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Publisher Summary Glycosphingolipids are classified primarily on the basis of their carbohydrate portion, which may vary from a single monosaccharide to large branched structures composed of >20 monosaccharide units. Techniques for glycosphingolipid purification have largely focused on separations based on saccharide differences, resulting in purification to oligosaccharide homogeneity. There is additional variation in the ceramide, which is composed of any of several long-chain bases (of which sphingosine is the most common in mammalian tissues), each of which is further substituted, through amide linkage, with any of a variety of fatty acids. Glycosphingolipid purification involves three steps—(1) extraction of lipids from a biological source, using organic solvents, (2) bulk separation from major lipid and nonlipid contaminants, and (3) chromatographic resolution of individual species. Isolation of glycosphingolipids requires repeated removal of solvents. For small samples this is most conveniently achieved under a stream of nitrogen in a heated block or water bath at ∼45°, using a commercial apparatus for multiple small samples. For large numbers of small samples containing volatile salts, which require vacuum for efficient removal, a SpeedVac concentrator is recommended.

Publisher Summary This chapter discusses the receptors for gangliosides and related glycosphingolipids on central and peripheral nervous system cell membranes. The chapter focuses on the detection and characterization of novel protein receptors for gangliosides and related glycosphingolipids in the nervous system. The incorporated strategy is to identify ganglioside (and related) binding activities using synthetic glycosphingolipid-based radioligands, to characterize the structural and tissue specificity of binding activities, and to purify the responsible protein receptors. The chapter also discusses the identification of a ganglioside receptor in the central nervous system and a receptor for sulfoglucuronyl glycosphingolipids in the peripheral nervous system. Through the use of semi-synthetic glycosphingolipidbased neoglycoconjugates—which combine multivalence—sequestering or removal of the lipid moiety, and facile radioiodination, research is successful in demonstrating unique binding activities for gangliosides and sulfoglucuronyl glycosphingolipids in the central nervous system (CNS) and peripheral nervous system (PNS) respectively. Although the identification of the functional roles of these glycosphingolipid- specific binding activities await further studies, their differential tissue and membrane distribution suggests that they may be involved in ‘trans' interactions between myelinating cells and axons.

Publisher Summary Nonspecific hydrophobic binding and membrane insertion limit the usefulness of radiolabeled gangliosides as high-affinity probes for carbohydrate-specific ganglioside receptors on cell membranes. To circumvent this limitation, the chapter discusses the synthesis of neoganglioproteins, which consists of gangliosides covalently linked, via the lipid moiety, to bovine serum albumin (BSA). Neoganglioproteins have the advantage of presenting the ganglioside determinants in a polyvalent array that may be important for high-affinity binding to complementary receptors. Ganglioside-protein conjugates are readily radiolabeled to high specific activity by standard protein radioiodination techniques allowing their use as radioligands to detect complementary ganglioside-bindging activities. Neoganglioprotein characterization involves determining the carbohydrate composition and structure and confirming the covalent nature of the carbohydrate–protein bond. Protein values are determined by any suitable protein microassay.

Publisher Summary Recent advances in carbohydrate chemistry and biochemistry afford the opportunity to develop bioactive complex carbohydrates, per se , as drugs or as lead compounds in drug development. Complex carbohydrates are unique among biopolymers in their inherent potential to generate diverse molecular structures. While proteins vary only in the linear sequence of their monomer constituents, individual monosaccharides can combine at any of several sites on each carbohydrate ring, in linear or branched arrays, and with varied stereochemistry at each linkage bond. This chapter addresses some salient features of mammalian glycoconjugate structure and biosynthesis, and presents examples of the biological activities of complex carbohydrates. The chapter presents selected examples that will provide an accurate introduction to their pharmacological potential. In addition to their independent functions, oligosaccharides can modify the activities of proteins to which they are covalently attached. Many glycoprotein enzymes and hormones require glycosylation for expression and function. The chapter discusses the ancillary role of carbohydrates that is of great importance to the use of engineered glycoproteins as pharmaceuticals.

Sucrose phosphorylase (EC 2.4.1.7) reaction was combined into one-pot galactosyltransferase reaction in which the enzyme scavenged for the accumulated inorganic phosphate and utilized it with sucrose to generate in situ glucose I-phosphate. that was the essential component for galactosylation.

Glutamate binds to both excitatory neurotransmitter binding sites and a Cl − -dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (N18-RE105) are susceptible to glutamate-induced cytotoxicity. The Cl − -dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Lowering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and Ca 2+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like N18-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Glutamate is thought to be a major excitatory neurotransmitter in the central nervous system. To study the glutamate receptor and its regulation under carefully controlled conditions, the specific binding of [3H]glutamate was characterized in washed membranes isolated from a neuroblastoma X retina hybrid cell line, N18-RE-105. [3H]Glutamate bound in a saturable and reversible fashion with an apparent dissociation constant, KD, of 650 nM and a maximum binding capacity, Bmax, of 16 pmol/mg of protein. Pharmacologic characterization of the site indicates that it closely resembles the Na+-independent binding site for glutamate found on brain membranes and thought to be an excitatory amino acid neurotransmitter receptor. Thus, while kainate, N-methyl-DL-aspartate, and nonamino acid ligands did not displace [3H]glutamate, quisqualate and ibotenate were potent inhibitors of specific binding. Furthermore, this binding site is regulated by ions in a manner which resembles that described in the hippocampus (Baudry, M., and Lynch, G. (1979) Nature (Lond.) 282, 748-750). Calcium (10 mM) increased the number of binding sites 2.6-fold with no change in receptor-ligand affinity. Lanthanum (1 mM) was the only other cation added which enhanced (3-fold) the binding of [3H]glutamate. Monovalent cations resulted in a decrease in the number of glutamate binding sites. Incubation of membranes in the presence of chloride ions caused a marked increased in [3H] glutamate binding, an effect which was synergistic with that of calcium incubation. Thus, N18-RE-105 cells possess a binding site for [3H]glutamate pharmacologically similar to an excitatory neurotransmitter binding site in brain and which exhibits regulatory properties resembling those previously described in hippocampal membranes, providing an excellent model for mechanistic studies.

Phosphomannan polysaccharides and fucoidan, a polymer of fucose 4-sulfate, have been demonstrated to inhibit adhesion of lymphocytes to tissue sections that contain high endothelial venules (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). We have investigated the potential cell surface carbohydrate receptors involved by quantitating adhesion of rat cervical lymph node lymphocytes to purified polysaccharides immobilized on otherwise inert polyacrylamide gels. One-sixth of the lymphocytes adhered specifically to surfaces derivatized with PPME (a phosphomannan polysaccharide prepared from Hansenula holstii yeast), whereas up to half of the cells adhered to surfaces derivatized with fucoidan. Several lines of evidence demonstrated that two distinct receptors were involved. Adhesion to PPME-derivatized gels was labile at 37 degrees C (decreasing to background levels within 120 min) whereas adhesion to fucoidan-derivatized gels was stable. Soluble PPME and other phosphomannans blocked adhesion only to PPME-derivatized gels; fucoidan and a structurally related fucan blocked adhesion to fucoidan-derivatized gels. Other highly charged anionic polysaccharides, such as heparin, did not block adhesion to either polysaccharide-derivatized gel. Adhesion to PPME-derivatized gels was dependent on divalent cations, whereas that to fucoidan-derivatized gels was not. The PPME-adherent lymphocytes were shown to be a subpopulation of the fucoidan-adhesive lymphocytes which contained both saccharide receptors. These data reveal that at least two distinct carbohydrate receptors can be found on peripheral lymphocytes.

To understand better the proximate mechanism involved in the excitotoxic response to l-glutamate (Glu), we have exploited the Glu receptor present in the N18-RE-105 neuroblastoma-embryonic retinal hybrid cell line. These cells undergo lysis dependent on extracellular Ca2+ when exposed to Glu. We now report that the depolarizing action of Glu is not responsible for its cytotoxic effects. Furthermore, depolarization of these cells with elevated K+, ouabain or veratridine does not cause cytotoxicity but rather protects against the cytotoxic effects of Glu. Our results may implicate a role for voltage-sensitive Ca2+ channels (VSCCs) in cytotoxicity, and depolarization-induced inactivation of VSCCs (Nature (Lond.), 316 (1985) 440–443.) as a protection against Glu receptor agonists. Our findings demonstrate a clear dissociation between depolarization and the neuronal degeneration caused by Glu.

A new and versatile method for linking biologically active ligands to a polyacrylamide matrix is reported. Active esters of acrylic acid (N-succinimicyl acrylate and N-phthalimidyl acrylate) were synthesized, then copolymerized with acrylamide and N,N'-methylenebisacrylamide. Displacement of the active ester in the gel thus formed by various ligands containing aliphatic amino groups resulted in the formation of stable amid bonds between the ligands and the polyacrylamide gel. The affinity gel thus prepared has the following advantages: (i) resistance to chemical and microbiological degradation, (ii) ease of control of ligand level and higher levels of ligand possible, (iii) ease of control of porosity, and (iv) total displacement of the active ester under suitable conditions. Efficacy of this system was tested by preparation of 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside derivative polyacrylamide gel by the described method. It was found to be more effective for purification of wheat germ agglutinin than the previously published affinity chromatography systems and the wheat germ hemagglutinin was obtained in crystalline form. In addition, partial resolution of isolectins was obtained from the affinity gel witha pH gradient.

We propose a model to investigate the relation between insertion and excision activities of polymerases involved in DNA synthesis, and the frequency of errors resulting from substituting either mismatched bases or base analogues into a DNA molecule. An analytical equation is derived which expresses the error frequency as a function of nucleotide insertion and removal rates. For the general case, given arbitrary rates of insertion and removal, and allowing the enzyme to peel back by excising previously incorporated nucleotides, we have developed a computer simulation for the synthesis of a DNA molecule. In the special case, where insertion and removal frequencies are within the biologically interesting range for spontaneous mutations, the effect of “peelback” on error correction can be obtained analytically. Our results suggest that the magnitude of the removal frequency (3′-exonuclease activity) is the parameter that exerts the greatest influence on error correction capability; the frequency of errors is less sensitive to either the specificity for removal of mismatched relative to correctly matched bases, or to peelback.

A derivative of ganglioside GT1b (IV3NeuAc,II3(NeuAc)2-GgOse4) with an active ester in its lipid portion was synthesized and covalently attached to bovine serum albumin (BSA). The conjugate, having four GT1b molecules per albumin molecule [GT1b)4BSA) was radioiodinated and used to probe rat brain membranes for ganglioside binding proteins. A ganglioside-specific, high affinity (KD = 2-4 nM), saturable (Bmax = 13-20 pmol/mg membrane protein) binding site for 125I-(GT1b)4BSA was demonstrated on detergent-solubilized rat brain membranes adsorbed to filters. 125I-(GT1b)4BSA binding was tissue-specific (more than 35-fold greater to brain than to liver membranes) and was nearly eliminated by pretreatment of brain membrane-adsorbed filters with trypsin (1 microgram/ml). Underivatized gangliosides added as mixed detergent-lipid micelles blocked 125I-(GT1b)4BSA binding to brain membranes; structurally related GQ1b, GT1b, and GD1b were the most potent (half-maximal inhibition at 70-110 nM), while half-maximal inhibition by other gangliosides (GD3, GD1a, GM3, GM2, and GM1) required 5-20-fold higher concentrations. Other sphingolipids, neutral glycosphingolipids, and glycoproteins were poor inhibitors, and treatment of (GT1b)4BSA with neuraminidase attenuated its binding. Although most phospholipids were noninhibitory, phosphatidylinositol and phosphatidylglycerol inhibited half-maximally at 400-600 nM. However, inhibition of 125I-(GT1b)4BSA binding by gangliosides was competitive and reversible while that by phosphatidylinositol and phosphatidylglycerol was not. Ganglioside-protein conjugate binding reveals ganglioside-specific brain membrane receptors.

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.

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.

Glutamate is thought to be a major excitatory neurotransmitter in the vertebrate brain. In the preceding paper (Malouf, A. T., Schnaar, R. L., and Coyle, J. T. (1984) J. Biol. Chem. 259, 12756-12762), we demonstrated specific binding of [3H]glutamate to membranes from a neuroblastoma hybrid cell line, N18-RE-105. These sites are pharmacologically and kinetically similar to those seen on rat brain membranes and are regulated by ions added to the isolated membranes. In the current paper, we describe an additional level of regulation for the glutamate receptor in this cell line. Long-term incubation (72 h) of intact N18-RE-105 cells with glutamate (10 mM) results in a 2- to 3-fold increase in [3H]glutamate binding. Scatchard analysis reveals that the increase in binding is due to an increase in the number of glutamate receptors without significant change in their affinity. The ability of glutamate analogs to induce such up-regulation mirrors their ability to compete for [3H]glutamate binding to isolated membranes, suggesting that up-regulation is receptor-mediated. Binding of [3H]glutamate to membranes isolated from cells grown in the presence of glutamate can be further up-regulated by brief exposure (10 min) of the isolated membranes to calcium ions. This suggests that agonist-induced and calcium-induced up-regulation occur via independent mechanisms. The short-term ion-induced up-regulation and the long-term agonist-induced up-regulation described in this paper may model two levels of synaptic potentiation reported to occur in the vertebrate hippocampus. The N18-RE-105 cell line may offer a homogeneous cell type in which to study the molecular mechanisms underlying these phenomena.

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.

Affinity chromatography ligands containing amino groups are commonly linked to cyanogen bromide-activated insoluble polysaccharides. This method introduces cationic charge into the insoluble products which may be sufficient to interfere with the use of these materials for affinity chromatography. The present studies were designed to measure and minimize this potential artifact. The cationic charge of the products was determined simply and rapidly by measuring the quantity of labeled orthophosphate bound by the beads. The pH used during activation was critical in determining the extent of ligand incorporation and the cationic charge of the product; high pH gave increasing cationic charge. However, the extent of ligand or charge incorporation varied with the ligand used in the coupling reaction. The pH and the concentration of the ligand in the coupling step also had marked but different effects on the results, depending on the ligand used. It was possible to select conditions for maximizing incorporation while minimizing cationic charge of the products for a given ligand.

meta- bolic inhibitors which block strengthening do not block the development of sugar-resistant adhesion. Data is presented to support the hypothesis that sugar-resist- ant adhesion involves modification of the carbohy- drate-derivatized surface by the cells. Appropriate car- bohydrate-derivatized gels were pretreated with cells and then subjected to boiling in the presence of sodium dodecyl sulfate. After thorough washing, the pre- treated gels supported sugar-resistant adhesion with- out the normal

The movement of cells up an adhesive substratum gradient has been proposed as a mechanism for directing cell migration during development and metastasis. Critical evaluation of this hypothesis (haptotaxis) benefits from the use of quantifiable, stable substratum gradients of biologically relevant adhesion molecules. We report covalent derivatization of polyacrylamide surfaces with quantifiable gradients of a nonapeptide containing the adhesive Arg-Gly-Asp sequence. Cell migration was studied by seeding derivatized surfaces evenly with B16F10 murine melanoma cells. Within 8 hr, cells on gradients redistributed markedly; higher cell densities were found at gel positions having higher immobilized peptide densities. In contrast, cells seeded on control gels with uniform concentrations of adhesive peptide did not redistribute. Redistribution occurred on gradients in both serum-free and serum-containing media. Experiments with uniform density peptide-derivatized gels demonstrated that redistribution on gradients was not due to preferential initial cell attachment or preferential growth on the higher density of immobilized peptide, but must have been due to cell translocation. Cells on exponential gradients of immobilized peptide migrated to a position on the gel surface corresponding to the highest immobilized peptide density, while cells on linear gradients of the same peptide migrated to a position of intermediate peptide density. These data suggest that the B16F10 cells respond to proportional changes in immobilized peptide density rather than to absolute changes, implying a sensing mechanism which utilizes adaptation. These results demonstrate that (1) a gradient of a small adhesive peptide is sufficient to generate redistribution of cell populations and (2) controlled quantifiable substratum gradients can be produced and used to probe the underlying cellular mechanisms of this behavior.

A rapid method for determination of ganglioside glycosyltransferase activity has been developed employing ion-exchange chromatography. Using 13-day chick brain as a source of uridine diphospho- N -acetyl- d -galactosamine: ganglioside GM 3 N -acetylgalactosaminyltransferase (ganglioside GM 2 synthetase), we were able to accurately measure transfer of N -[ 3 H]acetylgalactosamine (GalNAc) from UDP-[ 3 H]GalNAc to GM 3 by application of the reaction mixture to small columns of DEAE-Sepharose and elution of radiolabeled GM 2 reaction product with 10 m m potassium acetate in methanol. This method proved to be as reliable and sensitive as previously published assays but requires less time and fewer manipulations.

Tetanus toxin is known to bind neuronal tissue selectively. To study the interactions of this potent neurotoxin in an intact cell system, the binding of 125I-tetanus toxin was characterized in a neuroblastoma retina hybrid cell line, N18-RE-105. The binding of 125I-tetanus toxin to membranes prepared from N18-RE-105 cells showed many similarities to the interactions of 125I-toxin with rat synaptic membranes. The binding was decreased with increasing temperature, ionic strength, and pH. 125I- Toxin bound to membranes with high affinity: KD = 0.62 +/- 0.05 nM; Bmax = 196 +/- 45 pmol/mg protein. Quantitative thin-layer chromatography and acid-degradation analysis revealed that N18-RE-105 cells contained polysialogangliosides GD1a and GT1b in high concentrations. An assay was developed to quantitate surface-bound and internalized 125I-tetanus toxin by exploiting the observation that surface-bound 125I-toxin is susceptible to pronase digestion. When cells were incubated with 125I-tetanus toxin at 0 degree C, all of the bound 125I-toxin could be degraded with pronase. In contrast, when the incubations were performed at 37 degrees C, within 10 min about 50% of the total cell-associated 125I-toxin was pronase-resistant. Temperature pulse experiments demonstrated that 125I-tetanus toxin that was bound to cells at 0 degree C rapidly disappeared from the surface when the cells were warmed to 37 degrees C, as revealed by the appearance of pronase-resistant radioactivity. This internalization was sensitive to metabolic inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic nonapeptide (Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser), which includes the adhesive Arg-Gly-Asp (RGD) sequence, was covalently immobilized on chemically well-defined polyacrylamide gel surfaces utilizing N-succinimidyl active esters. The amount of peptide immobilized varied linearly with the concentration added to the gels. Immobilization was approximately 80% efficient (based on peptide added), resulting in up to 17.5 nmol peptide/cm2 gel surface. Balb/c 3T3 mouse fibroblast cells adhered readily to peptide-derivatized surfaces, even in the absence of serum. Furthermore, surfaces derivatized with 2 nmol peptide/cm2 gel supported long-term fibroblast growth at a rate and to an extent comparable to that on tissue culture plastic. Surfaces derivatized with a control nonapeptide having no RGD sequence were nonsupportive of cell attachment or growth. The immobilization technology used to derivatize the gel surfaces with adhesive nonapeptide can be modified to allow coderivatization with proteins, glycoproteins, glycosides, or other amine-containing compounds to test their effects on long-term cell behaviors.

Complex carbohydrates coat the surfaces of cells and have the potential to carry the information necessary for cell–cell recognition. Sugar-specific receptors (lectins) are also present on cells, and can interact with sugars on apposing cells. This may result in the adhesion of the two cells via carbohydrates and specific cell-surface receptors. Such carbohydrate-directed cell adhesion appears to be important in many intercellular activities including infection by bacteria and viruses, communication among cells of lower eukaryotes, specific binding of sperm to egg; and recirculation of lymphocytes, among others [50]. New approaches involving synthesis of chemically defined cell-surface analogs, in conjunction with inhibition experiments, are beginning to reveal the mechanics of a potential carbohydrate “language” involved in intercellular interactions.

Publisher Summary This chapter focuses on the adhesion of eukaryotic cells to immobilized carbohydrates. To study the carbohydrate-directed control of cell behaviors, methods have been developed to immobilize various carbohydrates on otherwise inert synthetic surfaces and to analyze the responses of intact cells in contact with these surfaces. Various carbohydrates (including low molecular weight glycosides, glycopeptides, glycoproteins, polysaccharides, and glycolipids) are covalently attached or noncovalently (but stably) adsorbed to plastic surfaces. Hydrophilic carbohydrates are readily soluble in aqueous media and must be covalently attached to artificial surfaces for use as cell surface analogs. In contrast, amphipathic glycolipids are stably adsorbed to hydrophobic plastic surfaces for this purpose or are probed directly on thin-layer chromatography (TLC) plates. The chapter concludes with a discussion of cell adhesion to glycosphingolipids, separated on TLC plates.

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition and adhesion. We report a method which detects carbohydrate-specific adhesion of intact eukaryotic cells directly to glycosphingolipids separated on thin-layer chromatography plates. Various glycosphingolipids were chromatographed on high-performance silica gel thinlayer chromatography plates, and the plates were coated with a thin film of poly(isobutyl methacrylate) and mounted in a specially designed plexiglass chamber. Metabolically radiolabeled cells were added to the chamber, which was then sealed and gently centrifuged to bring the cells into contact with the surface of the TLC plate. After incubation to allow adhesion to occur, the chamber was inverted and centrifuged to remove nonadherent cells from the plate surface. The plate was removed from the chamber, the adherent cells were fixed in place with glutaraldehyde, and the plate was dried and subjected to autoradiography. Chicken hepatocytes, which have a cell surface receptor for N-acetylglucosamine, adhered only to those areas of the plate to which appropriate glycosphingolipids (having that terminal sugar) had migrated. Cell adhesion was blocked by soluble N-acetylglucosamine (but not by other sugars) and was readily detectable using a variety of developing solvents. Cell adhesion to as little as 8 pmol of the appropriate lipid was readily detected. This method can be used to test glycosphingolipids as cell surface recognition markers for a variety of cell types.

Publisher Summary This chapter presents the procedures for preparation of polyacrylamide gels containing active esters. The matrices are formed by copolymerization of bifunctional molecules, containing acryloyl groups and N-succinimidyl esters, with acrylamide and bisacrylamide. The affinity gels thus prepared had the advantages that (1) ligands containing free amino groups readily displaced the active ester resulting in the formation of stable amide bonds between the ligands and the polyacrylamide gel, (2) the displacement reaction was readily followed by measuring cleavage of the esters, (3) the matrix was resistant to chemical and microbial degradation, (4) ligand levels were easily controlled, and high ligand concentrations were obtained, (5) porosity and shape of the gel matrix were easily controlled, and (6) the matrix was hydrophilic, but low in extraneous ionic charge. Carbohydrate ligands in the form of glycosides containing an amino group in the aglycon were immobilized by the reaction of the amino group and the active esters in the gel and used to demonstrate specific adhesion of intact cells to carbohydrates.

Monoethylcholine aziridinium ion (AF64A), which is generated from the precursor, acetylcholine mustard hydrochloride, exerts selective neurotoxic effects against brain cholinergic neurons when injected intracerebrally. Conditions associated with optimal generation of AF64A were examined. The results indicate that hydrolysis and cyclization of the precursor are optimal at 25 degrees C with the former occurring at pH 9.0 and the latter at pH 7.3. The aziridinium ion is best stabilized at pH 7.3 at 4 degrees C.

The antibiotic tunicamycin blocks the transfer of GlcNAc-1- P from UDP-GlcNAc to dolichol phosphate, thereby blocking the synthesis of N -linked oligosaccharide chains on glycoproteins. Its effect on the biosynthesis of gangliosides has not been reported. We report that tunicamycin caused a 70-80% reduction in incorporation of [ 3 H]GlcN into gangliosides and neutral glycosphingolipids of the neuroblastoma-glioma hybrid cell line NG 108-15 at antibiotic concentrations that caused a 90% reduction of the radiolabel incorporation into glycoproteins. The effect of tunicamycin on ganglioside biosynthesis was apparent after only 4 hr of incubation, and maximum inhibition was seen within 6 hr. When control or tunicamycin-treated (5 μg/ml) cells were collected and fractionated to separate glycoproteins, neutral glycosphingolipids, gangliosides, and nucleotide sugar-precursor pools, the following results were obtained: ( i ) UDP-GlcNAc and UDP-GalNAc pool sizes increased >3-fold, and specific activities decreased 50% upon treatment with tunicamycin; ( ii ) when corrected for this value, the percentage inhibition of GlcN incorporation into various glycoconjugates by tunicamycin in these cells was 82% for glycoproteins, 54% for neutral glycosphingolipids, and 50% for gangliosides; and ( iii ) the different gangliosides were affected differentially, with the most striking inhibition apparent in GM 3 biosynthesis, which was decreased 78% in the presence of tunicamycin. These data suggest that the effects of tunicamycin on glycosphingolipids as well as on glycoproteins must be considered when interpreting its effects on intact cells and organisms.

Membranes from the neuroblastoma x embryonic retina cell hybrid cell line, N18-RE-105, bind L-[3H]glutamate with a pharmacologic profile consistent with a 'quisqualate-type' brain L-glutamate receptor. We describe here the cytotoxic effect of L-glutamate receptor agonists on intact N18-RE-105 cells. Cytotoxicity was quantitated by measurement of the release of the cytosolic enzyme, lactate dehydrogenase, into the culture medium after addition of L-glutamate and its analogs to the cell culture medium. L-Glutamate (10 mM) and its confirmationally restricted analogs, quisqualate (1 mM) and ibotenate (10 mM), caused cell lysis. In contrast, similar analogs which do not bind to N18-RE-105 cell membranes (kainic acid, N-methyl-D,L-aspartic acid and gamma-aminobutyric acid) were not cytotoxic. L-Glutamate-induced cytotoxicity was eliminated when calcium-free medium was used. Addition of inorganic or organic calcium channel antagonists also reduced the cytotoxicity of L-glutamate, even when 1.8 mM calcium was present in the medium. Cadmium chloride (10 microM) completely blocked L-glutamate toxicity, whereas manganese chloride (150 microM) and lanthanum chloride (25 microM) reduced toxicity by greater than 50%. Dihydropyridine voltage-sensitive calcium channel agonists or antagonists, had little or no significant effect on L-glutamate-induced toxicity. In contrast, the verapamil derivatives, D600 and D888, and the diltiazem derivative, MDL 12,330A reduced L-glutamate toxicity by greater than 50%. These results suggest that a subtype of voltage-sensitive calcium channels is involved in the mechanism of L-glutamate receptor mediated cytotoxicity in this cell line.

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.

Publisher Summary This chapter presents procedures to synthesize polyacrylamide gels containing various copolymerized 6-acrylamidohexyl glycosides in which the sugar is attached to the polymer matrix at the terminal amino group in the aglycon. Glycosides of mono- and disaccharides containing ω-amino groups in the aglycon are prepared. Amino groups are determined using 2,4,6-trinitrobenzenesulfonic acid. Neutral sugars are determined by a phenol-sulfuric acid procedure. The solvent system for thin-layer chromatography is (v/v/v) ethyl acetate-acetic acid-water. The following groups were detected on TLC plates as indicated: sugars, 10% H2SO4 in 50% ethanol; amino groups, ninhydrin spray; acrylamido compounds, ultraviolet absorption or a permanganateperiodate spray. Polyacrylamide gels containing covalently attached carbohydrates have proved to be useful as affinity adsorbents for lectins and in studies with intact eukaryotic cells. Thin, fiat gel pieces have been used as synthetic culture surfaces to show that freshly isolated hepatocytes from chicken or rat will specifically bind to surfaces containing, respectively, N-acetylglucosamine z or galactose.

Complex carbohydrates on the surfaces of eukaryotic cells are thought to participate in a wide variety of cell-cell interactions. A model system has therefore been developed to study these processes. In the present experiments, the ability of chicken hepatocytes to recognize and adhere to sugars covalently linked to polyacrylamide gels was investigated. The gels were snythesized by two methods. Type I gels were prepared from a co-polymer of an active ester of acrylic acid (N-succinimidyl acrylate), acrylamide, and bisacrylamide. The "activated" polyacrylamide gel was then treated with the desired ligand containing an amino group, such as 6-aminohexyl O- or S-glycoside. Type II gels were formed by treating similar ligands with acryloyl chloride, followed by co-polymerization of the resulting N-substituted acrylamide with acrylamide and N,N'-methylenebisacrylamide. These polyacrylamide derivatives offer many advantages for studies with intact cells. They are not toxic to any cell type studied, can be cast in any desired shape, are transparent and stable over a wide range of pH values, and contain no cationic and low to negligible levels of anionic charge (charged groups can be introduced if desired), and the polyacrylamide matrix is stable to common biological agents such as bacteria and enzymes. In addition, type I gels can be synthesized using a broad range of molecules containing amino groups, such as glycopeptides, proteins, etc. The hepatocytes were prepared by collagenase perfusion of intact chicken livers. The rate and extent of adhesion of the cells to the derivatized gels was determined by measuring lactate dehydrogenase in these cells. This enzyme was also used to assay viability and cell "leakiness." At 37 degrees C, 70 to 100% of the cells adhered within 60 min to gels derivatized with N-acetylglucosamine, i.e. gels derivatized with 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (or the corresponding thioglycoside). By contrast, less than 5% of the cells adhered to polyacrylamide or to gels derivatized with 6-aminohexanol or the 6-aminohexyl glycosides of beta-D-glucose, beta-D-galactose, alpha-D-mannose, beta-D-maltose, beta-D-melibiose, beta-D-cellobiose, and (alpha or beta)-D-lactose. Kinetic studies with the chicken hepatocytes and N-acetylglucosamine gels showed that cell-gel binding was dependent upon Ca2+ and was decreased at low temperatures. Binding was inhibited by N-acetylglucosamine or by glycosides of this sugar, the most effective inhibitor being orosomucoid (alpha1-acid glycoprotein) pretreated with sialidase and beta-galactosidase. The cell surface receptor(s) involved in this interaction is not known, but may be related or identical to the chicken liver binding protein described by Lunney and Ashwell (Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 341--343). The present results suggest that this model system should prove useful in delineating cell surface interactions with carbohydrates.

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Background: Protein–carbohydrate interactions are believed to be important in many biological processes that involve cell–cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein. Results We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc α 2→3Gal β 1→3(4)GlcNAc or NeuAc α 2→3Gal β 1→3GalNAc, myelin-associated glycoprotein recognizes only NeuAc α 2→3Gal β 1→3GalNAc and CD22 binds specifically to NeuAc α 2→6Gal β 1→4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes. Conclusion Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family.

Important cellular characteristics, including selective adhesion, growth rate, motility, and differentiation, are controlled, in part, by signals received at the cell surface. The molecular mechanisms for the cell surface control of these cell behaviors are largely unknown. In order to probe the role of specific extracellular molecules in controlling cell function, we report the development of synthetic surfaces which generally support the long-term growth of cells yet can be readily derivatized with a wide variety of molecules of biological interest. Polyacrylamide gels containing a gradient of active ester groups were prepared and then the esters were displaced with ligands to generate a gradient of carboxylic acid, tertiary amine, or hydroxyl groups. When untransformed mouse fibroblasts (BALB/3T3) were seeded on the various surfaces, they attached and grew only on those derivatized with carboxylic acids or hydroxyl groups within narrow concentration ranges. Cell growth rate, density, and morphology on polyacrylamide gels containing the optimal concentration of carboxylic acid groups (approximately 30 mumol/ml) were comparable to those on tissue culture plastic, whereas growth on hydroxyl group-derivatized gels was less extensive. In contrast, short-term (90-min) adhesion to hydroxyl group-derivatized gels was greater than that to carboxylic acid-derivatized gels. Both short-term adhesion and long-term growth required serum. Growth-supportive polyacrylamide gels were readily derivatized with molecules of biological interest. The techniques reported here are applicable to other types of cell in culture since the nature and concentration of substratum functional groups can be easily varied and tested for support of long-term cell growth.

International audience; B- and c-series of gangliosides are over-expressed in neuro-ectoderm-related cancers, including breast cancer. It has been shown that GD3 ganglioside is over-expressed in about 50% of invasive ductal breast carcinoma and the GD3 synthase (GD3S) gene displays higher expression among estrogen receptor (ER) negative breast tumors. We previously showed that GD3S expression in MDA-MB-231 breast cancer cells induces the expression of GD2 and increased cell proliferation and migration via a GD2-dependent activation of c-Met receptor. Here, we show that in ER-positive MCF-7 breast cancer cells, GD3S expression resulted in an increase of GD1b, which was associated with a decrease of GM1a and GM2. Meanwhile, GD3S expressing MCF-7 cells exhibited an increased migration without any modification of proliferation rate. Therefore, GD3S expression can result in different modifications of both ganglioside profiles and cell phenotypes depending on breast cell types.; Les gangliosides des séries b- et c- sont surexprimés dans différents cancers d’origine neuro-ectodermique, dont le cancer du sein. Il a été montré que le ganglioside GD3 est surexprimé dans environ 50 % des carcinomes canalaires infiltrants et le gène de la GD3 synthétase (GD3S) est surexprimé dans les tumeurs estrogène-récepteur négatives. Nous avons précédemment démontré que l’expression de la GD3S dans les cellules cancéreuses mammaires MDA-MB-231 induisait l’expression du GD2 et augmentait la prolifération et la migration cellulaires par l’activation GD2-dépendante du récepteur c-Met. Nous montrons ici que l’expression de la GD3S dans les cellules MCF-7 provoque une accumulation du GD1b associée à une diminution du GM1a et du GM2. Parallèlement, les cellules MCF-7 GD3S positives montrent une augmentation de migration sans modification de la prolifération. Ce résultat montre que les modifications du profil gangliosidique et du phénotype induites par l’expression de la GD3S sont dépendantes du type cellulaire.