Showing papers in "Glycobiology in 2009"

Optimal and consistent protein glycosylation in mammalian cell culture.

Abstract: In the biopharmaceutical industry, mammalian cell culture systems, especially Chinese hamster ovary (CHO) cells, are predominantly used for the production of therapeutic glycoproteins. Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein. Obtaining a consistent glycoform profile in production is desired due to regulatory concerns because a molecule can be defined by its carbohydrate structures. An optimal profile may involve a spectrum of product glycans that confers a desired therapeutic efficacy, or a homogeneous glycoform profile that can be systemically screened for. Studies have shown some degree of protein glycosylation control in mammalian cell culture, through cellular, media, and process effects. Studies upon our own bioprocesses to produce fusion proteins and monoclonal antibodies have shown an intricate relationship between these variables and the resulting protein quality. Glycosylation optimization will improve therapeutic efficacy and is an ongoing goal for researchers in academia and industry alike. This review will focus on the advancements made in glycosylation control in a manufacturing process, as well as the next steps in understanding and controlling protein glycosylation.

Focus on antivirally active sulfated polysaccharides: from structure-activity analysis to clinical evaluation.

Abstract: In recent years, many compounds having potent antiviral activity in cell culture have been detected and some of these compounds are currently undergoing either preclinical or clinical evaluation. Among these antiviral substances, naturally occurring sulfated polysaccharides and those from synthetic origin are noteworthy. Recently, several controversies over the molecular structures of sulfated polysaccharides, viral glycoproteins, and cell-surface receptors have been resolved, and many aspects of their antiviral activity have been elucidated. It has become clear that the antiviral properties of sulfated polysaccharides are not only a simple function of their charge density and chain length but also their detailed structural features. The in vivo efficacy of these compounds mostly corresponds to their ability to inhibit the attachment of the virion to the host cell surface although in some cases virucidal activity plays an additional role. This review summarizes experimental evidence indicating that sulfated polysaccharides might become increasingly important in drug development for the prevention of sexually transmitted diseases in the near future.

Metabolic Glycoengineering: Sialic Acid and Beyond

Abstract: This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems - such as the human brain - through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous - and sometime quite unexpected - biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the "druggability" of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated "scaffold-dependent" activities, are summarized.

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn.

Abstract: High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (approximately 5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS-agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive beta-elimination and analyzed by nanoLC/mass spectrometry and MS(n). The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Gal beta 1-3/4-GlcNAc beta 1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were -Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.

Two distinct α-l-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates

Abstract: Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

Protein O-mannosylation: conserved from bacteria to humans.

Abstract: Protein O-mannosylation is an essential modification in fungi and animals. Different from most other types of O-glycosylation, protein O-mannosylation is initiated in the endoplasmic reticulum by the transfer of mannose from dolichol monophosphate-activated mannose to serine and threonine residues of secretory proteins. In recent years, it has emerged that even bacteria are capable of O-mannosylation and that the biosynthetic pathway of O-mannosyl glycans is conserved between pro- and eukaryotes. In this review, we summarize the observations that have opened up the field and highlight characteristics of O-mannosylation in the different domains/kingdoms of life.

Antiganglioside antibodies and their pathophysiological effects on Guillain–Barré syndrome and related disorders—A review

Abstract: Guillain-Barre syndrome (GBS) is an acute immune-mediated polyradiculoneuropathy which can cause acute quadriplegia. Infection with micro-organisms, including Campylobacter jejuni (C. jejuni), Haemophilus influenzae, and Cytomegalovirus (CMV), is recognized as a main triggering event for the disease. Lipooligosaccharide (LOS) genes are responsible for the formation of human ganglioside-like LOS structures in infectious micro-organisms that can induce GBS. Molecular mimicry of LOSs on the surface of infectious agents and of ganglioside antigens on neural cells is thought to induce cross-reactive humoral and cellular immune responses. Patients with GBS develop antibodies against those gangliosides, resulting in autoimmune targeting of peripheral nerve sites, leading to neural damage. Heterogeneity of ganglioside expression in the peripheral nervous system (PNS) may underlie the differential clinical manifestation of the GBS variants. Recent studies demonstrate that some GBS sera react with ganglioside complexes consisting of two different gangliosides, such as GD1a and GD1b, or GM1 and GD1a, but not with each constituent ganglioside alone. The discovery of antiganglioside complex antibodies not only improves the detection rate of autoantibodies in GBS, but also provides a new concept in the antibody-antigen interaction through clustered carbohydrate epitopes. Although ganglioside mimicry is one of the possible etiological causes of GBS, unidentified factors may also contribute to the pathogenesis of GBS. While GBS is not considered a genetic disease, host factors, particularly human lymphocyte antigen type, appear to have a role in the pathogenesis of GBS following C. jejuni infection.

The CMP-legionaminic acid pathway in Campylobacter: Biosynthesis involving novel GDP-linked precursors

Abstract: The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legion-aminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.

Stability of N-glycan profiles in human plasma

Abstract: Glycan heterogeneity was shown to be associated with numerous diseases and glycan analysis has a great diagnostic potential. Recently, we reported high biological variability of human plasma N-glycome at the level of population. The observed variations were larger than changes reported to be associated with some diseases; thus, it was of great importance to examine the temporal constancy of human N-glycome before glycosylation changes could be routinely analyzed in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during 5 days. N-Linked glycans, released from plasma proteins, were separated using hydrophilic interaction high-performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of N-glycome in a single individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1 were observed. The average coefficient of variation was 5.6%. These variations were comparable to those observed when analytical procedure was tested for its precision. Good stability of plasma N-glycome in healthy individuals implies that glycosylation is under significant genetic control. Changes observed in glycan profiles are consequence of environmental influences and physiologic responses and therefore have a significant diagnostic potential.

Comparative analysis of core-fucose-binding lectins from Lens culinaris and Pisum sativum using frontal affinity chromatography

Abstract: Lens culinaris lectin (LCA) is a useful probe for the detection in serum of a core-fucosylated alpha-fetoprotein, called AFP-L3 fraction, which is a well-known marker for the diagnosis and prognosis of hepatocellular carcinoma. Here we performed a systematic quantitative interaction analysis of LCA and its close homolog, Pisum sativum lectin (PSA), by frontal affinity chromatography with 143 pyridylaminated (PA) glycans including a series of core-fucosylated glycans. Both lectins showed binding affinity to core-fucosylated, mono- and bi-antennary N-glycans, but not to their tri- and tetra-antennary forms, indicating that the addition of the GlcNAc residue at the N-acetylglucosaminyltransferase IV position abrogates the binding affinity. However, their specificities are distinguishable: while LCA showed the highest affinity to the core-fucosylated, agalactosylated, bi-antennary N-glycan (K(a)=1.1 x 10(5) M(-1)), PSA showed the highest affinity to the core-fucosylated, trimannosyl structure (K(a)=1.2 x 10(5) M(-1)). Glycan-binding specificities of LCA and PSA were also analyzed by glycoconjugate microarray compared to other core-fucose-binding lectins from Aspergillus oryzae (AOL) and Aleuria auratia (AAL). LCA and PSA bound specifically to core fucose, whereas AOL and AAL exhibited broad specificity to fucosylated glycans. These results explain why LCA is appropriate as a specific probe for AFP-L3, which mainly contains a core-fucosylated, biantennary N-glycan, but not its highly branched forms.

Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa

Abstract: Glycoconjugates expressed on gastric mucosa play a crucial role in host-pathogen interactions. The FUT2 enzyme catalyzes the addition of terminal alpha(1,2)fucose residues, producing the H type 1 structure expressed on the surface of epithelial cells and in mucosal secretions of secretor individuals. Inactivating mutations in the human FUT2 gene are associated with reduced susceptibility to Helicobacter pylori infection. H. pylori infects over half the world's population and causes diverse gastric lesions, from gastritis to gastric cancer. H. pylori adhesion constitutes a crucial step in the establishment of a successful infection. The BabA adhesin binds the Le(b) and H type 1 structures expressed on gastric mucins, while SabA binds to sialylated carbohydrates mediating the adherence to inflamed gastric mucosa. In this study, we have used an animal model of nonsecretors, Fut2-null mice, to characterize the glycosylation profile and evaluate the effect of the observed glycan expression modifications in the process of H. pylori adhesion. We have demonstrated expression of terminal difucosylated glycan structures in C57Bl/6 mice gastric mucosa and that Fut2-null mice showed marked alteration in gastric mucosa glycosylation, characterized by diminished expression of alpha(1,2)fucosylated structures as indicated by lectin and antibody staining and further confirmed by mass spectrometry analysis. This altered glycosylation profile was further confirmed by the absence of Fucalpha(1,2)-dependent binding of calicivirus virus-like particles. Finally, using a panel of H. pylori strains, with different adhesin expression profiles, we have demonstated an impairment of BabA-dependent adhesion of H. pylori to Fut2-null mice gastric mucosa, whereas SabA-mediated binding was not affected.

Human noroviruses recognize sialyl Lewis x neoglycoprotein.

Abstract: The carbohydrate binding characteristics of a norovirus GII.3 (Chron1) and a GII.4 (Dijon) strain were investigated using virus-like particles (VLPs) and saliva samples from 81 individuals genotyped for FUT2 (secretor) and FUT3 (Lewis) and phenotyped for ABO and Lewis blood groups. The two VLPs showed a typical secretor-gene-dependent binding and bound significantly stronger to saliva from A, B, and AB than from O individuals (P < 0.0001 and P < 0.001) but did not bind to any samples from secretor-negative individuals. The GII.3 strain showed larger interindividual variation and bound stronger to saliva from B than from A(2) secretors (P < 0.01). When assaying for binding to neoglycoproteins, the GII.3 and GII.4 strains were compared with the Norwalk GI.1 prototype strain. Although all three strains bound to Lewis b (and H type 1 chain) glycoconjugates, only the two GII strains showed an additional binding to sialyl Lewis x. This novel binding was specific since the VLPs did not bind to structural analogs, e.g., Lewis x or sialyl Lewis a, but only to sialyl Lewis x, sialyl diLewis x and sialylated type 2 chain conjugates. In inhibition experiments, the sialyl Lewis x conjugate was the most potent inhibitor. The minimal requirement for this potential receptor structure is Neu5Ac alpha 3Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta- where Fuc is not absolutely necessary for binding. Our study shows that some human norovirus GII strains have at least two binding specificities: one secretor-gene-dependent related to alpha 1,2-fucosylated carbohydrates and another related to alpha 2,3-sialylated carbohydrates of the type 2 chain, e.g., sialyl Lewis x.

Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile.

Abstract: A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae.

Deletion polymorphism of SIGLEC14 and its functional implications.

Abstract: Human Siglec-14, a member of the Siglec family of sialic acid-binding lectins, shows extensive sequence similarity to human Siglec-5. To analyze respective expression patterns of Siglec-14 and Siglec-5, we developed specific antibodies against each of them. We found that the former was expressed on granulocytes and monocytes, while the latter was on granulocytes and B-cells. Surprisingly, some individuals lacked the expression of Siglec-14, while they all expressed Siglec-5. We found that a fusion between SIGLEC14 and SIGLEC5 genes, resulting in the functional deletion of SIGLEC14, underlies this phenotype. The presence of the "SIGLEC14 null" allele in all human populations we tested implies an ancient origin, while its allelic frequency is higher in Asians compared with Africans and Europeans. The forced expression of Siglec-14 in a monocytic cell line-enhanced TNF-alpha secretion elicited by lipopolysaccharide. These results imply that Siglec-14 may play some role in bacterial infection.

Structural insights into what glycan arrays tell us about how glycan-binding proteins interact with their ligands

Abstract: Screening of glycan arrays represents a powerful, high-throughput approach to defining oligosaccharide ligands for glycan-binding receptors, commonly referred to as lectins. Correlating results from such arrays with structural analysis of receptor-ligand complexes provide one way to validate the arrays. Using examples drawn from the family of proteins that contain C-type carbohydrate-recognition domains, this review illustrates how information from the arrays reflects the way that selectivity and affinity for glycan ligands is achieved. A range of binding profiles is observed, from very restricted binding to a small set of structurally similar ligands to binding of broad classes of ligands with related terminal sugars and even to failure to bind any of the glycans on an array. These outcomes provide insights into the importance of multiple factors in defining the selectivity of these receptors, including the presence of conformationally defined units in some oligosaccharide ligands, local and extended interactions between glycans and the surfaces of receptors, and steric factors that exclude binding of some ligands.

The effects of 4-methylumbelliferone on hyaluronan synthesis, MMP2 activity, proliferation, and motility of human aortic smooth muscle cells

Abstract: Extracellular matrix remodeling after proatherosclerotic injury involves an increase in hyaluronan (HA) that is coupled with vascular smooth muscle cell (SMC) migration, proliferation, and with neointima formation. As such events are dependent on HA, in this study we assessed the effects on SMC behavior of 4-methylumbelliferone (4-MU). As previously described in other cell types, 4-MU reduced HA in cultures of primary human aortic SMCs (AoSMCs) as well as the cellular content of the HA precursor UDP-glucuronic acid. We found that SMCs increased UDP-glucuronyl transferase 1 enzymes, which can reduce the cellular content of UDP-glucuronic acid confirming that the availability of the UDP-sugar substrates can regulate HA synthesis. Interestingly, we reported that 4-MU reduced the transcripts coding for the three HA synthases as well as UDP glucose pyrophosphorylase and dehydrogenase. As HA synthase transcript reduction is common to other cell types, the 4-MU effect on gene expression may be considered a mechanism for HA synthesis inhibition. Moreover, we showed that 4-MU strongly inhibits AoSMCs migration, which was restored by the addition of exogenous HA indicating that the rescuing depends on the interaction of HA with its receptor CD44. Besides the decrease in HA synthesis and cell migration, 4-MU reduced AoSMCs proliferation, indicating that 4-MU may exert a vasoprotective effect.

A mathematical model to derive N-glycan structures and cellular enzyme activities from mass spectrometric data.

Abstract: Effective representation and characterization of biosynthetic pathways of glycosylation can be facilitated by mathematical modeling. This paper describes the expansion of a previously developed detailed model for N-linked glycosylation with the further application of the model to analyze MALDI-TOF mass spectra of human N-glycans in terms of underlying cellular enzyme activities. The glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes. The use of a molecular mass cutoff and a network pruning method typically limits the model size to about 10,000 glycan structures. This allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. A synthetic mass spectrum from model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with experiment. The result of this process is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of 19 enzymes. In addition, a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. The method was applied to mass spectrometric data of normal human monocytes and monocytic leukemia (THP1) cells to derive glycosyltransferase activity changes underlying the differences in glycan structure between the normal and diseased cells. Model predictions could lead to a better understanding of the changes associated with disease states, identification of disease-associated biomarkers, and bioengineered glycan modifications.

The α-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

Abstract: Galectins are a sub-family of lectins, defined by their highly conserved beta-sandwich structures and ability to bind to beta-galactosides, like Gal beta1-4 Glc (lactose). Here, we used (15)N-(1)H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of beta1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via alpha1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (K(d)) of 10 x 10(-6) M, compared to 260 x 10(-6) M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the beta-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1-carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences.

The specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry.

Abstract: More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid called seminolipid. The galactosylation of the molecule is catalyzed by UDP-galactose:ceramide galactosyltransferase (CGT). Disruption of the CGT gene in mice results in male infertility due to the arrest of spermatogenesis, indicating that seminolipid plays an important role in reproductive function. Seminolipid molecules can be assigned to different molecular species based on the fatty acid composition. In this report, we investigated the localizations of the molecular species of seminolipid by imaging mass spectrometry and demonstrated that major molecule (C16:0-alkyl-C16:0-acyl) was expressed throughout the tubules: some (C16:0-alkyl-C14:0-acyl and C14:0-alkylC16:0-acyl)werepredominantlyexpressedinspermatocytes and the other (C17:0-alkyl-C16:0-acyl) was specifically expressed in spermatids and spermatozoa. This is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation.

Targeted glycoproteomic identification of cancer cell glycosylation

Abstract: GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewisx and Lewisy structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewisx antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Involvement of chondroitin sulfate E in the liver tumor focal formation of murine osteosarcoma cells

Abstract: Cell surface heparan sulfate plays a critical role in regulating the metastatic behavior of tumor cells, whereas the role of chondroitin sulfate/dermatan sulfate (CS/DS) has been little understood in this context. Here, we characterized CS/DS chains from the murine osteosarcoma cell line LM8G7, which forms tumor nodules in liver. Structural analysis of the CS/DS chains showed a higher proportion of GlcUA beta 1-3GalNAc(4,6-O-disulfate) (E-units) in LM8G7 (12%) than in its parental cell line LM8 (6%), which rarely forms tumors in the liver. Immunostaining with GD3G7, an antibody specific to E-units, confirmed the higher expression of the epitope in LM8G7 than LM8 cells. The tumor focal formation of LM8G7 cells in the liver in mice was effectively inhibited by the preadministration of CS-E (rich in E-unit) or the preincubation of the antibody GD3G7 with the tumor cells. CS-E or GD3G7 inhibited the adhesion of LM8G7 cells to a laminin-coated plate in vitro. In addition, the invasive ability of LM8G7 cells in vitro was also reduced by the addition of CS-E or the antibody. Further, CS-E or the antibody inhibited the proliferation of LM8G7 cells dose dependently. The binding of LM8G7 cells to VEGF in vitro was also significantly reduced by CS-E and GD3G7. Thus, the present study reveals the significance of highly sulfated CS/DS structures in the liver colonization of osteosarcoma cells and also provides a framework for the development of GAG-based anticancer molecules.

Analysis of lectin binding to glycolipid complexes using combinatorial glycoarrays

Abstract: Glycolipids are major components of the plasma membrane, interacting with themselves, other lipids, and proteins to form an array of heterogeneous domains with diverse biological properties Considerable effort has been focused on identifying protein binding partners for glycolipids and the glycan specificity for these interactions, largely achieved through assessing interactions between proteins and homogenous, single species glycolipid preparations This approach risks overlooking both the enhancing and attenuating roles of heterogeneous glycolipid complexes in modulating lectin binding Here we report a simple method for assessing lectin-glycolipid interactions An automatic thin-layer chromatography sampler is employed to create easily reproducible arrays of glycolipids and their heterodimeric complexes immobilized on a synthetic polyvinyl-difluoride membrane This array can then be probed with much smaller quantities of reagents than would be required using existing techniques such as ELISA and thin-layer chromatography with immuno-overlay Using this protocol, we have established that the binding of bacterial toxins, lectins, and antibodies can each be attenuated, enhanced, or unaffected in the presence of glycolipid complexes, as compared with individual, isolated glycolipids These findings underpin the wide-ranging influence and importance of glycolipid-glycolipid cis interactions when the nature of protein-carbohydrate recognition events is being assessed

Identification of a novel gene encoding the trehalose phosphate synthase in the cotton bollworm, Helicoverpa armigera

Abstract: Trehalose and trehalose metabolism are crucial for insect development. We measured the content of polyhydric compounds in the hemolymph of diapause- and nondiapause-destined individuals of Helicoverpa armigera. We found that the trehalose content is much higher in diapause-destined individuals than that in nondiapause individuals. The activity of trehalose-6-phosphate synthase (TPS) during H. armigera larval-pupal development is significantly higher in diapause-type individuals and is closely correlated with the changes in the trehalose content. The cDNA encoding TPS, which converts uridine-5'-diphosphoglucose and glucose-6-phosphate to trehalose-6-phosphate, was cloned from the fat body of H. armigera using rapid amplification of cDNA ends (RACE). The molecular characterization of the cDNA revealed that the mRNA encodes a precursor polypeptide of 826-amino-acid residues, containing Har-TPS at residues 6-507 and a putative trehalose-6-phosphate phosphatase, which converts trehalose-6-phosphate into free trehalose, at residues 512-783. The Har-TPS precursor polypeptide shows 73% identity with that of Drosophila melanogaster. The presence of a 2.8 kb transcript in the fat body and ovary was detected with a northern blot. The Har-TPS mRNA was detected at high levels in the late stage of sixth larval instar and the early middle stage of diapause-destined pupae, which are most likely to respond the changes in TPS activity and trehalose in the hemolymph. The Har-TPS protein was successfully overexpressed in the Bombyx mori baculovirus expression system, and the catalytic activity of Har-TPS was found to be approximately 5-fold higher in B. mori blood infected by the recombined-baculovirus than the control. When diapause is broken, the trehalose content drops significantly and glucose increases rapidly. These results suggest that trehalose is involved in regulating H. armigera pupal diapause.

Recognition of non-self-polysaccharides by C-type lectin receptors dectin-1 and dectin-2.

Abstract: The discovery of several transmembrane receptors expressed by antigen presenting cells, including those that detect and interact with specific sugar moieties on the surface of microbes, has improved our understanding of how immunity against infection is generated. This knowledge, in turn, prompted us to review such interactions with emphasis on C-type lectin receptors and a focus on the roles of dectin-1 and dectin-2 in anti-fungal immunity.

Roles of gastric mucin-type O-glycans in the pathogenesis of Helicobacter pylori infection

Abstract: Helicobacter pylori is a Gram-negative bacterium that infects over 50% of the world's population. This organism causes various gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer. H. pylori possesses lipopolysaccharides that share structural similarity to Lewis blood group antigens in gastric mucosa. Such antigenic mimicry could result in immune tolerance against antigens of this pathogen. On the other hand, H. pylori colonizes gastric mucosa by utilizing adhesins that bind Lewis blood group antigen-related carbohydrates expressed on gastric epithelial cells. After colonization, H. pylori induces acute inflammatory responses mainly by neutrophils. This acute phase is gradually replaced by a chronic inflammatory response. In chronic gastritis, lymphocytes infiltrate the lamina propria, and such infiltration is facilitated by the interaction between L-selectin on lymphocytes and peripheral lymph node addressin (PNAd), which contains 6-sulfo sialyl Lewis X-capped O-glycans, on high endothelial venule (HEV)-like vessels. H. pylori barely colonizes gland mucous cell-derived mucin where α1,4-GlcNAc-capped O-glycans exist. In vitro experiments show that α1,4-GlcNAc-capped O-glycans function as a natural antibiotic to inhibit H. pylori growth. These findings show that distinct sets of carbohydrates expressed in the stomach are closely associated with pathogenesis and prevention of H. pylori-related diseases, providing therapeutic potentialities based on specific carbohydrate modulation.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.

Abstract: Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

Salivary MUC7 is a major carrier of blood group I type O-linked oligosaccharides serving as the scaffold for sialyl Lewis x.

Abstract: Isolation of salivary MUC7 with gel electrophoresis allowed analysis by LC-MS and LC-MS(2) of released O-linked oligosaccharides and a thorough description of the glycosylation of this molecule, where high-molecular-weight oligosaccharides up to the size of 2790 Da and with up to three sialic acid residues were identified. A common theme of these novel high abundant oligosaccharides on MUC7 showed that the C-3 branch of the oligosaccharides consisted of branched I-antigen type structural epitopes (GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-), where the branch point was initiated on core 1 and core 2 galactose residues, and the branches were terminated by sialyl type 2 and sialyl Lewis x epitopes. Six sulfated sialylated oligosaccharides of low intensity were also identified, with the sulfate mainly on N-acetyl glucosamine residues located close to the reducing termini. One of these oligosaccharides was identified as a candidate for the high-affinity L-selectin ligand 6'-sulfo sialyl Lewis x. Neutral oligosaccharides and blood group antigens were found to be less abundant on MUC7 and the glycosylation appeared to be more preserved between individuals as compared to salivary MUC5B. This was illustrated by comparing the LC-MS spectra of MUC7 and MUC5B glycans from secretors (23 individuals) and nonsecretors (6 individuals). The data show that MUC7 provides a multivalent scaffold for sialylation, meeting the requirement for high-avidity binding via its glycosylation and mediator of the interaction between immune cells such as salivary neutrophils and oral bacteria.

AftD, a novel essential arabinofuranosyltransferase from mycobacteria.

Abstract: Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

QCM-D studies of human norovirus VLPs binding to glycosphingolipids in supported lipid bilayers reveal strain-specific characteristics.

Abstract: Susceptibility to norovirus infection has been linked to secretor status. Norovirus virus-like particles (VLPs; 0- 20 microg/mL) from the Norwalk (GI.1) and Dijon (GII.4) strains were assayed for binding to H type 1 and Lewis a pentaglycosylceramides, incorporated in laterally fluid supported lipid bilayers. Binding kinetics was monitored in real time in 40 microL stationary reaction chambers, using quartz crystal microbalance with dissipation (QCM-D) monitoring. Both strains displayed binding only to H type 1 and not to Lewis a glycosphingolipids, typical for epithelial cells of susceptible and resistant individuals, respectively. This binding specificity was confirmed by VLPs binding to the two glycosphingolipids chromatographed on TLC-plates. Experiments using bilayers with mixtures of H type 1 and Lewis a, with the total glycosphingolipid concentration constant at 10 wt%, showed that binding was only dependent on H type 1 concentrations and identical to experiments without additional Lewis a. Both strains showed a threshold concentration of H type 1 below which no binding was observable. The threshold was one order of magnitude higher for the Dijon strain (2 wt% versus 0.25 wt%) demonstrating that the interaction with a significantly larger number of glycosphingolipids was needed for the binding of the Dijon strain. The difference in threshold glycosphingolipid concentrations for the two strains suggests a lower affinity for the glycosphingolipid for the Dijon compared to the Norwalk strain. We propose that VLPs initially bind only a few glycosphingolipids but the binding is subsequently strengthened by lateral diffusion of additional glycosphingolipids moving into the interaction area.