Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules.

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highlysensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (-->8Neu5Acalpha2-->)(n), (-->9Neu 5Acalpha2-->)(n), (-->8Neu5Gcalpha2-->)(n), (-->5-O(glycolyl)-Neu5Gcalpha2-->)(n), and Neu5Gc9SO(4)alpha2-->(-->5-O(glycolyl)-Neu5Gcalpha2--> )(n), ()expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySiacompounds and confirmed that resolution of high polymers (DP >70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4, 5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.     

A new sialic acid analogue, 9-O-acetyl-deaminated neuraminic acid, and alpha -2,8-linked O-acetylated poly(N-glycolylneuraminyl) chains in a novel polysialoglycoprotein from salmon eggs

A new polysialoglycoprotein, designated PSGP(On), was isolated from the unfertilized eggs of the kokanee salmon, Oncorhynchus nerka adonis. 400-MHz 1H NMR analyses showed the O. nerka adonis PSGP contained alpha -2,8-linked oligo- and polysialic acid (polySia) chains that were made up of 4-O-Ac-, 7-O-Ac-, and 9-O-Ac esters of N-glycolylneuraminic acid (Neu5Gc) residues. The presence of a new sialic acid derivative, identified by 1H NMR as 9-O-acetyl-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (trivial name, 9-O-acetyldeaminated neuraminic acid; 9-O-Ac-KDN), was also shown to be present as a minor component. The O-acetylated KDN residues appear to cap the nonreducing termini of the O-acetylated poly(Neu5Gc) chains. The O-acetylated polySia chains were resistant to depolymerization by bacterial exosialidases and a bacteriophage-derived endo-N-acylneuraminidase that is specific for catalyzing the hydrolysis of alpha -2,8-linkages in polySia containing either N-acetylneuraminic acid or Neu5Gc residues. After de-O-acetylation by mild alkali, the polySia chains were sensitive to digestion by endo-N-acylneuraminidase, yet partially resistant to exosialidase. These data confirm the alpha -2,8-ketosidic linkage in these chains and the nonreducing terminal location of the KDN residues. These results extend further the range of structural diversity in polySia-containing glycoconjugates, and in the family of naturally occurring sialic acids. They also suggest that the O-acetylated Neu5Gc and 9-O-Ac-KDN residues may have an important role during oogenesis.     

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.     

Developmental profile of neural cell adhesion molecule glycoforms with a varying degree of polymerization of polysialic acid chains

More precise information on the degree of polymerization (DP) of polysialic acid (polySia) chains expressed on neural cell adhesion molecule (NCAM) and its developmental stage-dependent variation are considered important in understanding the mechanism of regulated polysialylation and fine-tuning of NCAM-mediated cell adhesion by polySia. In this paper, first we performed a kinetic study of acid-catalyzed hydrolysis of polySia and report our findings that (a) in (-->8Neu5Ac alpha 2-->)(n)-->8Neu5Ac alpha 2-->3Gal beta 1-->R, the proximal Neu5Ac residue alpha 2-->3 linked to Gal is cleaved about 2.5-4 times faster than the alpha 2-->8 linkages and (b) in contrary to general belief that alpha 2-->8 linkages in polySia are extremely labile, the kinetic consideration showed that they are not so unstable, and every ketosidic bond is hydrolyzed at the same rate. These findings are the basis of our strategy for DP analysis of polySia on NCAM. Second, using the recently developed method that provides base-line resolution of oligo/polySia from DP 2 to >80 with detection thresholds of 1.4 fmol per resolved peak, we have determined the DP of polySia chains expressed in embryonic chicken brains at different developmental stages. Our results support the presence of numerous NCAM glycoforms differing in DPs of oligo/polySia chains and a delicate change in their distribution during development.     

Isolation and characterization of low density detergent-insoluble membrane (LD-DIM) fraction from sea urchin sperm.

The low density detergent-insoluble membrane (LD-DIM) fraction was obtained by a sucrose-density gradient centrifugation from sperm of three sea urchin species, Hemicentrotus pulcherrimus, Strongylocentrotus purpuratus, and Anthocidaris crassispina. These LD-DIM preparations were characterized by enriched glycosphingolipids (GSL) including gangliosides and sulfatide (SLF), having more than 50% of the total amount of GSL present in these sperm. Interestingly, a minor component of H. pulcherrimus sperm (HO3S-->8Neu5Acalpha2-->8Neu5Acalpha2-->6Glcbeta1++ +-->Cer) was shown to be even more enriched in the LD-DIM as revealed by using monoclonal antibody (mAb.3G9) speific to this ganglioside. In addition to the GSL, phosphatidyl-serine (PS) and diacylglcerol (DG) were enriched in the LD-DIM. On the other hand, cholesterol (CL) and sphingomyelin (SM) were not so enriched, which contrasted with the LD-DIM from Madin-Darby canine kidney (MDCK) cells, where CL and SM were reported to be abundant. Because mammalian somatic cell-derived DIMs have been proposed to be associated with functional signal transduction, it seems possible that the ganglioside-enriched LD-DIM in sea urchin sperm can participate in binding to eggs and the subsequent egg activation process. To our knowledge this is the chemical characterization of the LD-DIM fraction of a gametic cell.     

Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses

Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.     

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.     

Co-expression of two distinct polysialic acids, α2,8- and α2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm

Naturally occurring polysialic acid (polySia) structures have a large diversity, primarily arising from the diversity in the sialic acid components as well as in the intersialyl linkages. In 2004, we demonstrated the presence of a new type of polySia, 8-O-sulfated N-acetylneuraminic acid (Neu5Ac) capped α2,9-linked polyNeu5Ac, on the O-glycans of a major 40-80 kDa sialoglycoprotein, flagellasialin, in sea urchin sperm. In this study, we demonstrated that another type of polySia, the α2,8-linked polyNeu5Ac, exclusively occurs on O-glycans of a 190 kDa glycoprotein (190 kDa-gp), whereas the α2,9-linked polyNeu5Ac is exclusively present on flagellasialin. The 190 kDa-gp is localized in both flagellum and head of sperm. We also demonstrated that polysialogangliosides containing the α2,8-linked polyNeu5Ac are present in sperm head. Thus, this study shows two novel features of the occurrence of polySia in nature, the co-localization of polySia with different intersialyl linkages, the α2,8- and α2,9-linkages, in a single cell and the occurrence of α2,8-linked polyNeu5Ac in glycolipids. Anti-α2,8-linked polyNeu5Ac antibody had no effect on fertilization, which contrasted with the previous results that anti-α2,9-linked polyNeu5Ac antibody inhibited sperm motility and fertilization. Based on these properties, distinct functions of α2,8- and α2,9-polySia structures are implicated in fertilization.     

Identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation

Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus minute virus of mice (MVM). We have utilized a glycan array consisting of approximately 180 different carbohydrate structures to identify the specific sialosides recognized by the prototype (MVMp) and immunosuppressive (MVMi) strains of MVM plus three virulent mutants of MVMp, MVMp-I362S, MVMp-K368R, and MVMp-I362S/K368R. All of the MVM capsids specifically bound to three structures with a terminal sialic acid-linked alpha2-3 to a common Galbeta1-4GlcNAc motif: Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-4Galbeta1-4GlcNAc (3'SiaLN-LN), Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (3'SiaLN-LN-LN), and Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)-GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc (sLe(x)-Le(x)-Le(x)). In addition, MVMi also recognized four multisialylated glycans with terminal alpha2-8 linkages: Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha ((Sia)(3)), Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GD3), Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GT3), and Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glc (GD2). Interestingly, the virulent MVMp-K368R mutant also recognized GT3. Analysis of the relative binding affinities using a surface plasmon resonance biospecific interaction (BIAcore) assay showed the wild-type MVMp and MVMi capsids binding with higher affinity to selected glycans compared with the virulent MVMp mutants. The reduced affinity of the virulent MVMp mutants are consistent with previous in vitro cell binding assays that had shown weaker binding to permissive cells compared with wild-type MVMp. This study identifies the sialic acid structures recognized by MVM. It also provides rationale for the tropism of MVM for malignant transformed cells that contain sLe(x) motifs and the neurotropism of MVMi, which is likely mediated via interactions with multisialylated glycans known to be tumor cell markers. Finally, the observations further implicate a decreased binding affinity for sialic acid in the in vivo adaptation of MVMp to a virulent phenotype.     

Chemical analysis of the developmental pattern of polysialylation in chicken brain. Expression of only an extended form of polysialyl chains during embryogenesis and the presence of disialyl residues in both embryonic and adult chicken brains

Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40-50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acalpha2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development.     

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.     

Chemoenzymatic synthesis of artificial glycopolypeptides containing multivalent sialyloligosaccharides with a gamma-polyglutamic acid backbone and their effect on inhibition of infection by influenza viruses

Highly water-soluble, artificial glycopolypeptides with a gamma-polyglutamic acid (gamma-PGA) backbone derived from Bacillus subtilis sp. and multivalent sialyloligosaccharide units have been chemoenzymatically synthesized as potential polymeric inhibitors of infection by bird and human influenza viruses. 5-Trifluoroacetamidopentyl beta-N-acetyllactosaminide and 5-trifluoroacetamidopentyl beta-lactoside were enzymatically synthesized from LacNAc and lactose, respectively, by cellulase-mediated condensation with 5-trifluoroacetamido-1-pentanol. After deacetylation, the resulting 5-aminopentyl beta-LacNAc and beta-lactoside glycosides were coupled to the alpha-carboxyl groups of the gamma-PGA side chains. The artificial glycopolypeptides carrying LacNAc and lactose were further converted to Neu5Acalpha2-(3/6)Galbeta1-4Glcbeta and Neu5Acalpha2-(3/6)Galbeta1-4GlcNAcbeta sialyloligosaccharide units by alpha2,3- and alpha2,6-sialyltransferase, respectively. The interaction of these glycopolypeptides with various influenza virus strains has been investigated by three different methods. Glycopolypeptides carrying Neu5Acalpha2,6LacNAc inhibited hemagglutination mediated by influenza A and B viruses, and their relative binding affinities for hemagglutinin were 10(2)- to 10(4)-fold higher than that of the naturally occurring fetuin control. A glycopolypeptide carrying Neu5Acalpha2,6LacNAc inhibited infection by A/Memphis/1/71 (H3N2) 93 times more strongly than fetuin, as assessed by cytopathic effects on virus-infected MDCK cells. The avian virus [A/duck/Hong kong/4/78 (H5N3)] bound strongly to Neu5Acalpha2,3LacNAc/Lac-carrying glycopolypeptides, whereas the human virus [A/Memphis/1/71 (H3N2)] bound to Neu5Acalpha2,6LacNAc in preference to Neu5Acalpha2,6Lac. Taken together, these results indicate that the binding of viruses to terminal sialic acids is markedly affected by the structure of the asialo portion, in this case either LacNAc or lactose, in the sugar chain of glycopolypeptides.     

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.     

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.     

Cross metathesis for the synthesis of novel C-sialosides

Cross metathesis of both anomers of C-allyl sialoside (3alpha/3beta) with styrene catalyzed by the second generation Grubbs or Hoveyda-Grubbs catalysts gave the corresponding aryl derivatives (4alpha/4beta) in virtually quantitative yields. The products were hydrogenated to model compounds 5alpha/5beta. Similarly, reaction of the alpha-anomer 3alpha with galactose derivative 8 gave the olefin-linked disaccharide mimetic 9. Following hydrogenation and deprotection, the ethylene-bridged Neu5Acalpha(2-->6)Gal analogue 11 could be obtained.     

Elderberry bark lectins evolved to recognize Neu5Ac alpha2,6Gal/GalNAc sequence from a Gal/GalNAc binding lectin through the substitution of amino-acid residues critical for the binding to sialic acid

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acalpha2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S(197), A(233) and Q(234), in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acalpha2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acalpha2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution.     

Rapid Trimming of Cell Surface Polysialic Acid (PolySia) by Exovesicular Sialidase Triggers Release of Preexisting Surface Neurotrophin

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation.     

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

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

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.     

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.