity was reinvestigated on a molecular basis. We determined the ligand specificity of wild-type and S128R E-selectin in a target-based competitive assay, a glycan array screen and cell-based binding assays under static and flow conditions. Regarding ligand-specificity, the binding properties of S128R E-selectin were identical to those of wt E-selectin, i.e., no mutant-specific binding of 3'-sialyl- N -acetyllactosamine, heparin, fetuin and K562 cells was observed. Additionally, the binding affinities of glycomimetic E-selectin antagonists were identical for wt and S128R E-selectin. Overall, the previous reports on carbohydrate ligand promiscuity of S128R E-selectin could not be confirmed.

Description: Phytohemagglutinin from Phaseolus vulgaris (PHA-E), a legume lectin, has an unusual specificity toward biantennary galactosylated N -glycan with bisecting N -acetylglucosamine (GlcNAc). To investigate the interaction in detail, we have solved the crystal structures of PHA-E without ligand and in complex with biantennary N -glycan derivatives. PHA-E interacts with the trisaccharide unit (Galβ1-4GlcNAcβ1-2Man) in a manner completely different from that of mannose/glucose-specific legume lectins. The inner mannose residue binds to a novel site on the protein, and its rotation is opposite to that occurring in the monosaccharide-binding site of other lectins around the sugar O3 axis. Saturation-transfer difference NMR using biantennary di-galactosylated and bisected glycans reveals that PHA-E interacts with both antennas almost equally. The unique carbohydrate interaction explains the glycan-binding specificity and high affinity.

Description: The syp locus includes four genes encoding putative regulators, six genes encoding glycosyltransferases, two encoding export proteins, and six other genes encoding unidentified functional proteins associated with biofilm formation and symbiotic colonization. However, the individual functions of the respective genes remain unclear. Amino acid alignment indicates that s ypQ is presumably involved in biosynthesizing poly- N -acetylglucosamine (PNAG), which is proposed to be a critical virulence factor in pathogen infection and is regarded as a target for protective immunity against a variety of Gram-negative/positive pathogens. However, no evidence showing that Vibrio parahaemolyticus also produces PNAG has been reported. Herein, the V. parahaemolyticus is confirmed to possess potential for producing PNAG for the first time. Our results indicated that gene sypQ is associated with PNAG biosynthesis and PNAG is involved in pathogen colonization. We propose that the function of pgaC in Escherichia coli could be taken over by sypQ from V. parahaemolyticus . We also tested whether PNAG can be used as a target against V. parahaemolyticus when it infects Pseudosciaena crocea . Our results showed that PNAG isolated from V. parahaemolyticus is an effective agent for decreasing V. parahaemolyticus invasion, implying that PNAG could be used to develop an effective vaccine against V. parahaemolyticus infection.

Description: The cell surface sialyl Lewis a (sLe a ) and sialyl Lewis x (sLe x ) antigens, which are built on the terminals of glyco-structures called poly- N -acetyllactosamine (LacNAc) chains, have been shown to play a critical role in the metastasis of colon cancer. In the present investigation, expression of the B3GNT7 gene, which encodes a β-1,3- N -acetylglucosaminyltransferase that mainly acts on and extends sulfated poly-LacNAc chains, was found to be markedly suppressed during the oncogenetic processes associated with colon cancer. DNA methylation in the promoter region of the B3GNT7 gene was found to play a significant role in the suppression of the B3GNT7 gene in colon cancer cells. The results obtained from Transwell experiments and the nude mice xenograft model demonstrated that ectopic expression of the B3GNT7 gene in colon cancer cells diminished the migration capability and the liver-metastasis potential, respectively, of colon cancer cells. Flow cytometric analysis showed that expression of cell surface sLe a and sLe x antigens was decreased in colon cancer cells when the B3GNT7 gene was ectopically expressed. Taken together, the results of the present investigation suggest a link between suppression of B3GNT7 gene expression and elevation of sLe a /sLe x antigen expressions on the surface of cells and that this consequently promotes the metastasis potential of cancer cells as part of the colon cancer oncogenetic process.

Description: Congenital disorders of glycosylation (CDGs) result from mutations in various N-glycosylation genes. The most common type, phosphomannomutase-2 (PMM2)-CDG (CDG-Ia), is due to deficient PMM2 (Man-6-P -〉 Man-1-P). Many patients die from recurrent infections, but the mechanism is unknown. We found that glycosylation-deficient patient fibroblasts have less intercellular adhesion molecule-1 (ICAM-1), and because of its role in innate immune response, we hypothesized that its reduction might help explain recurrent infections in CDG patients. We, therefore, studied mice with mutations in Mpi encoding phosphomannose isomerase (Fru-6-P -〉 Man-6-P), the cause of human MPI-CDG. We challenged MPI-deficient mice with an intraperitoneal injection of zymosan to induce an inflammatory response and found decreased neutrophil extravasation compared with control mice. Immunohistochemistry of mesenteries showed attenuated neutrophil egress, presumably due to poor ICAM-1 response to acute peritonitis. Since phosphomannose isomerase (MPI)-CDG patients and their cells improve glycosylation when given mannose, we provided MPI-deficient mice with mannose-supplemented water for 7 days. This restored ICAM-1 expression on mesenteric endothelial cells and enhanced transendothelial migration of neutrophils during acute inflammation. Attenuated inflammatory response in glycosylation-deficient mice may result from a failure to increase ICAM-1 on the vascular endothelial surface and may help explain recurrent infections in patients.

Description: Being recognized as an important constituent of the glycoprotein folding cycle, uridine diphosphate-glucose:glycoprotein glucosyltransferase (UGGT) has been a subject of intense study. Up to now, it is two isoforms, UGGT1 and 2 have been identified, which share ~50% amino acid identity. UGGT1 is a well-documented enzyme which functions as a folding sensor in the endoplasmic reticulum, by the virtue of its ability to transfer a glucose residue to non-glucosylated high-mannose-type glycans of immature glycoproteins exhibiting non-native conformation. On the other hand, direct evidence to support the glucosyltransferase activity of UGGT2 has been lacking, leaving it unclear as to whether it has any function in the glycoprotein folding process. This study aimed to reveal the property of human UGGT2 by using synthetic substrates such as fluorescently labeled glycans and N-glycosylated proteins. The analysis, for the first time, revealed the glucosyltransferase activity of UGGT2, whose specificity was shown to be quite similar to UGGT1, in terms of both glycan specificity and preferential recognition of proteins having non-native conformations. Finally, Sep15 was found to form the heterodimeric complex with both isoforms of UGGT and markedly enhanced its glucosyltransferase activity.

Description: β-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known β-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae -derived β-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble β-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of β-glucan.

Description: Aspergillus fumigatus is an opportunistic fungal pathogen that causes fatal invasive aspergillosis among immunocompromised patients. The cell wall β-1,3-glucan is mainly elongated by β-1,3-glucanosyltransferase Gel family, which is vital for growth and virulence of A. fumigatus . Although seven members of Gels have been annotated, only Gel1, Gel2 and Gel4 were characterized. In this study, the function of Gel7 was analyzed for the first time, by constructing gel7 , gel7 cwh41 and gel1 gel7 cwh41 separately. Disruption of gel7 alone did not result in any obvious phenotype except an abnormality in conidia formation, whereas gel7 cwh41 and gel1 gel7 cwh41 exhibited abnormal conidiogenesis, a heat-induced delay of germination and a severe decrease in β-1,3-glucan content. Our results suggested that the A. fumigatus β-1,3-glucanosyltransferase Gel7 was involved in conidiation and was compensated for the cell wall β-1,3-glucan defects when Gel1 and Gel2 lost their functions, especially at an elevated temperature.

Description: The structure of a antigen-binding fragment (Fab) from the bactericidal monoclonal antibody LPT3-1 specific to lipooligosaccharide (LOS) inner cores from Neisseria meningitidis has been solved in complex with an eight-sugar inner core fragment NmL3 galE lpt3 KOH to 2.69 Å resolution. The epitope is centered about an inner core N -acetylglucosamine residue unique to N. meningitidis and does not include the lipid A moiety, which is disordered in the structure, but is positioned to allow the binding of free and membrane-anchored full-length LOS. All the amino acid residues that contact antigen are of germline origin but, remarkably, two consecutive somatic mutations of serine to glycine in the heavy chain at residues 52 and 52a are positioned to deprive the antibody of advantageous interactions and so weaken binding. However, these mutations are key to allowing selective cross-reactivity with the HepII-3-PEtn inner core variant expressed by 70% of strains. Neisseria meningitidis is a leading cause of disease in the developed world and is especially dangerous to children, who lack the necessary protective antibodies. The structure of Fab LPT3-1 in complex with LOS provides insight into the antibody's selective ability to recognize multiple clinically relevant variations of the LOS inner core from N. meningitidis .

Description: Recent studies have explored the function of N -linked glycosylation in the nervous system, demonstrating essential roles of carbohydrate structures in neural development. The function of N -glycans in neural physiology remains less understood; however, increasing evidence indicates that N -glycans can play specific modulatory roles controlling neural transmission and excitability of neural circuits. These roles are mediated via effects on synaptic proteins involved in neurotransmitter release, transporters that regulate nerotransmitter concentrations, neurotransmitter receptors, as well as via regulation of proteins that control excitability and response to milieu stimuli, such as voltage-gated ion channels and transient receptor potential channels, respectively. Sialylated N -glycan structures are among the most potent modulators of cell excitability, exerting prominent effects on voltage gated Na + and K + channels. This modulation appears to be underlain by complex molecular mechanisms involving electrostatic effects, as well as interaction modes based on more specific steric effects and interactions with lectins and other molecules. Data also indicate that particular features of N -glycans, such as their location on a protein and structural characteristics, can be specifically associated with the effect of glycosylation. These features and their functional implications can vary between different cell types, which highlight the importance of in vivo analyses of glycan functions. Experimental challenges are associated with the overwhelming complexity of the nervous system and glycosylation pathways in vertebrates, and thus model organisms like Drosophila should help elucidate evolutionarily conserved mechanisms underlying glycan functions. Recent studies supported this notion and shed light on functions of several glycosylation genes involved in the regulation of the nervous system.

Description: The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Seattle, WA, in November 2011 in order to develop guidelines for reporting the qualitative and quantitative results obtained by diverse types of glycomics analyses, including the conditions and techniques that were applied to prepare the glycans for analysis and generate the primary data along with the tools and parameters that were used to process and annotate this data. These guidelines must address a broad range of issues, as glycomics data are inherently complex and are generated using diverse methods, including mass spectrometry (MS), chromatography, glycan array-binding assays, nuclear magnetic resonance (NMR) and other rapidly developing technologies. The acceptance of these guidelines by scientists conducting research on biological systems in which glycans have a significant role will facilitate the evaluation and reproduction of glycomics experiments and data that is reported in scientific journals and uploaded to glycomics databases. As a first step, MIRAGE guidelines for glycan analysis by MS have been recently published (Kolarich D, Rapp E, Struwe WB, Haslam SM, Zaia J., et al. 2013. The minimum information required for a glycomics experiment (MIRAGE) project – Improving the standards for reporting mass spectrometry-based glycoanalytic data. Mol. Cell Proteomics. 12:991–995), allowing them to be implemented and evaluated in the context of real-world glycobiology research. In this paper, we set out the historical context, organization structure and overarching objectives of the MIRAGE initiative.

Description: A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, -〉6)[α- d -Gal p A3Ac/4Ac-(1 -〉 3)]-α- d- Man p -(1 -〉 3)-α- d- Man p -(1 -〉 3)-β- d- Man p -(1 -〉 3)-α- d- Glc p NAc-(1-〉. The identification of the O-acetylation positions was efficiently performed by comparison of the 1 H, 13 C HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d -Gal p A residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.

Description: Galectin-1 (Gal-1), a member of a family of multifunctional lectins, plays key roles in diverse biological processes including cell signaling, immunomodulation, neuroprotection and angiogenesis. The presence of an unusual number of six cysteine residues within Gal-1 sequence prompted a detailed analysis of the impact of the redox environment on the functional activity of this lectin. We examined the role of each cysteine residue in the structure and function of Gal-1 using both experimental and computational approaches. Our results show that: (i) only three cysteine residues present in each carbohydrate recognition domain (CRD) (Cys2, Cys16 and Cys88) were important in protein oxidation, (ii) oxidation promoted the formation of the Cys16–Cys88 disulfide bond, as well as multimers through Cys2, (iii) the oxidized protein did not bind to lactose, probably due to poor interactions with Arg48 and Glu71, (iv) in vitro oxidation by air was completely reversible and (v) oxidation by hydrogen peroxide was relatively slow (1.7 ± 0.2 M –1 s –1 at pH 7.4 and 25°C). Finally, an analysis of key cysteines in other human galectins is also provided in order to predict their behaviour in response to redox variations. Collectively, our data provide new insights into the structural basis of Gal-1 redox regulation with critical implications in physiology and pathology.

Description: Heparin, a commonly used anticoagulant drug, is a mixture of highly sulfated polysaccharides with various molecular weights (MWs). The unique sulfation pattern dictates the anticoagulant activity of heparin. Commercial heparins are categorized into three forms according to their average MW: unfractionated heparin (UFH, MW avg 14,000), low-MW heparin (LMWH, MW avg 3500–6500) and the synthetic pentasaccharide (fondaparinux, MW 1508.3). UFH is isolated from porcine intestine while LMWH is derived from UFH by various methods of depolymerization, which generate a wide range of oligosaccharide chain lengths. Different degradation methods result in structurally distinct LMWH products, displaying different pharmacological and pharmacokinetic properties. In this report, we utilized a chemoenzymatic method to synthesize LMWH with the emphasis on controlling the size distribution of the oligosaccharides. A tetrasaccharide primer and a controlled enzyme-based polymerization were employed to build a narrow size oligosaccharide backbone. The oligosaccharide backbones were further modified by a series of sulfation and epimerization steps in order to obtain a full anticoagulation activity. Determination of the anticoagulation activity in vitro and ex vivo indicated that the synthetic LMWH has higher potency than enoxaparin, a commercial LMWH drug in clinical usage.

Description: Chondroitin sulfate proteoglycans (CSPGs) are the main component of the extracellular matrix in the central nervous system (CNS) and influence neuroplasticity. Although CSPG is considered an inhibitory factor for nerve repair in spinal cord injury, it is unclear whether CSPG influences the pathogenetic mechanisms of neuroimmunological diseases. We induced experimental autoimmune encephalomyelitis (EAE) in chondroitin 6- O -sulfate transferase 1-deficient (C6st1 –/– ) mice. C6ST1 is the enzyme that transfers sulfate residues to position 6 of N -acetylgalactosamine in the sugar chain of CSPG. The phenotypes of EAE in C6st1 –/– mice were more severe than those in wild-type (WT) mice were. In adoptive-transfer EAE, in which antigen-reactive T cells from WT mice were transferred to C6st1 –/– and WT mice, phenotypes were significantly more severe in C6st1 –/– than in WT mice. The recall response of antigen-reactive T cells was not significantly different among the groups. Furthermore, the number of pathogenic T cells within the CNS was also not considerably different. When EAE was induced in C6ST1 transgenic mice with C6ST1 overexpression, the mice showed considerably milder symptoms compared with those in WT mice. In conclusion, the presence of sulfate at position 6 of N -acetylgalactosamine of CSPG may influence the effecter phase of EAE to prevent the progression of pathogenesis. Thus, modification of the carbohydrate residue of CSPG may be a novel therapeutic strategy for neuroimmunological diseases such as multiple sclerosis.

Description: Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal β-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed β-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Description: Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2- O -sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2- O -sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (~〈5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate.

Description: Transferrin is an iron-transport protein which possesses N -glycans at Asn432 and Asn630 in humans. Transferrin glycoforms Tf-1 and Tf-2, previously identified in human cerebrospinal fluid, are defined as the lower and upper bands in gel electrophoresis, respectively. Importantly, the Tf-2/Tf-1 ratio is raised in idiopathic normal pressure hydrocephalus patients and is useful as a clinical marker. In order to gain insight into the relationship between transferrin glycoform and biological function, we performed comparative characterization of Tf-1, Tf-2 and serum transferrin (sTf). Mass spectrometric analyses confirmed that Tf-2 is modified with disialylated biantennary glycans at both of the two N -glycosylation sites, which are similar to the N -glycans of sTf. On the other hand, Tf-1 is site-specifically modified: Asn630 has biantennary agalacto-complex-type glycan with bisecting N -acetylglucosamine (GlcNAc) and core fucose while Asn432 is modified with complex/high mannose-type glycans and possibly single GlcNAc. Size exclusion chromatography and fluorescence correlation spectroscopy analysis revealed that the hydration volume of Tf-1 is slightly smaller than that of sTf. Our striking finding is that Tf-1 has an exposed hydrophobic surface as monitored by the fluorescence intensity and wavelength of a hydrophobic probe, 1-anilino-8-naphthalene sulfonate, whereas Tf-2 does not. These results suggest that the different N -glycan structure of Tf-1 lowers the apparent hydration volume and reveals a patch of hydrophobic surface on transferrin which is otherwise covered with sialoglycan in sTf and Tf-2. The carbohydrate deficiency in certain pathological conditions may also expose hydrophobic surface which may modulate the function and/or stability of transferrin.