Glycobiology - current issue

Meeting Announcements

2008-08-18

Publisher's Note

2008-08-18

Introduction to a mini-forum on "glyconutrients"

McClain, D. A — 2008-08-18

A "Glyconutrient Sham"

Schnaar, R. L, Freeze, H. H — 2008-08-18

The discipline of glycobiology contributes to our understanding of human health and disease through research, most of which is published in peer-reviewed scientific journals. Recently, legitimate discoveries in glycobiology have been used as marketing tools to help sell plant extracts termed "glyconutrients." The glyconutrient industry has a worldwide sales force of over half a million people and sells nearly half a billion dollars (USD) of products annually. Here we address the relationship between glyconutrients and glycobiology, and how glyconutrient claims may impact the public and our discipline.

Response to "A 'Glyconutrient Sham'"

Persinger, T. — 2008-08-18

Letter to the Glyco-Forum

Varki, A. — 2008-08-18

Wielding the sword of professional ethics against misleading dietary supplement claims

Torok, C. B, Murray, T. H — 2008-08-18

Strategies for carbohydrate recognition by the mannose 6-phosphate receptors

Dahms, N. M, Olson, L. J, Kim, J.-J. P — 2008-08-18

The two members of the P-type lectin family, the 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR), are ubiquitously expressed throughout the animal kingdom and are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The best-characterized function of the MPRs is their ability to direct the delivery of ~60 different newly synthesized soluble lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) on their N-linked oligosaccharides to the lysosome. In addition to its intracellular role in lysosome biogenesis, the CI-MPR, but not the CD-MPR, participates in a number of other biological processes by interacting with various molecules at the cell surface. The list of extracellular ligands recognized by this multifunctional receptor has grown to include a diverse spectrum of Man-6-P-containing proteins as well as several non-Man-6-P-containing ligands. Recent structural studies have given us a clearer view of how these two receptors use related, but yet distinct, approaches in the recognition of phosphomannosyl residues.

Human and mouse macrophage-inducible C-type lectin (Mincle) bind Candida albicans

2008-08-18

Candida albicans is a causative agent in mycoses of the skin, oral cavity, and gastrointestinal tract. Identification of receptors, and their respective ligands, that are engaged by immune cells when in contact with C. albicans is crucial for understanding inflammatory responses leading to invasive candidiasis. Mincle is a recently identified macrophage-expressed receptor that is important for host responses to C. albicans. The carbohydrate-recognition domain of human and mouse Mincle were expressed, purified under denaturing conditions, and successfully refolded. In addition to oligomers, there are isolatable monomeric and dimeric forms of the protein that occur under two different buffer solutions. The human and mouse homologues bound yeast extract, and the isolated dimeric and monomeric species also demonstrated the recognition of whole C. albicans yeast cells. The data are indicative of several functional states mediating the interaction of Mincle and yeast at the surface of the macrophage.

Multifunctionality of Campylobacter jejuni sialyltransferase CstII: Characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities

2008-08-18

CstII from bacterium Campylobacter jejuni strain OH4384 has been previously characterized as a bifunctional sialyltransferase having both 2,3-sialyltransferase (GM3 oligosaccharide synthase) and 2,8-sialyltransferase (GD3 oligosaccharide synthase) activities which catalyze the transfer of N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophosphate (CMP)-Neu5Ac to C-3' of the galactose in lactose and to C-8 of the Neu5Ac in 3'-sialyllactose, respectively (Gilbert M, Karwaski MF, Bernatchez S, Young NM, Taboada E, Michniewicz J, Cunningham AM, Wakarchuk WW. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem. 277:327–337). We report here the characterization of a truncated CstII mutant (CstII32^I53S) cloned from a synthetic gene whose codons are optimized for an Escherichia coli expression system. In addition to the 2,3- and 2,8-sialyltransferase activities reported before for the synthesis of GM3- and GD3-type oligosaccharides, respectively, the CstII32^I53S has 2,8-sialyltransferase (GT3 oligosaccharide synthase) activity for the synthesis of GT3 oligosaccharide. It also has 2,8-sialidase (GD3 oligosaccharide sialidase) activity that catalyzes the specific cleavage of the 2,8-sialyl linkage of GD3-type oligosaccharides and 2,8-trans-sialidase (GD3 oligosaccharide trans-sialidase) activity that catalyzes the transfer of a sialic acid from a GD3 oligosaccharide to a different GM3 oligosaccharide (3'-sialyllactoside). The donor substrate specificity study of the CstII32^I53S GD3 oligosaccharide synthase activity indicates that the enzyme is flexible in using different CMP-activated sialic acids and their analogs for the synthesis of GD3 oligosaccharides containing natural and nonnatural modifications at the terminal sialic acid.

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile

2008-08-18

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked Gal(1,3)βGal(1,4)βGlc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to Gal(1,3)βGal(1,4)βGlc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by Gal(1,3)βGal(1,4)βGlc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-Gal(1,3)βGal(1,4)βGlc sequences with similar avidities. Additional ES-MS experiments using chemically altered Gal(1,3)βGal(1,4)βGlc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

Characteristics of carbohydrate antigen binding to the presentation protein HLA-DR

Cobb, B. A, Kasper, D. L — 2008-08-18

Zwitterionic polysaccharide antigens (ZPSs) were recently shown to activate T cells in a class II major histocompatibility complex (MHCII)-dependent fashion requiring antigen presenting cell (APC)-mediated oxidative processing although little is known about the mechanism or affinity of carbohydrate presentation (Cobb BA, Wang Q, Tzianabos AO, Kasper DL. 2004. Polysaccharide processing and presentation by the MHCII pathway. Cell. 117:677–687). A recent study showed that the helical conformation of ZPSs (Wang Y, Kalka-Moll WM, Roehrl MH, Kasper DL. 2000. Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis. Proc Natl Acad Sci USA. 97:13478–13483; Choi YH, Roehrl MH, Kasper DL, Wang JY. 2002. A unique structural pattern shared by T-cell-activating and abscess-regulating zwitterionic polysaccharides. Biochemistry. 41:15144–15151) is closely linked with immunogenic activity (Tzianabos AO, Onderdonk AB, Rosner B, Cisneros RL, Kasper DL. 1993. Structural features of polysaccharides that induce intra-abdominal abscesses. Science. 262:416–419) and is stabilized by a zwitterionic charge motif (Kreisman LS, Friedman JH, Neaga A, Cobb BA. 2007. Structure and function relations with a T-cell-activating polysaccharide antigen using circular dichroism. Glycobiology. 17:46–55), suggesting a strong carbohydrate structure–function relationship. In this study, we show that PSA, the ZPS from Bacteroides fragilis, associates with MHCII at high affinity and 1:1 stoichiometry through a mechanism mirroring peptide presentation. Interestingly, PSA binding was mutually exclusive with common MHCII antigens and showed significant allelic differences in binding affinity. The antigen exchange factor HLA-DM that catalyzes peptide antigen association with MHCII also increased the rate of ZPS association and was required for APC presentation and ZPS-mediated T cell activation. Finally, the zwitterionic nature of these antigens was required only for MHCII binding, and not endocytosis, processing, or vesicular trafficking to MHCII-containing vesicles. This report is the first quantitative analysis of the binding mechanism of carbohydrate antigens with MHCII and leads to a novel model for nontraditional MHCII antigen presentation during bacterial infections.

Disruption of thymopoiesis in ST6Gal I-deficient mice

2008-08-18

Thymocyte development is accompanied by sequential changes in cell surface glycosylation. For example, medullary thymocytes have increased levels of 2,3-linked sialic acid and a loss of asialo core 1 O-glycans as compared to cortical thymocytes. Some of these changes have been linked to fine tuning of the T cell receptor avidity. We analyzed ST6Gal I transcript abundance and levels of 2,6-linked sialic acid across thymocyte subsets. We found that ST6Gal I transcript levels increased following T cell receptor β-selection suggesting that this sialyltransferase may influence the development of early thymocyte populations. Indeed, low levels of 2,6-linked sialic acid were found in the earliest T lineage cells, and then increased in T cell receptor β-selected cells. To determine whether ST6Gal I influences T cell development, we analyzed ST6Gal I-deficient mice for disruptions in thymocyte populations. We found reduced thymic cellularity in the ST6Gal I-deficient mice starting in the early thymocyte compartments.

Characterization of two different endo-{alpha}-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens

2008-08-18

Endo--N-acetylgalactosaminidase (endo--GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between -GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo--GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Galβ1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo--N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415–37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl--glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAcβ1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Galβ1-3(GlcNAcβ1-6)GalNAc, core 8 disaccharide Gal1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

2008-08-18

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin 1, 4, V, and β1, and that Colon26 cells express CD44, integrin 2, 5, V, and β1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.