GalNAc Beta Research Articles

A study on the mechanism of antigen–antibody reaction increased by ganglioside-complex in Guillain–Barré syndrome

Background & Objective: The mechanism of anti-ganglioside complex (GSC) antibody phenomenon in Guillain–Barré syndrome (GBS) is not revealed yet. In most case, one of the two gangliosides forming GSC is GM1, GD1b, or asialo-GM1 (GA1), which contains common neutral oligosaccharide structure, Gal(beta1-3)GalNAc, in non-reducing terminal, and patients shows clinical features of antibody against the other ganglioside. This suggests a possibility that the neutral oligosaccharide structure changes accessibility or affinity of the antibody to the pendant, not forming a novel conformational epitope.

Treatment resistant autoimmune autonomic ganglionitis associated with increased serum immunoglobulin light chains

Background & Objective: The mechanism of anti-ganglioside complex (GSC) antibody phenomenon in Guillain–Barre syndrome (GBS) is not revealed yet. In most case, one of the two gangliosides forming GSC is GM1, GD1b, or asialo-GM1 (GA1), which contains common neutral oligosaccharide structure, Gal(beta1-3)GalNAc, in non-reducing terminal, and patients shows clinical features of antibody against the other ganglioside. This suggests a possibility that the neutral oligosaccharide structure changes accessibility or affinity of the antibody to the pendant, not forming a novel conformational epitope. We studied the effect of the neutral oligosaccharide structure to the anti-GSC. Methods: Sera from patients with GBS or Fisher syndrome with antibodies against GSCs composed of GA1 and one of GD1a, GD1b, and GQ1b were studied on the effect of the replacement of GA1 in the GSCs with other neutral glycolipids or lipidwith partial common structure. Antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). The neutral glycolipids and lipid examined were following: asialoGM2(GA2), globoside, trihexosylceramide, paragloboside, glucosaminyl lactosylceramide, lactosylceramide, glucosylceramide and ceramide. Results: Four of 11 GA1/GD1a antibody-positive cases and four of 11 GA1/ GD1b antibody-positive cases also showed an enhancement of the antibody reaction by mixing with other neutral glycolipids instead of GA1. In these cases, the degree of the enhancementwasweaker compared to mixing with GA1. Nine of 16 GA1/GQ1b antibody-positive cases, also showed an enhancement of the antibody reaction by mixing with other neutral glycolipids. In five of them, the degree of the enhancement was equal or stronger than in GA1/GQ1b complex. Twelve cases showed enhancement bymixingwith ceramide. In these cases, ELISAwith reduced amount of the single ganglioside, which had been mixed with ceramide, showed decrement of the titer in seven cases and increment in three. Conclusions: There would be non-conformational, i.e. structure-nondependent, mechanism in enhancement of antigen–antibody reaction by mixing two gangliosides, as well as structure-dependent one. In the cases who showed enhanced reaction by reducing the quantity of ganglioside antigen, it is possibly considered that accessibility of antibody to antigen has improved by reduction of antigen density. One of the gangliosides in GSC might act as spacer separating true antigenic epitope in some cases.

Anti-Thomsen-Friedenreich-Ag anti-TF-Ag potential for cancer therapy

Thomsen-Friedenreich antigen (TF-Ag) is the disaccharide (Gal beta1-3 GalNAc alpha), which is also known as the core 1 structure. The presence of this disaccharide on the surface of approximately 90 percent of carcinomas is due to altered glycosylation in these tumors. TF-Ag plays a role in the adhesive properties of tumor cells involved in metastasis. Treatment of mice with JAA-F11, a monoclonal antibody to TF-Ag alpha inhibited lung metastasis and improved prognosis in a mouse breast cancer model. The presence of naturally occurring antibodies to TF-Ag in cancer patients is related to improved prognosis. The pancarcinoma expression of TF-Ag, combined with the evidence of a mechanistic role for TF-Ag in cancer spread, show that this target would have clinical utility. The presence of naturally occurring antibody to TF-Ag indicates that increasing the anti-TF-Ag antibody would be safe for the cancer patient and indicates that tolerance would not have to be broken to create this immune response. Finally, the prognostic improvements seen clinically and in animal models indicate that this is an important vaccine target.

Structural Variation of Chondroitin Sulfate and Its Roles in the Central Nervous System

Chondroitin sulfate is popular in the field of neuroscience, because the treatment of nervous tissues with chondroitinase ABC, which degrades chondroitin sulfate up to unsaturated disaccharides, causes severe changes in various aspects of neural development and functions. Chondroitinase ABC treatments of developing nervous tissue impair the growth and differentiation of neural progenitor cells, and cause various pathfinding errors of axons. After injury to the adult central nervous system, axon regeneration fails at scar regions expressing large amounts of chondroitin sulfate proteoglycans. However, after chondroitinase ABC treatment, many axons regenerate and traverse the damaged areas. Furthermore, it was revealed that chondroitin sulfate proteoglycans are involved in neural plasticity. These observations indicated that chondroitin sulfate proteoglycans as major components of the extracellular matrix and cell surface play pivotal roles in the development, regeneration, and plasticity of neuronal networks. Chondroitin sulfate shows highly diverse structural variation, and recent studies indicated that this glycosaminoglycan binds with various growth factors, chemokines and axon guidance molecules in a structure-dependent manner and regulates their activities. Notably, oversulfated structures such as D (GlcA(2-O-sulfate)beta 1-3GalNAc(6-O-sulfate)) and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the binding sites for many proteins, and play important roles in regulation of the growth of neural progenitors, neurite extension, and neuronal migration. The synthesis of these structures is strictly regulated by the chondroitin sulfate synthase family and many sulfotransferases, which should be useful therapeutic targets in neurological disorders.

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

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.

Differences in influenza virus receptors in chickens and ducks: Implications for interspecies transmission

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAalpha2,3-Gal receptors, we found widespread presence of both SAalpha2,6-Gal and SAalpha2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAalpha2,6-Gal was the dominant receptor type whereas in ducks SAalpha2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAalpha2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAalpha2,3-Gal receptors (both beta(1-4)GlcNAc and beta(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses.

Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human -1,3/4-fucosyltransferase in FVB/N mouse stomach

The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.

Helicobacter pylori binding to new glycans based on N-acetyllactosamine

Previously we reported binding of Helicobacter pylori to various nonacid and sialylated neolacto carbohydrate structures using a wide range of natural and chemically modified sequences. A novel nonsialylated neolacto-based binding epitope, GlcNAc beta 3Gal beta 4GlcNAc, and analogous structures carrying terminal GalNAc beta 3, GalNAc alpha 3, or Gal alpha 3 showed the binding activity (Miller-Podraza H, Lanne B, Angström J, Teneberg S, Abul-Milh M, Jovall P-A, Karlsson H, Karlsson K-A. 2005. Novel binding epitope for Helicobacter pylori found in neolacto carbohydrate chains. J Biol Chem. 280:19695-19703). The present work reports two other H. pylori-binding nonsialylated neolacto-based structures, GlcA beta 3Gal beta 4GlcNAc beta 3-R and Glc beta 3Gal beta 4GlcNAc beta 3-R, and two amide derivatives (N-methyl and N-ethyl) of GlcA beta 3Gal beta 4GlcNAc beta 3-R which were bound by H. pylori. The latter structures turned out to be more effective as H. pylori binders than the parent saccharide. New reducing-end variants of the neolacto epitope including species containing N-acetyllactosamine linked beta 6 to GlcNAc or Gal with similarity to branched polylactosamines and mucins were prepared and tested. The results extend our previous findings on binding specificities of H. pylori and show that this pathogen is able to interact with an array of N-acetyllactosamine/neolacto structures, which may be of importance for the in vivo interaction of the bacterium with human cells. The information gained in this work may also be of value for rational design of anti-H. pylori drugs.

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

Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-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-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (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-alpha-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-alpha-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, 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.

Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata)

In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal beta1,3 GalNAc and (GlcNAc beta1,4)(n) oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal beta1-3 GalNAc, Gal-alphaGal, and (GlcNAc beta1,4)(n) oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal beta1,3 GalNAc, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,6 or terminal alpha1,3 or alpha1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-alphaGal, Gal beta1-3 GalNAc and (GlcNAc beta1,4)(n)oligomers; Fuc linked alpha1,2 to Gal, alpha1,3 to GlcNAc in (poly)lactosamine chains and alpha1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H(+)K(+)-ATPase beta-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.

Conformational Studies on Five Octasaccharides Isolated from Chondroitin Sulfate Using NMR Spectroscopy and Molecular Modeling

Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(beta1-3)GalNAc(beta1- disaccharide units, partly O-sulfated at different positions. The presence of the disulfated disaccharide D-unit, GlcA2S(beta1-3)GalNAc6S, in the CS moiety of the proteoglycan DSD-1-PG/phosphacan, correlates with neurite outgrowth promotion. The binding of monoclonal antibody (mAb) 473HD to DSD-1-PG, reducing neuronal stimulation, is inhibited by shark cartilage CS-D. CS-D is also recognized by two other mAbs, MO-225 and CS-56. Conformational studies were performed using NMR spectroscopy and molecular modeling on five octasaccharides isolated from shark cartilage CS-D. These octasaccharides present different binding properties toward the three mAbs. The combination of the experimental and theoretical approaches revealed that the sulfate group at position 2 of GlcA in disaccharide D and the presence of an exocyclic negative tail in disaccharides C [GlcA(beta1-3)GalNAc6S] and DeltaC [Delta4,5HexA(alpha1-3)GalNAc6S] are important for antibody recognition.

Repeats of LacdiNAc and fucosylated LacdiNAc on N‐glycans of the human parasite Schistosoma mansoni

N-Glycans from glycoproteins of the worm stage of the human parasite Schistosoma mansoni were enzymatically released, fluorescently labelled and analysed using various mass spectrometric and chromatographic methods. A family of 28 mainly core-alpha1-6-fucosylated, diantennary N-glycans of composition Hex(3-4)HexNAc(6-12)Fuc(1-6) was found to carry dimers of N,N'-diacetyllactosediamine [LacdiNAc or LDN; GalNAc(beta1-4)GlcNAc(beta1-] with or without fucose alpha1-3-linked to the N-acetylglucosamine residues in the antennae {GalNAc(beta1-4)[+/-Fuc(alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)[+/-Fuc(alpha1-3)]GlcNAc(beta1-}. To date, oligomeric LDN and oligomeric fucosylated LDN (LDNF) have been found only on N-glycans from mammalian cells engineered to express Caenorhabditis elegansbeta4-GalNAc transferase and human alpha3-fucosyltransferase IX [Z. S. Kawar et al. (2005) J Biol Chem280, 12810-12819]. It now appears that LDN(F) repeats can also occur in a natural system such as the schistosome parasite. Like monomeric LDN and LDNF, the dimeric LDN(F) moieties found here are expected to be targets of humoral and cellular immune responses during schistosome infection.

Characterization of mucin‐type core‐1 β1‐3 galactosyltransferase homologous enzymes in Drosophila melanogaster

Mucin type O-glycosylation is a widespread modification of eukaryotic proteins. The transfer of N-acetylgalactosamine to selected serine or threonine residues is catalyzed by a family of polypeptide N-acetylgalactosaminyltransferases localized in the Golgi apparatus. The most abundant elongation of O-glycans is the addition of a beta1-3 linked galactose by the core-1 beta1-3 galactosyltransferase (core-1 beta3GalT), thereby building the T-antigen or core-1 structure Gal(beta1-3)GalNAc(alpha1-O). We have isolated four Drosophila melanogaster cDNAs encoding proteins structurally similar to the human core-1 beta3GalT enzyme and expressed them as FLAG-tagged proteins in Sf9 insect cells. The identity of these D. melanogasterbeta3GalT enzymes with a core-1 beta3GalT activity was confirmed by utilization of MUC5AC mucin derived O-glycopeptide acceptors. In addition to the core-1 beta3GalT activity toward O-glycoprotein substrates, one member of this enzyme family showed a strong activity towards glycolipid acceptors, thereby building the core-1 terminated Nz6 glycosphingolipid. Transcripts of the embryonically expressed core-1 beta3GalTs were found in the maternally deposited mRNA, in salivary glands and in the amnioserosa. The presence of multiple core-1 beta3GalT genes in D. melanogaster suggests an increased complexity of core-1 O-glycan expression, which is possibly related to multiple developmental and physiological functions attributable to this class of glycans.

Immune response to Thomsen–Friedenreich disaccharide and glycan engineering

Cancer-associated mucins show frequent alterations of their oligosaccharide chain profile, with a switch to unmask normally cryptic O-glycan backbone and core regions. Epithelial tumour cells typically show overexpression of the uncovered Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr (Core 1) structure, known as the T antigen or the Thomsen-Friedenreich antigen, the oligosaccharide chain of which is called the Thomsen-Friedenreich disaccharide (TFD). T antigen expression has been associated with immunosuppression, metastasis dissemination, and the proliferation of cancer cells. Several different strategies have been used to trigger a specific immune response to TFD. Natural T antigen and synthetic TFD residues have low immunodominance. In the T antigen, flexibility of the glycosidic bond reduces the immunogenicity of the sugar residue. Enhanced rigidity should favour certain glycan conformations and thereby improve TFD immunotargeting. We propose the term 'glycan engineering' for this approach. Such engineering of TFD should reduce the flexibility of its glycan moiety and thereby enhance its stability, rigidity and immunogenicity.

Pepsinogen and H,K-ATPase mediate acid secretion in gastric glands of Triturus carnifex (Amphibia, Caudata)

The gastric glands of Triturus carnifex (Amphibia, Caudata) have been examined by histochemical and immunohistochemical methods with particular regard to hydrochloric acid and pepsinogen secretion. Fundic glands consist of mucous neck cells, endocrine cells and oxynticopeptic cells producing both pepsinogen and hydrochloric acid. The neck cells showed an unexpected distribution pattern which was only observed in the oral fundus, and produced neutral mucins with glycosidic residues of GalNAc and Gal beta1,3GalNAc, and in this respect they differ from the neck cells of anuran amphibians. The secretion of pepsinogen and hydrochloric acid as demonstrated by immunolabelling with anti-H,K-ATPase and with anti-pepsinogen, respectively, seems not to vary significantly along the longitudinal axis of the stomach. The mechanism of gastric acid secretion seems to be mediated by an ATPase, having similar features to the mammalian gastric H,K-ATPase, and is localised in the luminal membrane and in the subapical cytoplasm of the oxynticopeptic cells. Unusually, the same cytoplasmic areas revealed binding specificity for the winged pea lectin (WPA) from Lotus tetragonolobus, even after beta elimination, indicating the presence of fucosyl residues in N-linked oligosaccharidic chains in glycoproteins of beta-H,K-ATPase subunits.

Lectinochemical studies on the glyco-recognition factors of a Tn (GalNAcα1→Ser/Thr) specific lectin isolated from the seeds of Salvia sclarea

The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc alpha1-->Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAcalpha1--> related oligosaccharides and glycopeptides. Thus, the requirements of functional groups in monosaccharide and high-density polyvalent carbohydrate structural units for SSL binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested for interaction, a high density of exposed Tn-containing glycoproteins such as in the armadillo salivary Tn glycoprotein and asialo ovine salivary glycoprotein reacted best with SSL. When the gps were tested for inhibition of SSL binding, which was expressed as 50% nanogram inhibition, the high density polyvalent Tn present in macromolecules was the most potent inhibitor. Among the monosaccharide and carbohydrate structural units studied, which were expressed as nanomole inhibition, GalNAc alpha1-->3GalNAc beta1-->3Gal alpha1-->4Gal beta1-->4Glc (Fp), GalNAc alpha1-->3Gal beta1-->4Glc (A(L)), GalNAc alpha1-->3GalNAc beta1-->Me (F beta), GalNAc alpha1-->3GalNAc alpha1-->Me (F alpha) and GalNAc alpha1--> Ser/Thr (Tn) were the most active ligands, being 2.5-5.0 x 10(3) and 1.25-2.5 times more active than Gal and GalNAc, respectively. From the results, it is suggested that the combining site of SSL is a shallow groove type, recognizing the monosaccharide of GalNAc as the major binding site or Tn up to the Forssman pentasaccharide (Fp). It can be concluded that the three critical factors for SSL binding are the -NH CH(3)CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc alpha1-->Ser/Thr) present in macromolecules. These results should assist in understanding the glyco-recognition factors involved in carbohydrate-lectin interactions in biological processes. The effect of the polyvalent F alpha, F beta and GalNAc beta1-->3Gal alpha1--> (P alpha) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.

Chondroitin sulphate structure affects its immunological activities on murine splenocytes sensitized with ovalbumin.

Chondroitin sulphate (CS) is a glycosaminoglycan widely distributed in animal tissues, which has anti-inflammatory and chondroprotective properties. We reported previously that chondroitin 4-sulphate (CS-A) up-regulates the antigen-specific Th1 immune response of murine splenocytes sensitized with ovalbumin in vitro, and that CS suppresses the antigen-specific IgE responses. We now demonstrate that a specific sulphation pattern of the CS polysaccharide is required for the Th1-promoted activity, as other polysaccharides such as dextran and dextran sulphate do not significantly induce this activity. While the presence of some O-sulpho groups appear to be essential for activity, CS-A, and synthetically prepared, partially O-sulphonated CS, induce higher Th1-promoted activity than synthetically prepared, fully O-sulphonated CS. CS-A induces an activity greater than chondroitin sulphate B (CS-B) or chondroitin 6-sulphate (CS-C). In addition, chondroitin sulphate E (CS-E) induces greater activity than CS-A or CS-D. These results suggest that the GlcA(beta1-3)GalNAc(4,6-O-disulpho) sequence in CS-E is important for Th1-promoted activity. Furthermore, rat anti-mouse CD62L antibody, an antibody to L-selectin, inhibits the Th1-promoting activity of CS. These results suggest that the Th1-promoted activity could be associated with L-selectin on lymphocytes. These findings describe a new mechanism for the anti-inflammatory and chondroprotective properties of CS that may be useful in designing new therapeutic applications for CS used in the treatment of immediate-type hypersensitivity.

LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.

Although Gal beta 1-4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAc beta 1-4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.

TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells.

Toll-like receptors (TLRs) mediate host responses to bacterial gene products. As the airway epithelium is potentially exposed to many diverse inhaled bacteria, TLRs involved in defense of the airways must be broadly responsive, available at the exposed apical surface of the cells, and highly regulated to prevent activation following trivial encounters with bacteria. We demonstrate that TLR2 is enriched in caveolin-1-associated lipid raft microdomains presented on the apical surface of airway epithelial cells after bacterial infection. These receptor complexes include myeloid differentiation protein (MyD88), interleukin-1 receptor-activated kinase-1, and TNF receptor-associated factor 6. The signaling capabilities of TLR2 are amplified through its association with the asialoganglioside gangliotetraosylceramide (Gal beta 1,2GalNAc beta 1,4Gal beta 1,4Glc beta 1,1Cer), which has receptor function itself for many pulmonary pathogens. Ligation of either TLR2 or asialoGM1 by ligands with specificity for either receptor, by Pseudomonas aeruginosa, or by Staphylococcus aureus stimulates IL-8 production through activation of NF-kappa B, as mediated by TLR2 and MyD88. Thus, TLR2 in association with asialo-glycolipids presented within the context of lipid rafts provides a broadly responsive signaling complex at the apical surfaces of airway cells to initiate the host response to potential bacterial infection.

Glycoconjugate histochemistry of the seminal vesicles of the Japanese miniature (Shiba) goat.

The present study localizes and characterizes complex glycoconjugates in the seminal vesicles of the Japanese Shiba goat, using several carbohydrate histochemical procedures, including lectin techniques at light and electron microscopic levels. Glandular epithelial cells and luminal secretions were shown to contain neutral and acidic glycoconjugates with various saccharide residues, such as alpha-D-Man, alpha-D-Glc, alpha-L-Fuc, beta-D-Gal, GalNAc, GlcNAc, and NeuAc (sialic acid). The terminal oligosaccharide chains of sialoglycoconjugates present in the seminal vesicles were NeuAc alpha 2-3Gal beta 1-3GalNAc and NeuAc alpha 2-3Gal beta 1-4GlcNAc. In addition, in lysosomes of the glandular epithelial cells alpha-D-Man, alpha-D-Glc, GlcNAc and NeuAc (sialic acid) residues could be detected, the secretory vesicles contained alpha-L-Fuc, and the endoplasmic reticulum exhibited alpha-D-Man and alpha-D-Glc residues. The complex glycoconjugates with various sugar residues found in the seminal vesicles of the goat may be involved in various fertilization-related events.