Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Myelin-associated Glycoprotein Interacts with Neurons via a Sialic Acid Binding Site at ARG118 and a Distinct Neurite Inhibition Site

Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid–dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid– dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid–dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.     

Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration

Gangliosides, sialylated glycosphingolipids which are the predominant glycans on vertebrate nerve cell surfaces, are emerging as components of membrane rafts, where they can mediate important physiological functions. Myelin associated glycoprotein (MAG), a minor constituent of myelin, is a sialic acid binding lectin with two established physiological functions: it is involved in myelin-axon stability and cytoarchitecture, and controls nerve regeneration. MAG is found selectively on the myelin membranes directly apposed to the axon surface, where it has been proposed to mediate myelin-axon interactions. Although the nerve cell surface ligands for MAG remain to be established, evidence supports a functional role for sialylated glycoconjugates. Here we review recent studies that reflect on the role of gangliosides, sialylated glycosphingolipids, as functional MAG ligands. MAG binds to gangliosides with the terminal sequence 'NeuAc alpha 3Gal beta 3GalNAc' which is found on the major nerve gangliosides GD1a and GT1b. Gangliosides lacking that terminus (e.g., GM1 or GD1b), or having any biochemical modification of the terminal NeuAc residue fail to support MAG binding. Genetically engineered mice lacking the GalNAc transferase required for biosynthesis of the 'NeuAc alpha 3Gal beta 3GalNAc' terminus have grossly impaired myelination and progressive neurodegeneration. Notably the MAG level in these animals is dysregulated. Furthermore, removal of NeuAc residues from nerve cells reverses MAG-mediated inhibition of neuritogenesis, and neurons from mice lacking the 'NeuAc alpha 3 Gal beta 3GalNAc' terminus have an attenuated response to MAG. Cross-linking nerve cell surface gangliosides can mimic MAG-mediated inhibition of nerve regeneration. Taken together these observations implicate gangliosides as functional MAG ligands.     

Conversion of cellular sialic acid expression from N-acetyl- to N-glycolylneuraminic acid using a synthetic precursor, N-glycolylmannosamine pentaacetate: inhibition of myelin-associated glycoprotein binding to neural cells

Sialic acids are prominent termini of mammalian glycoconjugates and are key binding determinants for cell-cell recog-nition lectins. Binding of the sialic acid-dependent lectin, myelin-associated glycoprotein (MAG), to nerve cells is implicated in the inhibition of nerve regeneration after injury. Therefore, blocking MAG binding to nerve cell sialoglycoconjugates might enhance nerve regeneration. Previously, we reported that certain sialoglycoconjugates bearing N-acetylneuraminic acid (NeuAc) but not N-glycolylneuraminic acid (NeuGc) support MAG binding (Collins et al., 1997a). We now report highly efficient conversion of sialic acids on living neural cells from exclusively NeuAc to predominantly NeuGc using a novel synthetic metabolic precursor, N-glycolylmannosamine pentaacetate (Man-NGc-PA). When NG108-15 neuroblastoma-glioma hybrid cells, which normally express only NeuAc (and bind to MAG), were cultured in the presence of 1 mM ManNGcPA, they expressed 80-90% of their sialic acid precursor pool as NeuGc within 24 h. Within 5 days, 80% of their ganglioside-associated sialic acids and 70% of their glycoprotein-associated sialic acids were converted to NeuGc. Consistent with this result, treatment of NG108-15 cells with ManNGcPA resulted in nearly complete abrogation of MAG binding. These results demonstrate that ManNGcPA treatment efficiently alters the sialic acid structures on living cells, with a commensurate change in recognition by a physiologically important lectin.     

Characterization and partial purification of beta-N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Urine from Sd(a+) individuals was found to contain a beta-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3'-sialyllactose and glycoproteins carrying the terminal NeuAc alpha-3Gal beta group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn2+ but not for detergents. The Km for UDP-GalNAc was 66 X 10(-6) M, using fetuin as an acceptor. Like beta-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [3H]GalNAc to 3'-sialyllactose, we propose that the enzyme attaches GalNAc in beta-anomeric configuration to O-4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a-) donor lacking beta-GalNAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the beta-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171-184) the urinary transferase is able to transfer beta-GalNAc to the NeuAc alpha-3Gal beta-3(NeuAc alpha-6)GalNAc chains bound to the native glycophorin.     

Myelin-associated glycoprotein (MAG): past, present and beyond

The myelin-associated glycoprotein (MAG) is a type I transmembrane glycoprotein localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths where it functions in glia-axon interactions. It contains five immunoglobulin (Ig)-like domains and is in the sialic acid-binding subgroup of the Ig superfamily. It appears to function both as a ligand for an axonal receptor that is needed for the maintenance of myelinated axons and as a receptor for an axonal signal that promotes the differentiation, maintenance and survival of oligodendrocytes. Its function in the maintenance of myelinated axons may be related to its role as one of the white matter inhibitors of neurite outgrowth acting through a receptor complex involving the Nogo receptor and/or gangliosides containing 2,3-linked sialic acid. MAG is expressed as two developmentally regulated isoforms with different cytoplasmic domains that may activate different signal transduction pathways in myelin-forming cells. MAG contains a carbohydrate epitope shared with other glycoconjugates that is a target antigen in autoimmune peripheral neuropathy associated with IgM gammopathy and has been implicated in a dying back oligodendrogliopathy in multiple sclerosis.     

Ganglioside GD1 alpha analogues as high-affinity ligands for myelin-associated glycoprotein (MAG).

The first systematic synthesis of ganglioside GD1 alpha analogues carrying N-acetyldeoxyneuraminic acids linked to C-6 of the GalNAc residue was accomplished. The suitably protected GM1b pentasaccharide derivative was regioselectively glycosylated with the phenyl 2-thioglycosides of 7-deoxy, 8-deoxy, and 9-deoxy-N-acetylneuraminic acid promoted by N-iodosuccinimide (NIS)-trifluoromethanesulfonic acid (TfOH) in acetonitrile, and the resulting hexasaccharides were converted to the target GD1 alpha analogues. All of the analogues retained excellent efficiency in supporting the adhesion to myelin-associated glycoprotein (MAG), raising the possibility that the internal sialic acid linked to the GalNAc residue may be replaced by other anionic substituents, in contrast to the terminal sialic acid, which is essential for MAG binding.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

Purification and Characterization of Glycoprotein from the Skin Mucus of the Rainbow Trout,Salmo gaivdneri

Mucus glycoprotein (RGP) was purified and characterized from the skin mucus of rainbow trout, Salmo gairdneri. RGP was found to contain 30.1% NeuAc, 26.0% GalNAc, 5.0% Gal, and 26.0% amino acids. The protein moiety of RGP is very rich in Thr (32.4 mol%). Neither NeuGc nor KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) was found in RGP. Alkaline borohydride treatment of RGP yielded a major disaccharide alditol, NeuAcα2→6GalNAc-ol and more than 4 minor oligosaccharide alditols including NeuAc→(GalNAcα1→)GalNAc-ol. It was evident that an average RGP molecule has approximately 500 NeuAc-containing oligosaccharide chains, which are attached to the Thr and Ser residues of the protein moiety and spaced at an average of 3 amino acids apart.     

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.     

Coexpression of cancer-associated carbohydrate antigens,Tn and sialyl Tn

The expression of cancer-associated antigens, Tn and sialyl Tn, was examined using monoclonal antibodies, MLS 128 and MLS 102, recognizing these two antigens, respectively. A cell lysate from a human carcinoma cell line, LS 180 cells, was analysed by Western blotting using these two antibodies. Three glycoprotein bands were discernible with each antibody, of which two, corresponding to 250 and 210 kDa, were reactive with both the antibodies. LS 180 cells were metabolically labelled with³H-glucosamine and then the lysate from these cells was applied to two immunoaffinity columns. Sixty-five per cent of the Tn antigenic glycoproteins, based on radioactivity, bound to the MLS 102 affinity column. On the other hand, 45% of the sialyl Tn antigenic glycoproteins bound to the MLS 128 affinity column. These results indicate that some Tn and sialyl Tn antigens were expressed on the same polypeptide chains.The presence of non-sialylated GalNAc residues on the polypeptide chain with many Sia-GalNAc residues appears to be due to the incapability of three consecutive moieties of GalNAc-Ser/Thr to accept sialic acid.Abbreviations PSMF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - GalNAc N-acetylgalactosamine - Sia sialic acid     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists

Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH(2)) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists.     

Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A

In animal models, transplantation of bone marrow stromal cells (MSC) into the spinal cord following injury enhances axonal regeneration and promotes functional recovery. How these improvements come about is currently unclear. We have examined the interaction of MSC with neurons, using an established in vitro model of nerve growth, in the presence of substrate-bound extracellular molecules that are thought to inhibit axonal regeneration, i.e., neural proteoglycans (CSPG), myelin associated glycoprotein (MAG) and Nogo-A. Each of these molecules repelled neurite outgrowth from dorsal root ganglia (DRG) in a concentration-dependent manner. However, these nerve-inhibitory effects were much reduced in MSC/DRG co-cultures. Video microscopy demonstrated that MSC acted as “cellular bridges” and also “towed” neurites over the nerve-inhibitory substrates. Whereas conditioned medium from MSC cultures stimulated DRG neurite outgrowth over type I collagen, it did not promote outgrowth over CSPG, MAG or Nogo-A. These findings suggest that MSC transplantation may promote axonal regeneration both by stimulating nerve growth via secreted factors and also by reducing the nerve-inhibitory effects of the extracellular molecules present.     

Thyroid-stimulating hormone binding to beef thyroid membranes. Role of N-acetylneuraminic acid.

The effect of sugars on 125I-thyroid-stimulating hormone binding to beef thyroid membranes was studied to determine their role in thyroid-stimulating hormone (TSH) binding. At 0.1 M concentration, N-acetylneuraminic acid produced a 3- to 7-fold increase in TSH binding, was the only sugar to enhance TSH binding, and did so whether binding was determined in the cyclase medium or under conditions of optimum binding. The enhanced TSH binding remained after the membranes were removed from the high NeuAc concentration and an effect was observed at concentrations of 10 mM NeuAc. NeuAc did not alter the kinetics of TSH binding but the pH optimum for TSH binding shifted from pH 5.5 to 7.5 in the presence of NeuAc. Incubation of the membranes with increasing concentrations of NeuAc resulted in increased sialic acid content of the membranes. The NeuAc concentration curve of membrane sialic acid and TSH binding were roughly parallel. The capacity of the low affinity site increased from 0.74 to 2.5 nmol/mg of protein in the presence of NeuAc. The apparent affinity (0.88 X 10(6) M-1) of this site was unaffected by NeuAc. With the high affinity site, NeuAc increased both the apparent affinity and capacity from 2.2 X 10(8)M-1 to 5.5 X 10(8) M-1 and 1.6 to 3.1 pmol/mg of protein, respectively. Neuraminidase or neuraminidase plus beta-galactosidase incubation of the membranes removed approximately 60% of the sialic acid from the membranes within 15 to 30 min but did not affect TSH binding. Large quantities of sialic acid were detected in the soluble fractions during isolation of the membranes, 4 to 5% of which was ultrafilterable and not associated with high molecular weight proteins. It is concluded that among the sugars tested, NeuAc exhibits an unique effect on TSH binding that may have physiological significance. The inability to alter TSH binding by enzymatic removal of endogenous sialic acid suggests that either NeuAc resistant to hydrolysis is sufficient to maintain TSH binding or that NeuAc important in TSH binding is removed during membrane preparation but is replaced by incubation with exogenous NeuAc.     

Polysialoglycoproteins of Salmonidae fish eggs. Complete structure of 200-kDa polysialoglycoprotein from the unfertilized eggs of rainbow trout (Salmo gairdneri)

Polysialoglycoproteins (PSGP) we first isolated from the unfertilized eggs of rainbow trout (Salmo gairderi) and now found to be a ubiquitous component of Salmonidae fish eggs are a novel type of glycoprotein. PSGP from rainbow trout has a molecular weight of 200 X 10(3), a low protein content (about 15% w/w), and a high sialic acid (N-glycolylneuraminic acid (NeuGc] content (about 60%, w/w). In any evaluation of the biological functions of PSGP, information about the complete structure of this unique macromolecular component is relevant. We have now completed the determination of the overall structural organization of the 200-kDa PSGP, and this is the first report of the complete structural analysis of this novel class of glycoprotein: (Asp)0-2-Ala-Thr*-Ser*-Glu-(Ala-Ala-Thr*-Gly-Pro-Ser-Gly-Asp-Asp-Ala-Thr *-Ser*- Glu)n-Ala-Ala-Thr*-Gly-Pro-Ser-Gly where * indicates the amino acid residues to which oligo- and/or polysialylglycan units are attached and n = 25. Thus the most outstanding structural features of PSGP isolated from the unfertilized eggs of rainbow trout are now the occurrence of (a) tandem repeats of a tridecapeptide and (b) an alpha-2----8-linked oligo(poly)sialyl group on each of the core oligosaccharide chains, i.e. GalNAc- beta 1----4(NeuGc alpha 2----3)GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n ----6)GalNAc alpha 1----Ser (or Thr), GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), and Gal beta 1----3[----8NeuGc alpha 2)n----6) GalNAc alpha 1----Ser (or Thr).     

Tissue-specific expression of sialyltransferases

Three sialyltransferases which attach terminal sialic acids to glycoprotein sugar chains are shown to exhibit striking differential expression in seven tissues of the rat. Using cDNA probes for the Gal beta 1,4GlcNAc alpha-2,6-sialytransferase which forms a NeuAc alpha 2-6Gal beta 1-4GlcNAc sequence on N-linked sugar chains, three different sized mRNAs are detected, two of which (4.7 and 4.3 kilobases (kb] have high homology along the full length, and a third (3.6 kb, in kidney) which is missing the 5' region corresponding to 45% of the NH2-terminal coding sequence. The 4.7- and 4.3-kb mRNAs exhibit differential expression of over 50-fold with the highest levels in liver and lowest in brain and heart. Assays for enzyme activity in tissue homogenates show high correspondence to the levels of mRNA. Evidence of tissue-specific expression was also obtained for two other sialyltransferases which form the NeuAc alpha 2-3Gal beta 1-4/3GlcNAc and NeuAc alpha 2-3Gal beta 1-3GalNAc sequences on N-linked and O-linked sugar chains, respectively. Comparison of the ratios of the three enzymes in several tissues suggests that they are expressed independently. The results are discussed for their relevance to cellular control of terminal glycosylation sequences on glycoproteins and glycolipids.     

The hemagglutinins of the human influenza viruses A and B recognize different receptor microdomains

A cryptically I-active sialylglycoprotein (glycoprotein 2) isolated from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y., Suzuki, T. and Matsumoto, M. (1983) J. Biochem. 93, 1621-1633) contains N-glycolylneuraminic acid (NeuGc) as its predominate sialic acid and exhibits poor receptor activity for a variety of influenza viruses. Enzymatic modification of asialoglycoprotein-2 to contain N-acetylneuraminic acid (NeuAc) in the NeuAc alpha 2-3Gal and NeuAc alpha 2-6Gal sequences using specific sialyltransferase resulted in the appearance of receptor activity toward human influenza viruses A and B. The biological responsiveness chicken erythrocytes treated with sialidase and then reconstituted with derivatized glycoprotein 2 showed considerable recovery to influenza virus hemagglutinin-mediated agglutination, low-pH fusion and hemolysis. Specific hemagglutination inhibition activity of derivatized glycoprotein 2 was 5-16-times higher than that of human glycophorin. A/PR/8/34 (H1N1) virus preferentially recognized derivatized glycoprotein 2 containing NeuAc alpha 2-3Gal sequence over that containing NeuAc alpha 2-6Gal while the specificity of A/Aichi/2/68 (H3N2) for the sialyl linkages was reversed. B/Lee virus recognized both sequences almost equally. The biological responsiveness to the viruses of the erythrocytes labeled with the derivatized glycoprotein 2 containing NeuGc was considerably lower than that of derivatized glycoprotein 2 containing NeuAc. The results demonstrate that the hemagglutinins of human isolates of influenza viruses A and B differ in the recognition of microdomains (NeuAc, NeuGc) of the receptors for binding and fusion activities in viral penetration and the sequence to which sialic acid (SA) is attached (SA alpha 2-3Gal, SA alpha 2-6Gal). Inner I-active neolacto-series type II sugar chains may be important in revealing the receptor activity toward the hemagglutinin of both human influenza viruses A and B.     

Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro

Myelin-associated glycoprotein (MAG, Siglec-4) is one of several endogenous axon regeneration inhibitors that limit recovery from central nervous system injury and disease. Molecules that block such inhibitors may enhance axon regeneration and functional recovery. MAG, a member of the Siglec family of sialic acid-binding lectins, binds to sialoglycoconjugates on axons and particularly to gangliosides GD1a and GT1b, which may mediate some of the inhibitory effects of MAG. In a prior study, we identified potent monovalent sialoside inhibitors of MAG using a novel screening platform. In the current study, the most potent of these were tested for their ability to reverse MAG-mediated inhibition of axon outgrowth from rat cerebellar granule neurons in vitro. Monovalent sialoglycans enhanced axon regeneration in proportion to their MAG binding affinities. The most potent glycoside was disialyl T antigen (NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc-R), followed by 3-sialyl T antigen (NeuAcalpha2-3Galbeta1-3GalNAc-R), structures expressed on O-linked glycoproteins as well as on gangliosides. Prior studies indicated that blocking gangliosides reversed MAG inhibition. In the current study, blocking O-linked glycoprotein sialylation with benzyl-alpha-GalNAc had no effect. The ability to reverse MAG inhibition with monovalent glycosides encourages further exploration of glycans and glycan mimetics as blockers of MAG-mediated axon outgrowth inhibition.