Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside,leading to inhibition of laminin-5-dependent cell motility

GM3 ganglioside inhibits tetraspanin CD9-facilitated cell motility in various cell lines (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421). We now report the following: (i) CD9 has the novel feature of being soluble in chloroform/methanol, and classifiable as "proteolipid"; (ii) CD9 and alpha(3) integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the alpha(3) expression ratio (value for cells grown under +Gal condition divided by the value for cells grown under -Gal condition) in GEM of ldlD/CD9 cells was higher than that in control ldlD/moc cells, suggesting that CD9 recruits alpha(3) in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of alpha(3) and CD9 in the total extract or membrane fractions from cells grown under +Gal versus -Gal condition were nearly identical, whereas alpha(3) expressed at the cell surface, probed by antibody binding in flow cytometry, was higher under -Gal than +Gal condition. These results suggest that GM3 synthesized under +Gal condition promotes interaction of alpha(3) with CD9, which restricts alpha(3) binding to its antibody. A concept of the alpha(3)/CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and alpha(3) under +Gal but not -Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and alpha(3) when GM3 was added exogenously to cells under -Gal condition, and (iii) the co-localization images of CD9 with alpha(3) and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of alpha(3)/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for alpha(3), the laminin-5/alpha(3)-dependent motility of ldlD/CD9 cells was found to be greatly enhanced under -Gal condition, but strongly inhibited under +Gal condition. Such a motility difference under +Gal versus -Gal condition was not observed for ldlD/moc cells. The inhibitory effect observed in ldlD/CD9 cells under +Gal condition was reversed upon addition of anti-alpha(3) antibody and is therefore based on interaction between alpha(3), CD9, and GM3 in GEM.     

Carbohydrate to carbohydrate interaction in development process and cancer progression

Two types of carbohydrate to carbohydrate interaction (CCI) have been known to be involved in biological processes. One is the CCI between molecules expressed on interfacing cell membranes of different cells to mediate cell to cell adhesion, and subsequently induce cell signaling, and is termed trans-CCI. It has been indicated that the Le^x to Le^x interaction at the morula stage in mouse embryos plays an important role in the compaction process in embryonic development. GM3 to Gg3 or GM3 to LacCer interaction has been suggested to be involved in adhesion of tumor cells to endothelial cells, which is considered a crucial step in tumor metastasis. The other is the CCI between molecules expressed within the same microdomain of the cell surface membrane, and is termed cis-CCI. The interaction between ganglioside GM3, and multi (>3) GlcNAc termini of N-linked glycans of epidermal growth factor receptor (EGFR), has been indicated as the molecular mechanism for the inhibitory effect of GM3 on EGFR activation. Also, the complex with GM3 and GM2 has been shown to inhibit the activation of hepatocyte growth factor (HGF) receptor, cMet, through its association with tetraspanin CD82, and results in the inhibition of cell motility. Since CCI research is still limited, more examples of CCI in biological processes in development, and cancer progression will be revealed in the future.     

Interaction of N-linked glycans, having multivalent GlcNAc termini, with GM3 ganglioside

GM3 ganglioside interacts specifically with complex-type N-linked glycans having multivalent GlcNAc termini, as shown for (1) and (2) below. (1) Oligosaccharides (OS) isolated from ConA-non-binding N-linked glycans of ovalbumin, whose structures were identified as penta-antennary complex-type with bisecting GlcNAc, having five or six GlcNAc termini (OS B1, B2), or bi-antennary complex-type having two GlcNAc termini (OS I). OS I is a structure not previously described. (2) Multi-antennary complex-type N-linked OS isolated from fetuin, treated by sialidase followed by β-galactosidase, having three or four GlcNAc termini exposed. These OS, conjugated to phosphatidylethanolamine (PE), showed clear interaction with ³H-labeled liposomes containing GM3, when various doses of OS-PE conjugate were adhered by drying to multi-well polystyrene plates. Interaction was clearly observed only with liposomes containing GM3, but not LacCer, Gb4, or GalNAcα1-3Gb4 (Forssman antigen). GM3 interaction with PE conjugate of OS B1 or B2 was stronger than that with PE conjugate of OS I. GM3 interacted clearly with PE conjugate of N-linked OS from desialylated and degalactosylated fetuin, but not native fetuin. No binding was observed to cellobiose-PE conjugate, or to OS-PE conjugate lacking GlcNAc terminus. Thus, GM3, but not other GSL liposomes, interacts with various N-linked OS having multiple GlcNAc termini, in general. These findings suggest that the concept of carbohydrate-to-carbohydrate interaction can be extended to interaction of specific types of N-linked glycans with specific GSLs. Natural occurrence of such interaction to define cell biological phenomena is under investigation. All solvent ratios are by volume. An erratum to this article can be found at     

Binding of synthetic sulfated ligands by human splenic galectin 1, a β-galactoside-binding lectin

The carbohydrate-binding site of galectin 1, a vertebrate β-galactoside-binding lectin, has a pronounced specificity for the βGal(1→3)- and βGal(1→4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6′-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3′-OSO3-LacNAc, 3′-OSO3-Galβ(1→3)GlcNAcβ1-OBzl and 3-OSO3-Galβ1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2′-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.     

Characterization of a novel type of chain-terminator Gal beta 1-6Gal beta 1-4)GlcNAc in an oligosaccharide related to N-glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical-ionization mass spectrometry and 400-MHz 1H-NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N-acetyllactosamine-type glycan: Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3Man beta 1-4GlcNAc. The second is unusual with a terminal disaccharide Gal beta 1-6Gal, which had not yet been described for glycans of the N-acetyllactosamine type: Gal beta 1-6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6Man beta 1-4GlcNAc.     

Hypersialylated type-I lactosamine-containing N-glycans found in Artiodactyla sera are potential xenoantigens

There is increasing interest in biologics, i.e. human-originated biological pharmaceutics. Most of the protein drugs developed so far, such as immunoglobulins and erythropoietin, are secreted glycoproteins; as a result, any non-human-type glycans, such as αGal and NeuGc, derived from animal cells and sera must be removed to circumvent undesirable immunogenic reactions. In this study, we made an extensive search for potential xenoantigenic glycans among a panel of mammalian sera. As a result, sera belonging to the order Artiodactyla, i.e. bovine, lamb and goat sera, were found to contain substantial amounts of hypersialylated biantennary glycans closely associated with a type-I lactosamine structure containing a unique tetrasaccharide, Siaα2-3Galβ1-3(Siaα2-6)GlcNAc. In all three Artiodactyla sera, the most abundant structure was Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-3[Siaα2-6Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc. A dually hypersialylated biantennary structure, Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-3[Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc, was also abundant (10%) in bovine serum. The amount of hypersialylated glycans among total sialylated glycans was 46, 26 and 23% in bovine, lamb and goat sera, respectively. On the other hand, such structures could not be detected in the sera of other animals including human. The biological functions and the immunogenicity of the hypersialylated glycans in these animals remain to be elucidated; however, it is worth noting that glycoproteins biosynthesized from Artiodactyla cells and those contaminated with bovine serum might enhance undesirable antigenicity in human patients.     

Specificity analysis of the C-type lectin from rattlesnake venom,and its selectivity towards Gal- or GalNAc-terminated glycoproteins

The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Galβ1-4GlcNAc (LacNAc), α-Gal or α-GalNAc non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans, terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a K_D for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with a ten-fold better K_D for GalNAc.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Sulfated oligosaccharides isolated from the respiratory mucins of a secretor patient suffering from chronic bronchitis

The most acidic carbohydrate chains released by alkaline borohydride treatment of the bulk of airway mucins secreted by a patient (blood group O, secretor) suffering from a mildly infected chronic bronchitis have been fractionated using high-performance anion-exchange chromatography (HPAEC) according to a protocol already described [Lo-Guidice et al., J. Biol. Chem. 269 (1994) 18794] and were analyzed using 1H-NMR spectroscopy and matrix-assisted laser-adsorption-time-of-flight (MALDI-TOF) spectrometry. Many fractions corresponded to mixtures of oligosaccharides. This confirmed the wide diversity of the post-translational processes involved in the biosynthesis of airway mucins, which had already been observed in bronchial diseases, such as chronic bronchitis and cystic fibrosis (CF). Seven fractions were directly purified by HPAEC, allowing their structural determination. Six of them corresponded to 3-O-sulfated oligosaccharide chains terminated by a sulfated N-acetyllactosamine, a sulfated Lewis X or a sulfated Lewis A determinant, and the last one corresponded to a 6-O-sulfated chain terminated by a sulfated H-2 determinant. Three oligosaccharides had core type 2 and the other four had core type 4: IIIc2-9: Gal(beta1-3)[HSO(3)-3-Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol, IIIc2-10: Gal(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)[HSO(3)-6-]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-4: Fuc(alpha1-2)Gal(beta1-3)[HSO(3)-3-Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-8: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3)-3-Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol, IIIc2-7: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[Gal(beta1-4)[HSO(3)-6-]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-3: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3)-3-Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]GalNAc-ol, IIIc1-4: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3) -3-Gal(beta1-3)[Fuc(alpha1-4)]GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol. Like previous data concerning the airway mucins from another patient (blood group O and non-secretor) suffering from chronic bronchitis [Lo-Guidice et al., Glycoconj. J. 14 (1997) 113], no disialylated oligosaccharide and no sialylated and sulfated oligosaccharide bearing sialyl Lewis X epitope could be isolated. This is in contrast with the data obtained with the airway mucins secreted by the patient severely infected by Pseudomonas aeruginosa and suffering from CF, suggesting that important differences occur in the biosynthesis of airway mucins secreted by patients suffering from different bronchial diseases with or without severe infection.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

Human serum contains a novel beta 1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo (N-acetyllactosaminoglycans).

Incubation of UDP-GlcNAc and radiolabeled GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc beta 1-3Gal (4) and the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc beta 1-6Gal (6), together with the trisaccharides GlcNAc beta 1-6Gal beta 1-4GlcNAc (7) and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta 1,4-galactosyltransferase and UDP-[6-3H]Gal gave the octasaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4GlcNAc beta 1-3([6-3H]-Gal beta 1-4GlcNAc beta 1-6)[U-14C] Gal beta 1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3Gal (18) and the branched pentasaccharide GlcNAc beta 1-3-([6-3H]Gal beta 1-4GlcNAc beta 1-6) [U-14C]Gal beta 1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (20). The serum beta 1,6-GlcNAc transferase catalyzed also the formation of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (11) from UDP-GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (10). The pentasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (16), too, served as an acceptor for the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

H (0) blood group determinant is present on soluble human L-selectin expressed in BHK-cells.

In the present study we show that the H (0) blood group determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1-R is present on N-linked glycans of soluble human L-selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1 was detected for bi-, 2,4 branched tri- and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.     

Multiple recognition systems adopting four different glycotopes at the same domain for the Agaricus bisporus agglutinin-glycan interactions

For the GalNAcα1→ specific Agaricus bisporus agglutinin (ABA) from an edible mushroom, the mechanism of polyvalent Galβ1→3/4GlcNAcβ1→ complex in ABA-carbohydrate recognition has not been well defined since Gal and GlcNAc are weak ligands. By enzyme-linked lectinosorbent and inhibition assays, we show that the polyvalent Galβ1→3/4GlcNAcβ1→ in natural glycans also play vital roles in binding and we propose that four different intensities of glycotopes (Galβ1-3GalNAcα1-, GalNAcα1-Ser/Thr and Galβ1-3/4GlcNAcβ1-) construct three recognition systems at the same domain. This peculiar concept provides the most comprehensive mechanism for the attachment of ABA to target glycans and malignant cells at the molecular level.     

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galβ1-3GlcNAc or Galβ1-3GalNAc as basic motifs was commonly better than that to canonical Galβ1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galβ1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.     

Identification of a novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase in the connective tissue of the snail Lymnaea stagnalis

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in beta 1-3 linkage to terminal GalNAc of GalNAc beta 1-4GlcNAc-R [R = beta 1-2Man alpha 1-O(CH2)8COOMe, beta 1-OMe, or alpha,beta 1-OH]. Using GalNAc beta 1-4GlcNAc beta 1-2Man alpha-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe (Vmax 140 nmol.h-1.mg protein-1; Km 1.02 mM), GalNAc beta 1-4GlcNAc (Vmax 105 nmol.h-1.mg protein-1; Km 0.99 mM), and GalNAc beta 1-4GlcNAc beta 1-OMe (Vmax 108 nmol.h-1.mg protein-1; Km 1.33 mM). The products formed from GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe and GalNAc beta 1-4GlcNAc beta 1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz 1H-NMR spectroscopy to be Gal beta 1-3GalNAc beta 1-4GlcNAc 1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNac beta 1-3 GalNAc alpha 1-OC6H5, GalNAc alpha 1--ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.     

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.     

Glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.     

Complex fucolipids of hog gastric mucosa. Structure of the ceramide eikosahexoside

The structure of a complex fucolipid from hog gastric mucosa containing twenty sugar residues and exhibiting blood-group (A + H) activity has been investigated. Based on the results of immunological assays, partial acid hydrolysis, sequential degradation with specific exoglycosidases, oxidation with periodate and chromium trioxide, and permethylation analysis, we suggest that the carbohydrate chain of this fucolipid contains four termini. One of the termini bears beta Gal1 leads to 4 beta GlcNAc disaccharide, two bear blood-group A determinant and one bears H determinant. Two of the branches, terminated by beta Gal1 leads to 4 beta GlcNAc and blood-group A determinant, and two terminated by blood-group A and H determinants, are linked through beta Gal1 leads to 4 beta GlcNAc1 leads to 3/6 and beta Gal1 leads to 4 beta GlcNAc1 leads to 4 beta GlcNAc1 leads to 3/6 to the galactose residue adjacent to glucosylceramide core.     

Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.