Sulfoglycolipids bind to adhesive protein amphoterin (P30) in the nervous system.

HNK-1 antibody reactive carbohydrate epitope carried by glycolipids and glycoproteins has been shown to be involved in cell to cell interactions. It has been proposed that the HNK-1 reactive 3-sulfoglucuronyl carbohydrate epitope in glycolipids may interact with a cell surface receptor to promote the biological response in the developing nervous system. The possible occurrence of such a receptor was examined in rat nervous system. A specific binding of sulfoglycolipids to a 30 kD protein from adult rat cerebellum is described. Little binding was found with neutral glycolipids and gangliosides. The 30 kD protein from cerebellum was partially purified on a sulfatide-octyl-Sepharose affinity column. Binding of sulfoglucuronyl glycolipids to a similar 30 kD protein from forebrain previously identified as amphoterin is also shown. Amphoterin is developmentally regulated and is involved in neural cell adhesion and neurite extension.     

On the dissection of binding epitopes on carbohydrate receptors for microbes using molecular modelling

The most common receptors for microbes on animal cells seem to be carbohydrates. One characteristic property of microbial protein-carbohydrate interaction is the recognition of sequences placed within an oligosaccharide chain. This leads to a series of isoreceptors defined as saccharides carrying the particular receptor sequence with different neighbouring groups. A microbial ligand may have different binding affinities for such isoreceptors depending upon steric hindrance from neighbouring groups upon access to the binding epitope. By a comparison of binding preferences to a series of isoreceptors with their calculated conformation, the binding epitope on a particular receptor sequence may be approximated by use of molecular modelling. This approach is illustrated for two bacteria recognising lactosylceramide. The potential importance of the procedure for further developments including drug design is briefly discussed.     

Aglycone modulation of glycolipid receptor function

The aglycone has been largely ignored in consideration of glycoconjugate function. Evidence is reviewed which suggests that the role of the lipid in glycolipid carbohydrate function may be particularly significant. The lipid moiety can promote or reduce carbohydrate exposure of membrane glycolipids. Theoretical calculation has indicated that the plane of the plasma membrane can restrict the permitted conformations of a given glycolipid oligosaccharide. Thus the lipid moiety may influence the relative conformation of such carbohydrate sequences. Evidence of ceramide regulation of glycolipid function can be found in studies of enzyme substrate specificity, antiglycolipid recognition and bacterial/host cell interactions. Studies of verotoxin binding to its glycolipid receptor globotriaosyl ceramide indicate that modulation of receptor function by glycolipid fatty acid content plays an important role inin vitro binding assays, cell cytotoxicity and intracellular routing.     

MHC interaction and T cell recognition of carbohydrates and glycopeptides.

The T cell independence of complex polysaccharide Ag has suggested the possibility that carbohydrates may be incapable of T cell recognition because of a failure to interact with MHC restriction elements and/or a failure of MHC/carbohydrate complexes to interact with and be recognized by Ag-specific TCR. We have used two approaches to obtain information about T cell recognition of carbohydrate. First, we have determined the capacity of a series of oligosaccharides and glycolipids to bind a murine class II MHC molecule, IAd. No significant binding was observed with the 26 compounds tested, but the limitation to these studies was that there was a relatively limited collection of synthetic carbohydrate and glycolipid structures of limited complexity available for analysis. The second approach involved the study of the effect of glycosylation of a known peptide T cell epitope (OVA 323-339) on MHC binding of the peptide and on T cell recognition. Three patterns of effects were observed: 1) no effect on either binding or T cell recognition. This pattern was observed when the carbohydrate was located at residues removed from the core MHC-binding region. When the carbohydrate was located within the core MHC-binding regions, either 2) glycosylation destroyed both MHC binding and T cell recognition; or 3) glycosylation did not ablate MHC binding or T cell recognition. In this latter instance, there was evidence to indicate that the carbohydrate moiety was an important part of the antigenic determinant recognized by T cells.     

Carbohydrate recognition in the peripheral nervous system: a calcium- dependent membrane binding site for HNK-1 reactive glycolipids potentially involved in Schwann cell adhesion

The carbohydrate determinants recognized by the HNK-1 antibody are potential cell-cell recognition ligands in the peripheral nervous system (PNS). The HNK-1 reactive sulfoglucuronylneolacto (SGNL) glycolipids specifically support Schwann cell adhesion, suggesting the presence of a cell surface receptor specific for SGNL-oligosaccharides. We directly probed PNS membranes for receptors complementary to SGNL determinants using a synthetic radioligand consisting of radioiodinated serum albumin derivatized with multiple SGNL-oligosaccharides. A high- affinity, saturable, calcium-dependent binding site for this ligand was found in PNS myelin membranes. Binding activity was carbohydrate- specific (most potently inhibited by SGNL-lipids compared to other glycolipids) and PNS-specific (absent from comparable central nervous system membranes). The SGNL-specific binding activity on PNS membranes reported here may be involved in peripheral myelination or myelin stabilization.     

Carbohydrates act as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis: a study of bacterial binding to glycolipids

In this study we show for the first time the use of carbohydrate chains on glycolipids as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis. Previous studies have shown that this bacterium has the ability to adhere to and invade the epithelial lining of the dental pocket. Which receptor(s) the adhesin of P. gingivalis exploit in the adhesion to epithelial cells has not been shown. Therefore, the binding preferences of this specific bacterium to structures of carbohydrate origin from more than 120 different acid and nonacid glycolipid fractions were studied. The bacteria were labeled externally with (35)S and used in a chromatogram binding assay. To enable detection of carbohydrate receptor structures for P. gingivalis, the bacterium was exposed to a large number of purified total glycolipid fractions from a variety of organs from different species and different histo-blood groups. P. gingivalis showed a preference for fractions of human and pig origin for adhesion. Both nonacid and acid glycolipids were used by the bacterium, and a preference for shorter sugar chains was noticed. Bacterial binding to human acid glycolipid fractions was mainly obtained in the region of the chromatograms where sulfated carbohydrate chains usually are found. However, the binding pattern to nonacid glycolipid fractions suggests a core chain of lactose bound to the ceramide part as a tentative receptor structure. The carbohydrate binding of the bacterium might act as a first step in the bacterial invasion process of the dental pocket epithelium, subsequently leading to damage to periodontal tissue and tooth loss.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc

We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.     

Characteristic binding of human plasma apolipoprotein B to gangliotetraosylceramide and gangliotriaosylceramide.

The binding of human plasma low-density lipoproteins (LDL), freshly prepared by discontinuous ultracentrifugation, to several neutral and acidic glycosphingolipids was examined by TLC immunostaining with the anti [apolipoprotein B (apoB)] antibody. ApoB was found to bind characteristically to the asialogangliosides, gangliotetraosylceramide (Gg4Cer) and gangliotriaosylceramide (Gg3Cer), the former being a more potent receptor than the latter, indicating that the sequences Gal beta 1-3GalNAc beta 1-4Gal and GalNAc beta 1-4Gal are involved in the binding of apoB. A weak positive reaction with fucosylgangliotetraosylceramide (IV2Fuc-Gg4Cer), which has the same internal recognition sequences, was also observed (the binding ability was only 1/7 of that in the case of Gg4Cer). No binding to other neutral glycosphingolipids, or glycosphingolipid sulfates (I3-SO3-GalCer) and gangliosides, was detected, and therefore substitution of the receptor glycolipid with sialic acid was thought to inhibit the binding. The results indicate that, along with the binding of apoB to the LDL-binding domain of the receptor glycoprotein, interaction with some carbohydrate chains in the receptor, or with glycolipids coexisting on the plasma membrane, may be important for the binding of apoB to cells.     

Characterization of two monoclonal antibodies specific for lacto-series type 1 chain Gal beta 1----3GlcNAc-terminal structures

Murine monoclonal antibodies, TE-1 and TE-3, generated by immunization with a biosynthetic reaction product containing a terminal Gal beta 1----3GlcNAc structure have been produced and found to react specifically with underivatized type 1 chain lacto-series carbohydrate structures. Detailed analysis of these antibodies, both IgM, indicates two differing classes of epitope specificity. Antibody TE-1 was found to bind preferentially to longer chain carbohydrate structures containing a terminal Gal beta 1----3GlcNAc disaccharide, indicating that optimal antibody binding involved more than recognition of this disaccharide. In contrast, antibody TE-3 was found to bind strongly carbohydrate structures containing terminal Gal beta 1----3GlcNAc structures irrespective of chain length. Modification of core chain structures by addition of fucose and/or sialic acid residues completely abolished antibody binding with either antibody. TLC immunostaining of neutral glycolipids isolated from a variety of human colonic adenocarcinoma cell lines indicated intensely stained bands, particularly with antibody TE-3, which correlated with the level of expression of type 1 chain based glycolipid derivatives. These antibodies are applied to the detailed study of the regulation of synthesis of lacto-series type 1 chain based carbohydrate structures.     

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.     

Detection of the L2/HNK-1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.     

Perspectives for establishment and reorganization of carbohydrate-directed CD antibodies. Report of the carbohydrate section

Complex glycosylated glycoproteins, glycolipids and proteoglycans are expressed on the cell surface and are also found as constituents of the extracellular matrix (ECM). Interactions of the carbohydrate moiety of these macromolecules with specific receptors (lectins) are involved in many functions of immune cells such as cell-cell or cell-ECM adhesion, recognition, and neutralization of pathogens and regulation of apoptosis. For studies on live cells mAbs recognizing distinct oligosaccharide structures are useful tools because in contrast to other analytical methods of carbohydrate biochemistry they are able to react with glycans in the complex sterical context of the cell surface. In general expression patterns of carbohydrate mAbs depend on (i) the number and type of carriers to which the glycans are linked (glycoproteins, glycolipids), (ii) the steric situation on the cell surface, and (iii) modifications of the basic glycotope (different branching, chain length, masking by sialylation, sulphation or fucosylation).     

Expression and biological functions of sulfoglucuronyl glycolipids (SGGLs) in the nervous system—A review

Sulfoglucuronyl carbohydrate linked to neolactotetraose reacts with HNK-1 antibody. The HNK-1 carbohydrate epitope is found in two major glycolipids, several glycoproteins and in some proteoglycans of the nervous system. Most of the HNK-1 reactive glycoproteins so far identified are neural cell adhesion molecules and/or are involved in cell-cell interactions. HNK-1 carbohydrate is highly immunogenic. Several HNK-1-like antibodies, including IgM of some patients with plasma cell abnormalities and having peripheral neuropathy, have been described. This article summarizes published work mainly on sulfoglucuronyl glycolipids, SGGLs and covers: structural requirements of the carbohydrate epitope for binding to HNK-1 and human antibodies, expression of the lipids in various neural areas, stage and region specific developmental expression in CNS and PNS, immunocytochemical localization, loss of expression in Purkinje cell abnormality murine mutations, biosynthetic regulation of expression by a single enzyme N-acetylglucosaminyl transferase, identification of receptor-like carbohydrate binding neural proteins (lectins), and perceived role of the carbohydrate in physiological functions. The latter includes role in: pathogenesis of certain peripheral neuropathies, in migration of neural crest cells, as a ligand in cell-cell adhesion/interaction and as a promoter of neurite outgrowth for motor neurons. Multiple expression of HNK-1 carbohydrate in several molecules and in various neural cell types at specific stages of nervous system development has puzzled investigators as to its specific biological function, but this may also suggest its importance in multiple systems during cell differentiation and migration processes.Special issue dedicated to Dr. Marjorie B. Lees.     

Collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system

Collectins and ficolins represent two important groups of pattern recognition molecules, which bind to oligosaccharide structures on the surface of microorganisms, leading to the killing of bound microbes through complement activation and phagocytosis. Collectins and ficolins bear no significant sequence homology except for the presence of collagen-like sequences over the N-terminal halves of the polypeptides that enable the assembly of these molecules into oligomeric structures. Collectins and ficolins both contain lectin activities within the C-terminal halves of their polypeptides, the C-type carbohydrate recognition domain (CRDs) and fibrinogen beta/gamma (homology) (FBG) domain, respectively. These domains form trimeric clusters at the ends of the collagen triple helices emanating from a central hub, where the N-terminal ends of the polypeptides merge. The collectins and ficolins seem to have evolved to recognize the surface sugar codes of microbes and their binding, to these arrays of cell surface carbohydrate molecules, targets the microbe for subsequent clearance by phagocytic cells.     

Determination of structural elements of the L2/HNK-1 carbohydrate epitope required for its function

The L2/HNK-1 carbohydrate epitope has been shown to carry an unusual 3-sulfoglucuronic acid linkedO-glycosidically through a neolactosyl-type back bone to a ceramide residue. Using monoclonal antibodies, the same or a closely related epitope has also been detectedN-glycosidically linked to glycoproteins, amongst them several neural cell adhesion molecules. We used synthetic glycolipids carrying sulfated or non-sulfated glucuronic acid attached to ceramide through glycans of different length to show that not only the sulfated glucuronic acid but also the neolactosyl-type backbone is essential for the recognition of the L2/HNK-1 carbohydrate by a monoclonal antibody, its binding to laminin and its role in neural cell migration and outgrowth of processes from neurons and astrocytes.Abbreviations mab monoclonal antibody - TLC thin layer chromatography - HRP horseradish peroxidase - glcA glucuronic acid - gal galactose - glcNAc N-acetyl-glucosamine - man mannose     

Close-up of the immunogenic α1,3-galactose epitope as defined by a monoclonal chimeric immunoglobulin E and human serum using saturation transfer difference (STD) NMR

Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen α1,3-Gal (α-Gal) are associated with a delayed type of anaphylaxis, providing evidence for the clinical relevance of carbohydrate epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction, and fine epitope of α-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety of an α-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed by mediator release assays, surface plasmon resonance (SPR), and saturation transfer difference NMR analyses. The α-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The α1,3-Gal epitope fine structures of both the recombinant IgE and affinity-purified serum were defined by saturation transfer difference NMR, revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying α1,3-Gal-mediated anaphylaxis. The complementary data of affinity and fine specificity may help to elucidate the recognition of carbohydrates by the adaptive immune response and the molecular requirements of carbohydrate-based anaphylaxis.     

Understanding TR binding to pMHC complexes: how does a TR scan many pMHC complexes yet preferentially bind to one

Understanding the basis of the binding of a T cell receptor (TR) to the peptide-MHC (pMHC) complex is essential due to the vital role it plays in adaptive immune response. We describe the use of computed binding (free) energy (BE), TR paratope, pMHC epitope, molecular surface electrostatic potential (MSEP) and calculated TR docking angle (θ) to analyse 61 TR/pMHC crystallographic structures to comprehend TR/pMHC interaction. In doing so, we have successfully demonstrated a novel/rational approach for θ calculation, obtained a linear correlation between BE and θ without any "codon" or amino acid preference, provided an explanation for TR ability to scan many pMHC ligands yet specifically bind one, proposed a mechanism for pMHC recognition by TR leading to T cell activation and illustrated the importance of the peptide in determining TR specificity, challenging the "germline bias" theory.     

Sweet Tooth, a novel receptor protein-tyrosine kinase with C-type lectin-like extracellular domains

A gene encoding a novel type of receptor protein-tyrosine kinase was identified in Hydra vulgaris. The extracellular portion of this receptor (which we have named Sweet Tooth) contains four C-type lectin-like domains (CTLDs). Comparison of the sequences of these domains with the sequences of the carbohydrate recognition domains of various vertebrate C-type lectins shows that Sweet Tooth CTLD1 and CTLD4 have amino acids in common with those shown to be involved in carbohydrate binding by the lectins. Comparison of sequences encoding CTLD1 from the Sweet Tooth genes from different species of Hydra shows variation in some of the conserved residues that participate in carbohydrate binding in C-type lectins. The Sweet Tooth gene is expressed widely in the Hydra polyp, and expression is particularly high in the endoderm of the tentacles. Treatment of polyps with peptides corresponding to sequences in the Sweet Tooth CTLDs results in the disintegration of the animal. These same peptides do not block adhesion or morphogenesis of Hydra cell aggregates.     

Identification of a structure on human melanoma cells recognized by CTL exhibiting anomalous killer cell function

The structures involved in the recognition of melanoma cells by nonspecific cytotoxic T lymphocytes (CTL) activated in mixed lymphocyte culture were investigated with monoclonal antibodies (MAb) which blocked this anomalous killer (AK) function. Of over 2000 MAb raised against melanoma cells, only three inhibited killing; one of these, an IgMk termed Leo Me13, was investigated in detail. In antibody-binding studies using a large range of cultured tumor cells, it was shown that Leo Me13 was relatively specific for melanoma cells. Of more importance, Leo Me13 inhibited conjugate formation between AK cells and melanoma target cells by 60 to 80% and caused an eight- to 10-fold reduction in killing. The MAb did not immunoprecipitate protein from melanoma cells surface-labeled with 125I, and thin-layer chromatography followed by immunoblotting of the separated glycolipids from melanoma cells indicated that the epitope was on acidic glycolipids migrating between GM1 and GD1a; moreover, treatment of melanoma cells with neuraminidase resulted in complete loss of binding of Leo Me13 but not of other anti-melanoma antibodies which did not inhibit AK cell-mediated lysis. Other melanoma-reactive MAb of the same isotype as Leo Me13 did not block killing of melanoma cells, but one documented antibody, R24, an IgG3 with specificity for the ganglioside GD3, was found to inhibit this function. These data suggest that the AK cells recognize and bind to melanoma cells by a secondary "lectin-type" receptor for a carbohydrate moiety.