Synthesis of partially O-acetylated N-acetylneuraminic acid using regioselective silyl exchange technology.

Postglycosylation acetylation of sialic acid imparts unique roles to sialoglycoconjugates in mammalian immune response making structural and functional understanding of these analogues important. Five partially O-acetylated Neu5Ac analogues have been synthesized. Reaction of per-O-silylated Neu5Ac ester with AcOH and Ac2O in pyridine promotes regioselective silyl ether/acetate exchange in the following order: C4 (2°) > C9 (1°) > C8 (2°) > C2 (anomeric). Subsequent hydrogenolysis affords the corresponding sialic acid analogues as useful chemical biology tools

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal αGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or αGalNAc, Neu5Acα2,6Gal/ GalNAc, Neu5AcGalβ1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal αMan/αGlc, βGlcNAc and terminal αGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Galβ1,4GlcNAc, Neu5Ac-GalNAc were seen in nonciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct

Differential expression of 9-O-acetylated sialoglycoconjugates on leukemic blasts: a potential tool for long-term monitoring of children with acute lymphoblastic leukemia

Earlier studies have demonstrated overexpression of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) on lymphoblasts, concomitant with high titers of anti-9-O-AcSG antibodies in childhood acute lymphoblastic leukemia (ALL). Our aim was to evaluate the correlation between expression of different 9-O-AcSGs during chemotherapeutic treatment. Accordingly, expression of 9-O-AcSGs on lymphoblasts of ALL patients (n = 70) were longitudinally monitored for 6 years (1997-2002), using Achatinin-H, a 9-O-acetylated sialic acid (9-O-AcSA) binding lectin with preferential affinity for 9-O-AcSGs with terminal 9-O-AcSAα2→6GalNAc. Western blot analysis of patients (n = 30) showed that 3 ALL-specific 9-O-AcSGs (90, 120 and 135 kDa) were induced at presentation; all these bands disappeared after treatment in patients (n = 22) who had disease-free survival. The 90 kDa band persisted in 8 patients who subsequently relapsed with reexpression of the 120 kDa band. FACS analysis revealed that at presentation (n = 70) 90.1 ± 5.0% cells expressed 9-O-AcSGs, which decreased progressively with chemotherapy, remained <5% during clinical remission and reappeared in relapse (80 ± 10%, n = 18). Early clearance of 9-O-AcSG+ cells, during 4-8 weeks of treatment showed a good correlation with low risk of relapse. Sensitivity of detection of 9-O-AcSG+ cells was 0.1%. Numbers of both high- and low-affinity binding sites were maximum at presentation, decreased with treatment and increased again in clinical relapse. We propose that close monitoring of 90 and 120 kDa 9-O-AcSGs may serve as a reliable index for long-term management of childhood ALL and merits therapeutic consideration

Distribucija sialoglikokonjugata - gangliozida i PSA-NCAM u mozgu dviju zmija otrovnica: Vipera ammodytes i Vipera berus bosniensis

The Bosnian adder (Vipera berus bosniensis) and the horned viper (Vipera ammodytes) are two venomous snake species with different ecological preferences. The Bosnian adder occurs in a range of habitats and is endemic to the Balkan Peninsula, while the horned viper thrives in dry, rocky areas with little vegetation. The horned viper is best known for its highly venomous venom, making it the most dangerous of the European vipers. The aim of this study was to compare the expression and distribution of complex gangliosides and to identify migratoryzones in the brain of Bosnian adder and horned viper. Immunohistochemistry was performed using specific antibodies for the major brain gangliosides (GM1, GD1a, GD1b, GT1b) and PSA NCAM and analysed in different brain regions. Both snake species showed expression of all four complex gangliosides with similar distribution patterns. GD1b was the most prominent ganglioside expressed in all brain structures, while GM1 showed varying distribution between the species. The strongest expression of PSA NCAM was observed in the periventricular zones of the telencephalon, suggesting that these areas are associated with neurogenesis, whereas other regions with lower expression may serve as migratory zones. In addition, it is important to note that the specific distribution of gangliosides and PSA NCAM may be influenced by factors such as brain region, developmental stage, and species-specific characteristics.Bosanska riđovka (Vipera berus bosniensis) i poskok (Vipera ammodytes) dvije su otrovne vrste zmija različitih ekoloških preferencija. Bosanska se riđovka javlja u različitim staništima i endem je Balkanskog poluotoka, dok poskok obitava u suhim, stjenovitim područjima s malo vegetacije i najpoznatija je zmija po vrlo otrovnom otrovu, što ga čini najopasnijom od europskih zmija. Cilj je ovog rada bio usporediti ekspresiju i distribuciju složenih gangliozida i identificirati migracijske zone u mozgu bosanske riđovke i poskoka. Imunohistokemija je provedena pomoću specifičnih protutijela za glavne gangliozide mozga (GM1, GD1a, GD1b, GT1b) i PSA NCAM čija je ekspresija i distribucija analizirana u različitim regijama mozga. Obje vrste zmija pokazale su ekspresiju sva četiri složena gangliozida sa sličnim obrascima distribucije. GD1b je bio najistaknutiji gangliozid izražen u svim moždanim strukturama, a GM1 je pokazao različitu raspodjelu između dviju vrste. Najjača ekspresija PSA NCAM uočena je u periventrikularnim zonama telencefalona, a to sugerira da su ta područja povezana s neurogenezom, dok druge regije s nižom ekspresijom predstavljaju migracijske zone. Važno je napomenuti da na specifičnu distribuciju gangliozida i PSA NCAM mogu utjecati i drugi čimbenici kao što su: regija mozga, razvojna faza životinje i karakteristike specifične za vrstu. Stoga proučavanje njihove distribucije u različitim životinjskim vrstama pruža uvid u raznolikost i evoluciju sialoglikokonjugata u kontekstu razvoja i funkcije neurona

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 amannose (Man) and/or aglucose (Glc) during oestrus and pregnancy and a moderate presence of oligosaccharides terminating in afucose (Fuc) during oestrus; cilia exhibited a more complex glycoconjugate pattern for the presence of oligosaccharides terminating in N-acetylgalactosamine (GalNAc), GalNAca1,3 GalNAca1,3galactose(Gal)b1,4Galb1,4N-acetylglucosamine( GlcNAc), Fuc, sialic acid (Neu5Ac)-aGalNAc belonging or not to the GalNAca1,3GalNAca1,3 Galb1,4 Galb1, 4GlcNAc sequence, and.aGalNAc and Neu5Aca 2,6Gal/GalNAc increased during oestrus. Cilia displayed terminal Galb1,3 GalNAc in pregnancy, terminal aGal 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 aGalNAc, Neu5Aca2,6Gal/GalNAc, Neu5Ac GalNAca 1,3GalNAca1,3Galb1,4Galb1,4GlcNAc during the investigated stages, as well as GlcNAc in anoestrus and pregnancy. The supra-nuclear zone of non-ciliated cells exhibited oligosaccharides with terminal Galb1,4GlcNAc and internal Man during oestrus and pregnancy as well as terminal aGal and Fuc in oestrus and Neu5Ac-Galb1,3GalNAc in pregnancy. The luminal surface of non-ciliated cells showed glycans terminating with aGalNAc and/or Neu5Ac GalNAca1,3 GalNAca1,3Galb1,4Galb1,4GlcNAc in all specimens, oligosaccharides with terminal Galb1,4GlcNAc and internal Man during oestrus and pregnancy, Neu5Ac a2,6Gal/GalNAc in anoestrus and oestrus, and glycans terminating with Galb1,3GalNAc, Neu5A aca2,3 Galb1, 4GlcNac, Neu5ac- Galb1,3GalNAc, Neu5Ac-Galb1,4 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

Monoclonal antibodies differentially affect the interaction between the hemagglutinin of H9 influenza virus escape mutants and sialic receptors

AbstractTo determine the receptor binding properties of various H9 influenza virus escape mutants in the presence and absence of antibody, sialyloligosaccharides conjugated with biotinylated polyacrylamide were used. A mutant virus with a L226Q substitution showed an increased affinity for the Neu5Acα2-3Galβ1-4Glc. Several escape mutants viruses carrying the mutation N193D bound to Neu5Acα2-6Galβ1-4GlcNAc considerably stronger than to Neu5Acα2-6Galβ1-4Glc. Several monoclonal antibodies unable to neutralize the escape mutants preserved the ability to bind to the hemagglutinin as revealed by enzyme-linked immunosorbent assay. In each case, the bound monoclonal antibodies did not prevent the binding of the mutant HA to high affinity substrates and did not displace them from the virus binding sites. Together, these data suggest that amino acid changes selected by antibody pressure may be involved in the specificity of host-cell recognition by H9 hemagglutinin and in the ability of viruses with these mutations to escape the neutralizing effect of antibodies in a differential way, depending on the specificity of the host cell receptor. It may be important in the natural evolution of the H9 subtype, a plausible candidate for the agent likely to cause a future pandemic

Glycobiology of Leishmania donovani

Leishmania donovani, the causative organism of visceral leishmaniasis (VL) is one of the deadliest of the entire known Leishmania species. This protozoan parasite displays immense adaptability to survive under extremely harsh conditions. Cell surface glycoconjugates play a pivotal role in parasite virulence and infectivity. This review mainly highlights on the importance of these molecules and their reported roles with special emphasis on L. donovani sialobiology. The recently evolved information reported by our group regarding the identification and characterization of sialoglycans and their possible mode(s) of acquisition as also the detailed identification, characterization of anti-O-acetylated sialic acid (anti-OAcSA) antibodies and their emerging biological roles, notably as molecules that may aid in host defense against the pathogen has been vividly discussed in this review

Down regulation of membrane-bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces apoptosis suppression of of neoplastic cells

Membrane-linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans-membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down-regulation of Neu3 in terms of both gene expression (-30 to 40%) and enzymatic activity toward ganglioside GD1a (-25.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL-cell line, MOLT-4, led to a significant increase of ceramide (+66%) and to a parallel decrease of lactosylceramide (-55%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/Bax ratio, the accumulation of Neu3 transfected cells in the sub G0-G1 phase of the cell cycle, the enhanced annexin-V positivity, the higher cleavage of procaspase-3. Therefore, the reduced expression of Neu3 in ALL could help lymphoblasts to survive, maintaining the cellular content of ceramide below a critical level. Interestingly, we found that Neu3 activity varied in relation to disease progression, increasing in clinical remission after chemotherapy, and decreasing again in patients that relapsed. In addition, a negative correlation was observed between Neu3 expression and the percentage of the ALL marker 9-OAcGD3 positive cells. Consequently, Neu3 could represent a new potent biomarker in childhood ALL, to assess the efficacy of therapeutic protocols and to rapidly identify an eventual relapse