Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans.

Leukocytes are coated with a layer of heterogeneous carbohydrates (glycans) that modulate immune function, in part by governing specific interactions with glycan-binding proteins (lectins). Although nearly all membrane proteins bear glycans, the identity and function of most of these sugars on leukocytes remain unexplored. Here, we characterize the N-glycan repertoire (N-glycome) of human tonsillar B cells. We observe that naive and memory B cells express an N-glycan repertoire conferring strong binding to the immunoregulatory lectin galectin-9 (Gal-9). Germinal center B cells, by contrast, show sharply diminished binding to Gal-9 due to upregulation of I-branched N-glycans, catalyzed by the ß1,6-N-acetylglucosaminyltransferase GCNT2. Functionally, we find that Gal-9 is autologously produced by naive B cells, binds CD45, suppresses calcium signaling via a Lyn-CD22-SHP-1 dependent mechanism, and blunts B cell activation. Thus, our findings suggest Gal-9 intrinsically regulates B cell activation and may differentially modulate BCR signaling at steady state and within germinal centers.

SHIP-1 inhibits CD95/APO-1/Fas-induced apoptosis in primary T lymphocytes and T leukemic cells by promoting CD95 glycosylation independently of its phosphatase activity.

SHIP-1 (SH2 (Src homology 2)-containing inositol 5'-phosphatase-1) functions as a negative regulator of immune responses by hydrolyzing phosphatidylinositol-3,4,5-triphosphate generated by phosphoinositide-3 (PI 3)-kinase activity. As a result, SHIP-1 deficiency in mice results in myeloproliferation and B-cell lymphoma. On the other hand, SHIP-1-deficient mice have a reduced T-cell population, but the underlying mechanisms are unknown. In this work, we hypothesized that SHIP-1 plays anti-apoptotic functions in T cells upon stimulation of the death receptor CD95/APO-1/Fas. Using primary T cells from SHIP-1(-/-) mice and T leukemic cell lines, we report that SHIP-1 is a potent inhibitor of CD95-induced death. We observed that a small fraction of the SHIP-1 pool is localized to the endoplasmic reticulum (ER), in which it promotes CD95 glycosylation. This post-translational modification requires an intact SH2 domain of SHIP-1, but is independent of its phosphatase activity. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired death-inducing signaling complex (DISC) formation and downstream apoptotic cascade. These results uncover an unanticipated inhibitory function for SHIP-1 and emphasize the role of glycosylation in the regulation of CD95 signaling in T cells. This work may also provide a new basis for therapeutic strategies using compounds inducing apoptosis through the CD95 pathway on SHIP-1-negative leukemic T cells.

Prevalence and factors associated with it, of birds dead on arrival at the slaughterhouse and other rejection conditions in broiler chickens.

1. Standardised data on husbandry were recorded for a flock of birds in one house on each of 150 broiler farms in the UK during the 4 d prior to slaughter. 2. For each flock, the incidence of birds found dead on arrival (DoAs) and the Meat Hygiene Service carcase rejection records were recorded at the slaughterhouse. 3. The mean percentage of birds in each flock found DoA was 0.12% (range 0-0.64%) and the mean percentage of Total Carcase Rejects (TCRs) for each flock was 1.23% (range 0.07-5.51%). 4. A general linear model was developed to examine factors associated with flock percentage DoAs. Assuming a linear relationship, all other factors remaining the same, a one percentage point (PP) increase in small/emaciated birds will result in a 0.155 PP increase in DoAs and a 1 PP increase in wheat in diet 4 will result in a 0.003 PP decrease. An increase by one in the total number of vaccines administered will cause a 0.029 PP decrease in DoAs, a 1 g increase in live weight at slaughter will be associated with a 0.000043 PP increase and a 1 PP increase in mortality on farm would be associated with a 0.000044 PP increase. A 1 PP increase in Ross birds decreases DoAs by 0.0004 PPS: there is also a seasonal effect. 5. The model developed for flock percentage TCRs found that a 1 PP increase in wheat in diet 3 will result in a 0.052 PP decrease in TCRs and a 1 PP increase in Ross birds will cause a 0.009 PP decrease. A 1 PP increase in birds culled on farm will be associated with a 0.03 PP increase in TCRs and the diagnosis of disease during the flock cycle increases TCRs by 0.397 PPs. A one day increase in age at slaughter will result in a 0.046 PP increase in TCRs.

Factors affecting the prevalence of foot pad dermatitis, hock burn and breast burn in broiler chicken.

1. Standardised data on flock husbandry were recorded on 149 broiler farms during the 4 d prior to slaughter. 2. Birds were examined at the slaughterhouse for contact dermatitis lesions. Foot pad dermatitis score (FPDS) and hock burn score (HBS) were measured on five point scales. Carcase rejection data were also collected. 3. The mean percentage of birds in each flock with: moderate or severe foot lesions was 11.1% (range 0-71.5%); moderate or severe hock burn was 1.3% (range 0-33.3%); and, breast burn was 0.02%. 4. A general linear model was developed to examine factors associated with mean flock FPDS. Assuming a linear relationship, within the range of data collected and with all other factors remaining the same, every 1% increase in the proportion of Genotype A birds in the flock was associated with an increase in mean FPDS of 0.003, every one-point increase in litter score was associated with a 0.326 increase in mean FPDS and every one-point increase in flock mean HBS was associated with a 0.411 increase in mean FPDS. Flock mean FPDS was associated with feed supplier and was higher in winter. 5. The general linear model developed for flock mean HBS, found that every one-point increase in mean FPDS increased mean HBS by 0.090, every one-point increase in litter score increased HBS by 0.119 and, every 1% increase in small/emaciated birds decreased mean HBS by 0.333. Reduced HBS was also associated with increased final litter depth, younger slaughter age and an increased percentage of dietary wheat. For every 1% increase in Genotype A birds, a decrease in flock mean HBS of 0.003 would be expected. 6. An effect of hatchery was also identified.

Preliminary study to examine the utility of using foot burn or hock burn to assess aspects of housing conditions for broiler chicken.

1. Eleven broiler chicken farms, representing 4 production system types, were visited during the last 5 d of the flock cycle: bird and flock details were recorded. Litter friability was assessed at 9 sites within the house, atmospheric ammonia was measured at three sites and bird cleanliness was assessed on a numerical rating scale. 2. For these flocks, hock burn, foot burn and breast burn were measured at the processing plant by standardised assessors. 3. Significant correlations were identified between the percentage of birds with foot burn and average litter score, average house ammonia concentrations and feather score. 4. No correlation was found between the percentage of birds with hock burn or breast burn and average litter scores, average ammonia concentrations or feather score. 5. No correlation was found between stocking density and foot burn, hock burn or breast burn.6. If confirmed, these findings may have implications for the draft EU Broiler Directive, for which it is proposed that permitted stocking density on farm may be determined by the incidence and severity of contact dermatitis measured on plant.

Hallmarks of Caenorhabditis elegans N-glycosylation: complexity and controversy.

Caenorhabditis elegans has become one of the most widely used model organisms for a range of molecular cell biological applications and is being increasingly used by glycobiologists. However, a major problem has been the lack of knowledge of the structure of the protein-linked glycans from this organism. In recent years several groups have published structural data, particularly N-glycan structural data. However, some of these data are contradictory. In this review we critically assess all the N-glycan structural data and consider how close we are in our goal of defining the glycome of C. elegans.

Isolation and structural determination of novel sulfated hexasaccharides from squid cartilage chondroitin sulfate E that exhibits neuroregulatory activities.

Squid cartilage chondroitin sulfate E (CS-E) exhibits various biological activities, including anticoagulant activities, lymphoid regulatory activities, and neuroregulatory activities [Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413]. These activities are expressed through molecular interactions with specific proteins, including heparin cofactor II, selectins, CD44, chemokines, and the heparin-binding growth factor midkine. Hence, the sugar sequence information is essential for a better understanding of the CS-E functions. Previously, several novel tetrasaccharides containing the unreported 3-O-sulfated glucuronic acid (GlcA) were isolated after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, hexasaccharides were isolated to obtain more detailed sequence information, especially around the GlcA(3-O-sulfate) residue, and were characterized by fast atom bombardment mass spectrometry and 500 or 600 MHz (1)H NMR spectroscopy. The findings demonstrate one tetrasulfated and five pentasulfated hexasaccharide sequences, five of them being novel. They were composed of three disaccharide building units of either A [GlcA(beta1-3)GalNAc(4-O-sulfate)], E [GlcA(beta1-3)GalNAc(4,6-O-disulfate)], K [GlcA(3-O-sulfate)(beta1-3)GalNAc(4-O-sulfate)], L [GlcA(3-O-sulfate)(beta1-3)GalNAc(6-O-sulfate)], or M [GlcA(3-O-sulfate)(beta1-3)GalNAc(4,6-O-disulfate)], forming E-A-A, M-A-A, K-L-A, E-E-A, K-K-A, and A-M-A hexasaccharide sequences. The K-L tetrasaccharide sequence is to date unreported. The isolated sequences appear to indicate the occurrence of an unreported GlcA 3-O-sulfotransferase specific for chondroitin sulfate. The obtained sequence information will be useful for investigating the structure-function relationship and biosynthesis of CS-E.

Mass spectrometric strategies: providing structural clues for helminth glycoproteins.

Here we review current knowledge of helminth glycans and introduce parasitologists to the power of the mass spectrometric techniques that have been largely responsible for defining their carbohydrate moieties. A brief overview of glycosylation in other eukaryotes is presented, with a focus on mammalian glycosylation, to facilitate understanding of how parasite structures might be recognized as 'self' or 'foreign' by the immune system of the host.

Phosphorylcholine-containing N-glycans of Trichinella spiralis: identification of multiantennary lacdiNAc structures.

Although the presence of phosphorylcholine (PC) in Trichinella spiralis is well established, the precise structure of the PC-bearing molecules is not known. In this paper, we report structural studies of N-glycans released from T.spiralis affinity-purified antigens by peptide N-glycosidase F. Three classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and eight N-acetylhexosamine residues. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine.

Characterization of the N-linked oligosaccharides of megalin (gp330) from rat kidney.

Megalin (gp 330) is a large cell surface receptor expressed on the apical surfaces of epithelial tissues, that mediates the binding and internalization of a number of structurally and functionally distinct ligands. In this paper we report the first detailed structural characterization of megalin-derived oligosaccharides. Using strategies based on mass spectrometric analysis, we have defined the structures of the N-glycans of megalin. The results reveal that megalin glycoprotein is heterogeneously glycosylated. The major N-glycans identified belong to the following two classes: high mannose structures and complex type structures, with complex structures being more abundant than high mannose structures. The major nonreducing epitopes in the complex-type glycans are: GlcNAc, Galbeta1-4GlcNAc (LacNAc), NeuAcalpha2-6Galbeta1-4GlcNAc (sialylated LacNAc), GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc (Sd(a)) and Galalpha1-3Galbeta1-4GlcNAc. Most complex structures are characterized by the presence of (alpha1,6)-core fucosylation and the presence of a bisecting GlcNAc residue.

Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode.

This paper reports the first rigorous evidence for the existence of N-linked oligosaccharides in Dictyocaulus viviparus, an economically important nematode that parasitises cattle. Structural strategies based upon fast atom bombardment mass spectrometry were employed to examine detergent extracts of homogenised adult D.viviparus for their N-glycan content. These revealed that detergent-soluble material is rich in high mannose, truncated and complex-type families of N-linked oligosaccharides. Importantly, the most abundant antennae in the complex-type structures were shown to carry the Lewis(x)epitope (Galbeta1-4(Fucalpha1-3)GlcNAc). Although the Lewis(x)moiety occurs in other helminths such as schistosomes, nematodes have previously been thought to lack this epitope. The Lewis(x)epitopes in D.viviparus are carried on bi-, tri-, and tetraantennary glycans and are therefore candidates for recognition events requiring multivalent ligands. There is compelling evidence from schistosome research that glycoconjugates containing Lewis(x)structures are immunomodulators. We propose that the Lewis(x)-rich glycans identified in this study might similarly be involved in D.viviparus host interactions.

Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Immunogenic glycoconjugates implicated in parasitic nematode diseases.

Parasitic nematodes infect billions of people world-wide, often causing chronic infections associated with high morbidity. The greatest interface between the parasite and its host is the cuticle surface, the outer layer of which in many species is covered by a carbohydrate-rich glycocalyx or cuticle surface coat. In addition many nematodes excrete or secrete antigenic glycoconjugates (ES antigens) which can either help to form the glycocalyx or dissipate more extensively into the nematode's environment. The glycocalyx and ES antigens represent the main immunogenic challenge to the host and could therefore be crucial in determining if successful parasitism is established. This review focuses on a few selected model systems where detailed structural data on glycoconjugates have been obtained over the last few years and where this structural information is starting to provide insight into possible molecular functions.

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]

Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers.

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

Structural analysis of sequences O-linked to mannose reveals a novel Lewis X structure in cranin (dystroglycan) purified from sheep brain.

The Lewis X epitope, Galbeta1-4(Fucalpha1-3)GlcNAc-R, has been implicated in cell-cell recognition events in a number of systems including the central nervous system and is expressed on diverse glycoconjugates including cell adhesion molecules, glycolipids, and the proteoglycan phosphacan. Although Lewis X sequences 3-linked to mannose have been described within proteoglycan fractions of mammalian brain, these have not been reported in other contexts and have been widely believed to be peculiar constituents of brain proteoglycans. In the present paper, we confirm the existence of Lewis X structures O-linked to mannose within the mammalian brain, demonstrate that these structures are present on a well defined mucin-like glycoprotein, cranin (dystroglycan), and report studies suggesting that the linkages involved may be predominantly 2-linked to mannose. Mannose-linked Lewis X is the latest in an increasing list of oligosaccharide recognition "tags" that have been shown to be expressed on cranin (dystroglycan) purified from brain.