Glycobiology - recent issues

Meeting Announcements

2008-04-14

Erratum

2008-04-14

Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry

2008-04-14

In an effort to understand the role of molecular glycosylation in cancer a murine model has been used to characterize and fingerprint malignancies in established cell lines that manifest all the hallmarks of metastatic disease: spontaneous development, local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Using astrocyte cell controls, we compared N-linked glycosylation from a nonmetastatic brain tumor cell line and two different metastatic brain tumor cells. Selected ions in each profile were disassembled by ion trap mass spectrometry (MSⁿ) which exhibited multiple structural differences between each tissue. These unique structures were identified within isomeric compositions as pendant nonreducing termini of di- and trisaccharide fragments, probably transparent to a tandem MS approach but distinctively not to sequential ion trap MSⁿ detection.

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

Manthri, S., Guther, M L. S, Izquierdo, L., Acosta-Serrano, A., Ferguson, M. A J — 2008-04-14

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man₉GlcNAc₂ from Man₉GlcNAc₂-PP-Dol while others receive Man₅GlcNAc₂ from Man₅GlcNAc₂-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the -mannosyltransferase that converts Man₅GlcNAc₂-PP-Dol to Man₆GlcNAc₂-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man₉GlcNAc₂ and processed no further than Man₅GlcNAc₂ in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man₅GlcNAc₂-PP-Dol is the only route to complex N-glycans in T. brucei and that Man₉GlcNAc₂-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man₅GlcNAc₂ or Man₉GlcNAc₂ transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.

Different affinity of galectins for human serum glycoproteins: Galectin-3 binds many protease inhibitors and acute phase proteins

2008-04-14

Here we report the first survey of galectins binding to glycoproteins of human serum. Serum was subjected to affinity chromatography using immobilized galectins, and the bound glycoproteins were analyzed by electrophoresis, Western blotting, and mass spectrometry. Galectins-3, -8, and -9 bound a much broader range of ligands in serum than previously known, galectin-1 bound less, and galectins-2, -4, and -7 bound only traces or no serum ligands. Galectin-3 bound most major glycoproteins, including alpha-2-macroglobulin and acute phase proteins such as haptoglobin. It bound only a selected minor fraction of transferrin, and bound none or little of IgG. Galectins-8 and -9 bound a similar range of glycoproteins as galectin-3, but in lower amounts, and galectin-8 had a relative preference for IgA. Galectin-1 bound mainly a fraction of alpha-2-macroglobulin and only traces of other glycoproteins. The binding of galectin-3 to serum glycoproteins requires affinity for LacNAc, since a mutant (R186S), which has lost this affinity, did not bind any serum glycoproteins. The average affinity of galectin-3 for serum glycoproteins was estimated to correspond to K_d ~1–5 µM by modeling of the affinity chromatography and a fluorescence anisotropy assay. Since galectins are expressed on endothelial cells and other cells exposed to serum components, this report gives new insight into function of galectins and the role of their different fine specificity giving differential binding to the serum glycoproteins.

The AP-2{alpha} transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene

2008-04-14

Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking region of the PTEN gene showed that the region between –1175 and –1077 from the translational initiation site, which contains the AP-2 binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2 is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2 diminished the enhancement of AP-2 and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2 also results in the induction of PTEN transcription in AP-2-negative colon cancer cells. Additionally, GM3 induced AP-2-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2 transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene.

Roles of complex gangliosides in the development of experimental autoimmune encephalomyelitis

Miyamoto, K., Takada, K., Furukawa, K., Furukawa, K., Kusunoki, S. — 2008-04-14

We induced experimental autoimmune encephalomyelitis (EAE) in GM2/GD2 synthase knockout mice (GM2/GD2^–/–), which cannot synthesize complex gangliosides, such as GM1, GD1a, GD1b, GT1b, and GQ1b, to investigate the roles of complex gangliosides in the pathogenesis of this disease. We used myelin-oligodendrocyte glycoprotein (MOG) as an immunogen. In active immunization EAE, the severity of clinical score was not different but the disease onset was significantly delayed in GM2/GD2^–/– compared with those in wild-type mice. When we transferred MOG-reactive T cells from GM2/GD2^–/– or wild-type mice to wild-type mice, no significant differences were observed between the two groups. In contrast, when we transferred MOG-reactive T cells from wild-type mice to GM2/GD2^–/– or to wild-type mice, the onset of EAE in GM2/GD2^–/– mice was delayed. The recall response of MOG-specific T cells, the function of antigen presenting cells, or the expression of several adhesion molecules in the endothelium were not significantly different between GM2/GD2^–/– and wild-type mice. On the other hand, quantitative analysis of cellular infiltration in the central nervous system (CNS) on day 9 of active immunization EAE showed that the CD4⁺ cell number in the CNS isolated from GM2/GD2^–/– mice was significantly less than that from wild-type mice. It indicated that the delayed onset of EAE in GM2/GD2^–/– mice was due to the delay of the migration of pathogenic T cells into the CNS. Thus, the complex gangliosides may be involved in the T cell–endothelial cell interaction in the pathogenetic process of EAE.

A new type of plant chitinase containing LysM domains from a fern (Pteris ryukyuensis): Roles of LysM domains in chitin binding and antifungal activity

Onaga, S., Taira, T. — 2008-04-14

Chitinase-A (PrChi-A), of molecular mass 42 kDa, was purified from the leaves of a fern (P. ryukyuensis) using several column chromatographies. The N-terminal amino acid sequence of PrChi-A was similar to the lysin motif (LysM). A cDNA encoding PrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1459 nucleotides and encoded an open-reading frame of 423-amino-acid residues. The deduced amino acid sequence indicated that PrChi-A is composed of two N-terminal LysM domains and a C-terminal catalytic domain, belonging to the group of plant class IIIb chitinases, linked by proline, serine, and threonine-rich regions. Wild-type PrChi-A had chitin-binding and antifungal activities, but a mutant without LysM domains had lost both activities. These results suggest that the LysM domains contribute significantly to the antifungal activity of PrChi-A through their binding activity to chitin in the cell wall of fungi. This is the first report of the presence in plants of a family-18 chitinase containing LysM domains.

Meeting Announcements

2008-03-25

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells

2008-03-25

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan (HA), a large polysaccharide abundant in the extracellular matrix of vertebrate tissues. Six genes have been predicted to encode hyaluronidases in humans, but the protein products of only SPAM1, HYAL1, and HYAL2 have been characterized. We have now expressed the mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in Baby Hamster Kidney (BHK) cells and demonstrated the presence of multiple forms of Hyal3 ranging from ~45 to 56 kDa in expression lysates. Complete and partial digestions of the expressed protein with PNGase F showed three N-linked oligosaccharides accounted for all forms of Hyal3 detected in expression lysates. Most of these oligosaccharides were Endo H sensitive, indicating that they were high mannose or hybrid N-linked oligosaccharides. Subcellular fractionation of Hyal3-expressing BHK cells by density gradient centrifugation revealed most Hyal3 in a low-density vesicular population. Low levels of Hyal3 were detected in higher density vesicles, but no colocalization with the late endosomal/lysosomal marker Lamp1 was found by immunofluorescence microscopy. BHK cells stably expressing Hyal3 had increased acid-active hyaluronidase activity, but no such activity was detected when Hyal3 was transfected into Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpression of Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels, suggesting that the increased hyaluronidase activity in these cells was due to Hyal1 rather than Hyal3. The results indicate that Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidase activity and that Hyal3 may contribute to HA metabolism by augmenting the activity of Hyal1.

A novel {alpha}1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

Zheng, Q., Van Die, I., Cummings, R. D — 2008-03-25

The 1,2-fucosyltransferase family (1,2FT) is the largest family of glycosyltransferases in the genome of the free-living nematode Caenorhabditis elegans, and early evidence suggests that each member may have a unique activity. Here we describe a C. elegans gene (designated CE2FT-2) encoding an 1,2FT that has the potential to generate the sequence Fuc1-2Galβ1-3GalNAc-R, which is the H-type 3 blood group structure. The CE2FT-2 cDNA encodes a putative transmembrane protein that shows ~42% amino acid identity to a previously cloned C. elegans 1,2FT (termed CE2FT-1), but has a very low identity (~16–20%) to 1,2FT sequences in humans, rabbits, and mice. A recombinant form of CE2FT-2 expressed in human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP, but unexpectedly, the enzyme is inactive toward the acceptor Galβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTs previously described from animals that all utilize Galβ-O-phenyl. CE2FT-2 is expressed at all stages of worm development, but remarkably, promoter analysis of the CE2FT-2 gene using green fluorescent protein reporter constructs indicates that the CE2FT-2 is expressed exclusively in pharyngeal cells of the worm from embryo to an adult stage. Because pharyngeal cells are known to secrete their glycoconjugates to the nematode surface, these results may indicate that products of CE2FT-2 contribute to interactions of the nematode with its environment or are used as ligands for bacterial attachment. These findings, along with those on other 1,2FTs in C. elegans, suggest that each 1,2FT in this organism may have a unique acceptor specificity, expression pattern, and biological function.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains

Chigira, Y., Oka, T., Okajima, T., Jigami, Y. — 2008-03-25

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure–function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: A case study on galectins-1 and -3 and the impact of assay setting

2008-03-25

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes’ carbohydrate headgroup and the density of unsubstituted β-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for 1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.

Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase

2008-03-25

The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F^– releasing activity from -xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F^– releasing activity. As a result of the high F^– releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney

2008-03-25

The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.

N-Glycoproteomics - An automated workflow approach

Joenvaara, S., Ritamo, I., Peltoniemi, H., Renkonen, R. — 2008-03-25

Glycan decorations dictate protein functions and thus have crucial importance in life sciences. Previously glycoprotein analysis was mainly focused on the analysis of the liberated glycans allowing detailed structural, but lacking positional information. Analysis of intact glycopeptides required purified glycoproteins and manual interpretation of spectra. We developed an approach where mixtures of native glycopeptides were analyzed with tandem mass spectrometry and the spectra were analyzed with automated in silico workflows. The latter included combination of the original spectra, generation of a human N-glycopeptide library, matching the glycopeptide spectra to the theoretical peptide fragments, scoring the observations, predicting the glycan composition, which were then matched against the observed spectra, statistical validation of the results with target–decoy filtering, and finally the calculation of glycan structures. We verified this approach with the 150 serotransferrin glycopeptide spectra, where we automatically generated 10⁵ putative interpretations from >10⁹ theoretical glycopeptides. After scoring 62 glycopeptide spectra obtained validated interpretation with concomitant amino acid sequences, glycan compositions, and structures. When applying this method to an unknown mixture of human plasma glycoproteins we identified 80 glycopeptides with their glycan compositions or structures. Instead of weeks and months of interpretation work of mass spectrometry files our automated workflow can be executed in few hours and provide information concomitantly from both the amino acid and glycan moieties of intact glycopeptides in mixtures. No advanced computational skills were needed to use these preformed and tested workflows. In case users want to add complexity to the analysis they are allowed to alter all parameters and rebuild the workflows.

Meeting Announcements

2008-03-06

In memory of Roger W. Jeanloz, a pioneer glycobiologist (1917-2007)

Sharon, N., Glick, M. C., Hughes, R C. — 2008-03-06

The Roy L. Whistler International Award in Carbohydrate Chemistry 2008

Kamerling, J. P. — 2008-03-06

Free oligosaccharide regulation during mammalian protein N-glycosylation

Chantret, I., Moore, S. E H — 2008-03-06

During protein N-glycosylation in mammalian cells, free oligosaccharides (fOS) are generated from lipid-linked oligosaccharides by a pyrophosphatase activity and oligosaccharyltransferase and from misfolded glycoproteins by peptide:N-glycanase in both the ER and cytoplasm. Trafficking machinery comprising oligosaccharide-specific ER and lysosomal transporters, an endo-β-N-acetyl-glucosaminidase, and the cytosolic M2C1 mannosidase drives a flux of fOS from the ER to cytoplasm and from the cytoplasm into lysosomes where fOS are degraded. Transport of fOS out of the ER is normally efficient and if inhibited causes fOS to be secreted via the Golgi apparatus. By contrast, fOS clearance from the cytosol into lysosomes is less efficient resulting in low micromolar concentrations of fOS in the cytoplasm. Structural analysis of cytosolic fOS reveals oligosaccharide families whose relative abundance highlights the importance of different ER-associated degradation (ERAD) pathways for misfolded glycoproteins and suggests that in liver cells substantial amounts of glycoproteins destined for ERAD may transit early compartments of the Golgi apparatus. Glycoprotein quality control and ERAD are controlled by N-glycan/lectin interactions and the fOS trafficking pathway would seem to ensure that fOS do not interfere with these processes which occur in both the ER and cytoplasm. Although Saccharomyces cerevisiae strains harbouring mutations in genes of the yeast fOS metabolic pathway do not display obvious phenotypes, mammalian fOS are quantitatively more important and the processes leading to their regulation are more complex, raising the possibility that distinct phenotypes will be seen in mammalian cells or animals in which fOS metabolism is modified.

Antithrombin activity and disaccharide composition of dermatan sulfate from different bovine tissues

Osborne, S. A., Daniel, R. A., Desilva, K., Seymour, R. B. — 2008-03-06

Dermatan sulfate is a glycosaminoglycan that selectively inhibits the action of thrombin through interaction with heparin cofactor II. Unlike heparin it does not interact with other coagulation factors and is able to inhibit thrombin associated with clots. This property has made dermatan sulfate an attractive candidate as an antithrombotic drug. Previous studies have showed that dermatan sulfate derived from porcine/bovine intestinal mucosa/skin or marine invertebrates is capable of stimulating heparin cofactor II-mediated thrombin inhibition in vitro. This biological activity is reported for the first time in this study using dermatan sulfate derived from mammalian tissues other than intestinal mucosa or skin. Ten different bovine tissues including the aorta, diaphragm, eyes, large and small intestine, esophagus, skin, tendon, tongue, and tongue skin were used to prepare dermatan sulfate-enriched fractions by anion exchange chromatography and acetone precipitation. Heparin cofactor II/dermatan sulfate-mediated thrombin inhibition measured in vitro revealed activity comparable to or higher than the commercial standard with 2-fold differences observed between some tissues. Analysis of the extracted dermatan sulfate using fluorophore-assisted carbohydrate electrophoresis revealed significant differences in the relative percentage of all the mono-sulfated disaccharides, in particular the predominant mammalian disaccharide uronic acid->N-acetyl-d-galactosamine-4-O-sulfate, confirming previous reports regarding variations in sulfation in dermatan sulfate from different tissues. Overall, these findings demonstrate that dermatan sulfate extracted from a range of bovine tissues exhibits in vitro antithrombin activity equivalent to or higher than that observed for porcine intestinal mucosa, identifying additional sources of dermatan sulfate as potential antithrombotic agents.

A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits

2008-03-06

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.

Embryonic stem cells deficient in I {beta}1,6-N-acetylglucosaminyltransferase exhibit reduced expression of embryoglycan and the loss of a Lewis X antigen, 4C9

Muramatsu, H., Kusano, T., Sato, M., Oda, Y., Kobori, K., Muramatsu, T. — 2008-03-06

Embryoglycan is a class of branched high-molecular-weight poly-N-acetyllactosamines characteristically expressed in early embryonic cells and has been shown to be involved in the intercellular adhesion of early embryonic cells in vitro. Branching of poly-N-acetyllactosamine chains is performed by β1,6-N-acetylglucosaminylation of the galactosyl residue. We previously knocked out the gene encoding I β1, 6-N-acetylglucosaminyltransferase (IGnT), and the resultant deficient mice were born without any abnormality, although the mice exhibited various deficits in later life. In the present investigation, we produced embryonic stem (ES) cells from IGnT-deficient embryos. The mutant ES cells exhibited a reduced capability in embryoglycan synthesis. Thus, IGnT is a major enzyme involved in the branching of poly-N-acetyllactosamine chains in embryoglycan. Since ES cells are equivalent to multipotential cells of the embryonic ectoderm in early postimplantation embryos, this result indicates that an abundance of embryoglycan in these cells is not essential for normal embryogenesis. The IGnT-deficient ES cells continued to express SSEA-1, but lacked the expression of 4C9 antigen, although the epitope of 4C9 antigen was confirmed to be Lewis X by a transfection experiment. The result establishes the distinct nature of 4C9 antigenicity, which requires either Lewis X epitope on I-branch or clustering of Lewis X epitope, best accomplished by poly-N-acetyllactosamine branching. 6-Integrin was newly identified as a carrier of embryoglycan. The IGnT-deficient ES cells adhered to dishes coated with laminin, which is a ligand for 6-integrin, significantly less than wild-type ES cells, raising the possibility that embryoglycan in ES cells enhances 6-integrin-dependent adhesion in vitro.

A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum

2008-03-06

The green algae of the genus Codium have recently been demonstrated to be an important source of sulfated galactans from the marine environment. Here, a sulfated galactan was isolated from the species Codium isthmocladum and its structure was studied by a combination of chemical analyses and NMR spectroscopy. Two fractions (SG 1, ~14 kDa, and SG 2, ~20 kDa) were derived from this highly polydisperse and heterogeneous polysaccharide. Both exhibited similar structures in ¹H 1D NMR spectra. The structural features of SG 2 and its desulfated derivative were analyzed by COSY, TOCSY, DEPT-HSQC, HSQC, and HMBC. This sulfated galactan is composed preponderantly of 4-sulfated, 3-linked β-d-galactopyranosyl units. In minor amounts, it is sulfated and glycosylated at C-6. Pyruvate groups are also found, forming five-membered cyclic ketals as 3,4-O-(1'carboxy)-ethylidene-β-d-galactose residues. A comparison of sulfated galactans from different marine taxonomic groups revealed similar backbones of 3-β-d-Galp-1.

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its {Delta}galU mutant: glycine residue location in the inner core of the lipopolysaccharide

2008-03-06

Shigella flexneri is a Gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a l,d-Hep-(1 ->7)-l,d-Hep-(1 ->3)-l,d-Hep-(1 ->5)-[Kdo-(2 ->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.

Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

2008-03-06

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a β-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (/)₅ for CS (chondroitin lyases AC) and β-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located ~12 Å from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.