Structural basis for the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.     

Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides

Sialyl-linkage specificity of sialidases of the human influenza A virus strains, A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) were studied using natural and synthetic gangliosides. The sialidase of the A/Aichi/2/68 strain hydrolyzed the terminal Neu5Acalpha2-3Gal sequence but not the Neu5Acalpha2-3 linkage on the inner Gal of GM1a, which is a ganglioside that has the gangliotetraose chain (Galbeta1-3GalNAcbeta1-4- (Neu5Acalpha2-3)Galbeta1++ +-4Glcbeta1-Cer). The sialidase hydrolyzed the Neu5Ac on the inner Gal of GM2, which had a shorter gangliotriose chain. GM4, which had the shortest chain (Neu5Acalpha2-3Galbeta1-Cer) of the gangliosides, had a lower substrate specificity. The N1 and N2 sialidase subtypes of the human influenza A virus had no significant variation in their substrate specificity for the gangliosides. Analysis of 11 synthetic gangliosides, which contained various ceramide or sialic acid moieties, demonstrated that A/Aichi/2/68 (H3N2) sialidase recognized the ceramide and sialic acid moiety and the length and structure of the sialyl sugar chain.      

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

New ganglioside analogs that inhibit influenza virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac alpha(2-S-6)Glc beta(1-1)Ceramide (1) and the GM3 analog Neu5Ac alpha(2-S-6)Gal beta(1-4)Glc beta(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent Ki value of 2.8 x 10(-6) and 1.5 x 10(-5) M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac alpha(2-S-6)Gal beta(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (Ki = 1.0 x 10(-4) M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were non-hydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases from Clostridium perfringens and Arthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.     

Evidence of Regional Differences in the Lectin Histochemistry Along the Ductus Epididymis of the Lizard, Podarcis Sicula Raf.

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)α(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Acα(2,6)Galβ(1,3)GalNAc, Neu5Acα(2,6)Galβ (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Acα(2,3)Galβ (1,4)GlcNAc, Neu5Acα(2,6)Galβ(1,3)GalNAc, Galβ (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Galβ(1,4)GlcNAc and αGalNAc, the luminal surface by αGalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Acα(2,3)Galβ (1,4)GlcNAc, Neu5Acα(2,6)Galβ (1,3)GalNAc, Neu5Acα(2,6)Galβ (1,4)GlcNAc, and internal GlcNAc, expressed terminal Galβ (1,4)GlcNAc and αGalNAc in the caput, and terminal β GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal βGalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal αGalNAc only in the corpus.     

Fractionation of sialylated oligosaccharides, glycopeptides, and glycoproteins on immobilized elderberry (Sambucus nigra L.) bark lectin

A new plant lectin from elderberry (Sambucus nigra L.) bark, which was shown by immunochemical techniques to bind specifically to terminal Neu5Ac(alpha 2-6)Gal/GalNAc residues of glycoconjugates, was immobilized onto Sepharose 4B (SNA-Sepharose) and its carbohydrate binding properties was determined using a series of standard compounds. Oligosaccharides, glycopeptides, or glycoproteins containing terminal Neu5Ac(alpha 2-6)Gal/GalNAc sequences bound to SNA-Sepharose and were eluted with 50-100 mM lactose, whereas those with Neu5Ac(alpha 2-3)Gal/GalNAc failed to bind to this column. Furthermore, the SNA-Sepharose column was capable of resolving two oligosaccharides/glycopeptides based on the number of Neu5Ac(alpha 2-6)Gal units present in each molecule. Application of this technique to two glycoproteins, fetuin and orosomucoid, revealed the presence of microheterogeneity. It was also shown that esterification of the carboxyl group of Neu5Ac units, or branching at the O-3 of the subterminal GalNAc (probably also Gal) destroyed the binding ability of the molecule.     

2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)- and N-acetylneuraminic acid-cleaving sialidase (KDN-sialidase) and KDN-cleaving hydrolase (KDNase) from the hepatopancreas of oyster, Crassostrea virginica.

KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.     

A novel sialidase which releases 2,7-anhydro-alpha-N-acetylneuraminic acid from sialoglycoconjugates

The leech (Macrobdella decora) was found to contain two sialic acid-cleaving enzymes: an ordinary sialidase and a novel sialic acid-cleaving enzyme. This novel enzyme released 2,7-anhydro-alpha-N-acetylneuraminic acid (Neu2,7-anhydro5Ac) instead of alpha-N-acetylneuraminic acid (Neu5Ac) from 4-methylumbelliferyl-Neu5Ac, glycoproteins, and gangliosides. We have partially purified this novel sialidase from M. decora. We have also isolated Neu2,7-anhydro5Ac released from 4-methylumelliferyl-Neu5Ac and whale nasal keratan sulfate in pure form. The novel sialidase produced Neu2,7-anhydro5Ac only from sialoglycoconjugates, but not from free Neu5Ac. The structure of Neu2,7-anhydro5Ac produced by the novel sialidase was established by chemical analysis, mass spectrometry, and NMR spectroscopy. NMR analysis showed that instead of the original 2C5 conformation, the pyranose ring of Neu2,7-anhydro5Ac was in the 5C2 conformation, which makes the formation of the 2,7-anhydro bridge possible.     

Synthesis of Neu5Ac(alpha2-->6)[Gal(alpha1-->3)]GalNAcalpha and Neu5Ac(alpha2-->6)[Gal(beta1-->3)]GalNAcalpha trisaccharides as protected trifluoroacetamidopropyl glycosides

Protected sialo-containing trisaccharides, fragments of oligosaccharide chains of mucin glycoproteins, were synthesized. Regioselective sialylation of the primary hydroxyl group of (3-fluoroacetamidopropyl)-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-ben zyl)-alpha -D-galactopyranosyl)-alpha-D-galactopyranoside with methyl ester of peracetyl-beta-ethylthioglycoside of N-acetylneuraminic acid in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) yielded 39 and 25% of alpha- and beta-sialyl-(2-->6)biosides, respectively. Catalytic hydrogenolysis of the azide and benzyl groups of the alpha-anomer followed by N- and O-acetylation gave target trifluoroacetamidopropyl glycoside, Neu5Ac(alpha 2-->6)[Gal(alpha 1-->3)]GalNAc alpha-OSp, as a peracetate. An analogous coupling of the sialyl donor with (3-fluoroacetamidopropyl)-2-acetamido-2-deoxy-3-O- (2,3,4,6-tetra-O-acetyl)-beta-D-galactopyranosyl)-alpha-D-galactopyranos ide affords acetylated trifluoroacetamidopropyl glycoside Neu5Ac(alpha 2-->6)[Gal(beta 1-->3)]GalNAc alpha-OSp in a yield of 15% and the corresponding Neu5Ac(beta 2-->6)-anomer in a yield of 12%. After O-deacetylation and N-detrifluoroacetylation, these sialylbiosides can be used as ligands in preparing neoglycoconjugates.     

Occurrence of a unique sialyl tetrasaccharide in colostrum of a bottlenose dolphin (Tursiops truncatus)

Crude oligosaccharides were recovered from bottlenose dolphin (Tursiops truncatus) colostrum after chloroform/methanol extraction of lipids and protein precipitation, and purified using gel filtration, anion exchange chromatography and high performance liquid chromatography (HPLC). Their chemical structures characterized by NMR spectroscopy were as follows: GalNAc(beta1-4)[Neu5Ac(alpha2-3)]Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc and Gal(alpha1-4)Gal(beta1-4)Glc. The monosialyltetrasaccharide and neutral trisaccharide have not previously been found as free forms in any natural sources including milk or colostrum, although these structures have been found in the carbohydrate units of glycosphingolipids GM2 and Gb3.     

Identification of sialic acids on Leishmania donovani amastigotes

The presence of Neu5Ac on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, has been reported recently. Here we report the occurrence of Neu5Ac as a major component on amastigotes, as well as Neu5Gc, Neu5,9Ac2 and Neu9Ac5Gc as indicated by fluorimetric high performance liquid chromatography and gas liquid chromatography/electron impact mass spectrometry. Furthermore, binding studies with Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and various Siglecs, showed the presence of both (alpha2 --> 6)- and (alpha2 --> 3)-linked sialic acids; their binding was reduced after sialidase pretreatment. Western blotting of amastigote membrane glycoproteins with SNA demonstrated the presence of two sialoglycoconjugates of Mr values of 164000 and 150000. Similarly, binding of MAA demonstrated the presence of five distinct sialoglycans corresponding to molecular masses of 188, 162, 136, 137 and 124 kDa. Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid (alpha2 --> 6)-linked to GalNAc, demonstrated the occurrence of two 9-O-acetylated sialoglycans with Mr 158000 and 150000, and was corroborated by flow cytometry; this binding was abolished by recombinant 9-O-acetylesterase pretreatment. Our results indicate that Neu5Ac [(alpha2 --> 6)- and (alpha2 --> 3)-linked], as well as Neu5Gc and their 9-O-acetyl derivatives, constitute components of the amastigote cell surface of L. donovani.     

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a K_D in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14⁺ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.     

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

Differential expression of sialic acid on porcine organs during the maturation process

Sialylated structures play important roles in cell communication, and change in a regulated manner during development and differentiation. In this work, we report the main glycosidic modifications that occur during the maturation of porcine tissues, involving the sialylation process as determined with lectins. Sialic acids were identified at several levels in a broad range of cell types of nervous, respiratory, genitourinary and lymphoid origin. Nevertheless, the most contrasting was the type of glycosidic linkage between 5-N-acetyl-neuraminic acid (Neu5Ac) and galactose (Gal) expressed in central nervous system (CNS). Newborn CNS abundantly expressed Neu5Acalpha2,3Gal, but weakly or scarcely expressed Neu5Acalpha2,6Gal/GalNAc. Maturation of CNS induced drastic changes in sialic acid expression. These changes include decrease or complete loss of NeuAcalpha2,3Gal residues, mainly in olfactory structures and brain cortex, which were replaced by their isomers Neu5Acalpha2,6Gal/GalNAc. In the brain cortex and cerebellum, the increase of Neu5Acalpha2,6Gal/GalNAc molecules was paralleled by an increase of 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2). In addition, terminal Gal and N-acetyl-D-galactosamine (GalNAc) residues also increased their expression in adult CNS tissues, but this was more significant in structures forming the encephalic trunk. Our results show that sialylation of porcine CNS is finely modulated throughout the maturation process.     

Solution conformations of GM3 gangliosides containing different sialic acid residues as revealed by NOE-based distance mapping, molecular mechanics, and molecular dynamics calculations.

The conformation of the GM3 ganglioside, Neu5Ac alpha 2-3Gal beta 1-4Glc beta 1-1 Cer, and its analogs containing the Neu5Gc or Neu4Ac5Gc residues (Gc = glycolyl, CH2OHCO) was investigated in Me2SO-d6 solution with the aid of a distance-mapping procedure based on rotating-frame NOE contacts, with hydroxyl protons being used as long-range sensors defining the distance constraints. A pronounced flexibility found for both the Neu-Gal and Gal-Glc linkages was confirmed by 1000-ps molecular dynamics simulations. Similar results, although based on a smaller number of NOE constraints, were obtained for GM3 gangliosides anchored in mixed D2O/dodecylphosphocholine-d38 micelles and for the Neu5Ac-, Neu5Gc-, and Neu5,9Ac2-sialyllactoses dissolved in D2O. No noteworthy differences in conformational behavior of the glycan chains of the three gangliosides or sialyllactoses were observed in either of the media.     

Recovery of function and mass of endogenous beta-cells in streptozotocin-induced diabetic rats treated with islet transplantation

We found that the hepatopancreas of oyster, Crassostrea virginica, contained a sialidase capable of releasing Neu5Gc from the novel polysialic acid chain (-->5-O(glycolyl)Neu5Gcalpha2-->)n more efficiently than from the conventional type of polysialic acid chains, (-->8Neu5Acalpha2-->)n, or (-->8Neu5Gcalpha2-->)n. We have partially purified this novel sialidase and compared its reactivity with that of microbial sialidases using four different sialic acid dimers, Neu5Gcalpha2-->5-O(glycolyl)Neu5Gc (Gg2), Neu5Acalpha2-->8Neu5Ac (A2), Neu5Gcalpha2-->8Neu5Gc (G2), and KDNalpha2-->8KDN (K2) as substrates. Hydrolysis was monitored by high performance anion-exchange chromatography with a CarboPac PA-100 column and pulsed amperometric detection, the method by which we can accurately quantitate both the substrate (sialiac acid dimers) and the product (sialic acid monomers). The oyster sialidase effectively hydrolyzed Gg2 and K2, whereas A2 and G2 were poor substrates. Neu5Ac2en but not KDN2en effectively inhibited the hydrolysis of Gg2 by the oyster sialidase. Likewise, the hydrolysis of K2 by the oyster sialidase was inhibited by a cognate inhibitor, KDN2en, but not by Neu5Ac2en. Using the new analytical method we found that Gg2 was hydrolyzed less efficiently than A2 but much more readily than G2 by Arthrobacter ureafaciens sialidase. This result was at variance with the previous report using the thiobarbituric acid method to detect the released free sialic acid [Kitazume, S., et al. (1994) Biochem. Biophys. Res. Commun. 205, 893-898]. In agreement with previous results, Gg2 was a poor substrate for Clostridium perfringens sialidase, while K2 was refractory to all microbial sialidases tested. Thus, the oyster sialidase is novel and distinct from microbial sialidases with regards to glycon- and linkage-specificity. This finding adds an example of the presence of diverse sialidases, in line with the diverse sialic acids and sialic acid linkages that exist in nature. The new sialidase should become useful for both structural and functional studies of sialoglycoconjugates.     

Distribution of sialoglycoconjugates in the oviductal isthmus of the horse during anoestrus, oestrus and pregnancy: a lectin histochemistry study

The distribution of sialic acid residues as well as other glycosidic sugars has been investigated in the horse oviductal isthmus during anoestrus, oestrus and pregnancy by means of lectin and pre-lectin methods. Ciliated cells and non-ciliated (secretory) cells exhibited different lectin binding profiles that were found to change during the investigated stages. Ciliated cells did not show any reactivity in the basal cytoplasm, while the supra-nuclear cytoplasm displayed a few of oligosaccharides with terminal and internal alphamannose (Man) and/or alphaglucose (Glc) during oestrus and pregnancy and a moderate presence of oligosaccharides terminating in alphafucose (Fuc) during oestrus; cilia exhibited a more complex glycoconjugate pattern for the presence of oligosaccharides terminating in N-acetylgalactosamine (GalNAc), GalNAcalpha1,3 GalNAcalpha1,3galactose(Gal)beta1,4Galbeta1,4N-acetylglucosamine(GlcNAc), Fuc, sialic acid (Neu5Ac)-aGalNAc belonging or not to the GalNAca1,3GalNAca1,3 Galb1,4 Galb1, 4GlcNAc sequence, and. alphaGalNAc and Neu5Aca 2,6Gal/GalNAc increased during oestrus. Cilia displayed terminal Galbeta1,3 GalNAc in pregnancy, terminal alphaGal in anoestrus and pregnancy and terminal or internal D-GlcNAc during anoestrus and pregnancy, respectively. The whole cytoplasm of non-ciliated cells showed oligosaccharides terminating with alphaGalNAc, Neu5Aca2,6Gal/GalNAc, Neu5Ac GalNAca 1,3GalNAcalpha1,3Galbeta1,4Galbeta1,4GlcNAc during the investigated stages, as well as GlcNAc in anoestrus and pregnancy. The supra-nuclear zone of non-ciliated cells exhibited oligosaccharides with terminal Galbeta1,4GlcNAc and internal Man during oestrus and pregnancy as well as terminal alphaGal and Fuc in oestrus and Neu5Ac-Galbeta1,3GalNAc in pregnancy. The luminal surface of non-ciliated cells showed glycans terminating with alphaGalNAc and/or Neu5Ac GalNAcalpha1,3 GalNAcalpha1,3Galbeta1,4Galbeta1,4GlcNAc in all specimens, oligosaccharides with terminal Galbeta1,4GlcNAc and internal Man during oestrus and pregnancy, Neu5Ac alpha2,6Gal/GalNAc in anoestrus and oestrus, and glycans terminating with Galbeta1,3GalNAc, Neu5A acalpha2,3 Galbeta1, 4GlcNac, Neu5ac-Galbeta1,3GalNAc, Neu5Ac-Galbeta1,4 GlcNAc in pregnancy. These findings show the presence of sialoglycoconjugates in the oviductal isthmus of the mare as well as the existence of great modifications in the glycoconjugates linked to different physiological conditions.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and structural characterization of sialic-acid-containing glycopeptides of the O-glycosidic type from the urine of two patients with an hereditary deficiency in alpha-N-acetylgalactosaminidase activity

Glycopeptides have been isolated from the urine of two patients, aged 5 and 6, with a new lysosomal storage disease characterized by a deficiency in alpha-N-acetylgalactosaminidase activity. Isolation of these glycopeptides was achieved using gel filtration and ion-exchange chromatography. Structural determination was done using one- and two-dimensional 500 MHz 1H-NMR spectroscopy and FAB mass spectrometry of native and derivatized glycopeptides. The following structures were inferred as being present: Glycopeptide A (up to 140 mg/l urine) (1)-(3) Neu5Ac alpha 2-3Gal beta 1-3 (Neu5Ac alpha 2-6)GalNAc alpha 1-R A1: R = Ser A2: R = Thr A3: R = Thr-Pro Glycopeptide B (up to 80 mg/l urine) (4)-(6) Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6 (Neu5Ac alpha 2-3-Gal beta 1-3) GalNAc alpha 1-R B1: R = Ser B2:R = Thr B3: R = Thr-Pro     

Saturation transfer difference (STD) 1H-NMR experiments and in silico docking experiments to probe the binding of N-acetylneuraminic acid and derivatives to Vibrio cholerae sialidase

Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.