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 location and structural specificities of sialic acid-beta-D-Gal sequences belonging to sialoderivatives of rabbit oviduct under hormonal treatment

Sialoderivatives expressed in the rabbit oviduct under hormonal treatment have been investigated in situ by lectin histochemistry with a view to specifying further regional and temporal specializations which enable ampulla and isthmus to play distinct roles in the reproductive events. Application of MAL II and SNA lectins allowed sialoglycoconjugates containing Sia(alpha2,3)Gal and Sia(alpha2,6)Gal groups to be discriminated. Sialic acid residues linked to Gal(beta1,3)-D-GalNAc sequences were identified using PNA combined with sialidase digestion. Information on structural features of sialic acids were acquired by deacetylation and differential oxidation pretreatments. In both oviductal portions, Sia(alpha2,6) groups were restricted to the luminal surface of the lining epithelium while Sia(alpha2,3) groups were specifically located in the secretory, non-ciliated cells. In the ampullary epithelium, non-acetylated sialic acids alpha2,3-linked to Gal(beta1,3)-D-GalNAc sequences were largely present. Only at ovulation time were sialic acid residues containing acetyl substituents on C4 also found. A great variety of sialic acids were found in the isthmic epithelium which showed the highest expression of acetylated forms at the first hours after the hormonal treatment. The heterogeneity of sialoderivatives differently expressed in the ampulla and isthmus as well as their distinct cycle-dependent modulation suggest that sialylated components may contribute to the molecular and functional specificities within the oviductal epithelium.     

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of<Emphasis Type="Italic"> Fonsecaea pedrosoi</Emphasis>

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing 2,3- and 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.     

Sialoglycoproteins in Morphological Distinct Stages of Mucor polymorphosporus and their Influence on Phagocytosis by Human Blood Phagocytes

The possible role of sialic acids in host cells–fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.     

Differential distribution of sialic acid in alpha2,3 and alpha2,6 linkages in the apical membrane of cultured epithelial cells and tissues.

We used lectin cytochemistry and confocal microscopy to examine the distribution of sialic acid in epithelial cells. Maackia amurensis lectin and Sambuccus nigra agglutinin were used to detect alpha2,3 and alpha2,6 sialic acid, respectively. In Caco-2, HT-29 5M12, and MCF-7 cells, which express sialic acid mainly in one type of linkage, the majority of the signal was observed in the apical membrane. In cells that bound both lectins, alpha2,3 sialic acid was distributed apically, whereas alpha2,6 sialic acid showed a broader distribution. In IMIM-PC-1 cultures, alpha2,3 sialic acid was detected mainly in the apical membrane, whereas alpha2,6 sialic acid was more abundant in the basolateral domain of polarized cells. In these cells, treatment with GalNAc-O-benzyl led to reduced alpha2,3 levels and to an increase and redistribution of alpha2,6 to the apical domain. Similarly, sialic acid was predominantly expressed apically in all epithelial tissues examined. In conclusion, (a) sialic acid is mainly distributed to the apical membrane of epithelial cells, (b) there is a hierarchy in the distribution of sialic acids in polarized epithelial cells, i.e., alpha2,3 is preferred to alpha2,6 in the apical membrane, and (c) IMIM-PC-1 cells are a good model in which to study the regulation of the levels and distribution of sialic acids.     

Characterization of carbohydrate chains of C1-inhibitor and of desialylated C1-inhibitor.

Carbohydrate chains of C1-inhibitor were identified with a binding assay using different lectins. Lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for sialic acids bound to C1-inhibitor. Lectin from Datura stramonium (DSA) reacted also with the inhibitor indicating complex and hybrid sugar structures. C1-inhibitor was enzymatically desialylated and reexamined for lectin binding. SNA and MAA did not react anymore, but in addition to DSA, peanut agglutinin, which can bind to carbohydrate chains after sialic acids are removed, bound to desialylated C1-inhibitor. C1-inhibitor contains about 30 sialic acid residues per molecule. SDS-polyacrylamide gel electrophoresis showed that desialylated C1-inhibitor had a faster mobility than native C1-inhibitor. The N-terminal sequence of desialylated C1-inhibitor was the same as of native C1-inhibitor and no change in the inhibition of human plasma kallikrein was observed.     

Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions.

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.     

Lectin histochemistry for in situ profiling of rat colon sialoglycoconjugates

The growing interest in glycoconjugates expressed and released by the epithelium of the intestinal mucosa is tightly related to the multiple functional roles attributed to sialic acid and its derivatives. In the present work, biotin and HRP conjugated lectins were used to detect the sialylation pattern and to identify specific structural features of sialoderivatives in the rat colon. In particular, the occurrence and distribution of sialic acids linked alpha2,6 to D-Gal/D-GalNAc and alpha2,3 to D-Gal were directly demonstrated with SNA and MAL II binding, respectively. In addition, in order to by-pass the specificity problems of SNA and MAL II as histochemical reagents, as well as to look for additional and complementary information about acetylation degree and sites, we combined sialidase digestion, potassium hydroxide deacetylation, and differential periodate oxidation with PNA and DBA binding. The data showed the distribution and structure of sialic acid-beta-D-Gal(1-3)-D-GalNAc and sialic acid-D-GalNac sequences, which proved to be widely distributed as cellular components or secretory products in surface goblet cells and crypt cells of the colonic epithelium. A high degree of O-acetylation, with acetyl groups mainly at 9 and 4 positions, was found, showing an increasing gradient from the proximal to distal portion of the colon. These results, which largely reproduce the sialylation pattern in other species, contribute new insights in defining the tissue specific expression of sialoderivatives in the colonic mucosa, and testify to their high heterogeneity which the wide range of sialic acid functional correlates in the intestinal tract depend on.     

Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic insect cells

Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in Mimic insect cells, that are commercial stably transformed Spodoptera frugiperda (Sf9) cells expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex-type monosialylated N-glycans. We previously showed that it exhibited no in vivo bioactivity although expressing full in vitro bioactivity, and it was suspected that this was because of insufficient sialylation of eLH/CG N-glycans. Lectin binding analyses were performed with recombinant dimeric eLH/CG or its alpha subunit, secreted in the serum-containing supernatant of infected Sf9 and Mimic cells. Two types of specific lectin affinity assays (blot analyses and enzyme-linked immunosorbent assay) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins. In natural equine chorionic gonadotropin (eCG), complex-type N-glycans terminating with both Siaalpha2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Siaalpha2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Siaalpha2-6Gal. In eLH/CG and its alpha subunit produced by Sf9 cells, N-glycans were found to be terminated by mannosyl residues (based on Galanthus nivalis agglutinin [GNA] binding), whereas those produced in Mimic cells were terminated by galactoses (based on binding to Ricinus communis agglutinin I [RCA I] , but not to SNA or MAA). This is in agreement with the fact that the nucleotide donor substrate of sialic acid is not naturally synthesized in insect cells. On the basis of binding to Arachis Hypogaea agglutinin [PNA], O-glycans exhibited the Galbeta1-3GalNAc structure in recombinant-free alpha and eLH/CG from both Sf9 and Mimic cell lines. Both N- and O-linked carbohydrate side chains synthesized in Mimic cells should thus be amenable to further acellular sialylation.     

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an 2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-¹⁴C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat 2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003.     

Construction of an in vitro trans-sialylation system: surface display of Corynebacterium diphtheriae sialidase on Saccharomyces cerevisiae

Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.     

Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro

Transfer of terminal alpha 2,6-linked sialic acids to N-glycans is catalyzed by beta-galactoside alpha 2,6-sialyltransferase (ST6Gal I). Expression of ST6Gal I and its products is reportedly increased in colon cancers. To investigate directly the functional effects of ST6Gal I expression, human colon cancer (HT29) cells were transfected with specific antisense DNA. ST6Gal I mRNA and protein were virtually undetectable in six strains of transfected HT29 cells. ST6Gal activity was reduced to 14% of control (P<0.005) in transfected cells. Expression of terminal alpha 2,6- and alpha 2,3-linked sialic acids, and unmasked N-acetyllactosamine oligosaccharides, respectively, was assessed using flow cytometry and fluoresceinated Sambucus nigra, Maackia amurensis and Erythrina cristagalli lectins. Results indicated a major reduction in expression of alpha 2,6-linked sialic acids and counterbalancing increase in unmasked N-acetyllactosamines in antisense DNA-transfected cells, without altered expression of alpha 2,3-linked sialic acids or ganglioside profiles. The ability of transfected cells to form colonies in soft agar and to invade extracellular matrix material (Matrigel), respectively, in vitro was reduced by approx. 98% (P<0.0001) and more than 3-fold (P<0.005) compared to parental HT29 cells. These results indicate that N-glycans bearing terminal alpha 2,6-linked sialic acids may enhance the invasive potential of colon cancer cells.     

Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells

Tumor-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of carcinoma cells. The aim of this study was to examine the effect of alpha 2,6-sialylation on the adhesion properties of breast carcinoma cells. To this end mammary carcinoma cells, MDA-MB-435, were sense-transfected with sialyltransferase ST6Gal-I cDNA or antisense-transfected with a part of the ST6Gal-I sequence. Sense transfectants showed an enhanced ST6Gal-I mRNA expression and enzyme activity and an increased binding of the lectin Sambucus nigra agglutinin (SNA), specific for alpha 2,6-linked sialic acid. Transfection with ST6Gal-I in the antisense direction resulted in less enzyme activity and SNA reactivity. A sense-transfected clone carrying increased amounts of alpha 2,6-linked sialic acid adhered preferentially to collagen IV and showed reduced cell-cell adhesion and enhanced invasion capacity. In contrast, antisense transfection led to less collagen IV adhesion but enhanced homotypic cell-cell adhesion. In another approach, inhibition of ST6Gal-I enzyme activity by application of soluble antisense-oligodeoxynucleotides was studied. Antisense treatment resulted in reduced ST6 mRNA expression and cell surface 2,6-sialylation and significantly decreased collagen IV adhesion. Our results suggest that cell surface alpha 2,6-sialylation contributes to cell-cell and cell-extracellular matrix adhesion of tumor cells. Inhibition of sialytransferase ST6Gal-I by antisense-oligodeoxynucleotides might be a way to reduce the metastatic capacity of carcinoma cells.     

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.     

Sialic acid-binding lectins: submolecular specificity and interaction with sialoglycoproteins and tumour cells

We examined the specificity of limulin,Limax flavus agglutinin (LFA) andSambucus nigra agglutinin I (SNA I) at the submolecular level of sialic acid, and characterized their interactions with a panel of structurally distinct sialoglycoproteins. In haemagglutination inhibition assays NeuAc--glycosides were stronger inhibitors for limulin and LFA than nativeN-acetylneuraminic acid (NeuAc). TheN-acetyl of NeuAc was crucial for binding to both lectins. N-thioacetylated NeuAc lost affinity for LFA, but still bound to limulin. Thus, distinct intermolecular interactions are involved in binding of sialic acid to the lectins. The glyceryl side chain was required for interaction with LFA, but not with limulin. SNA I specifically bound NeuAc2 6Gal1 4Glc, but not monomeric sialic acids. Limulin and LFA strongly interacted with O-chain glycoproteins, whereas SNA I preferred N-chain proteins that carry NeuAc2 6 residues. The lectins were compared with those fromCepaea hortensis andTachypleus tridentatus (TTA) and to wheat-germ agglutinin, and were then used to probe tumour cell lines for cell surface sialylation. With the exception of TTA, all lectins interacted with the tumour cells. Limulin distinguished between the low (Eb) and highly (ESb) metastatic mouse lymphoma lines by selectively agglutinating sialidase-treated ESb cells.Abbreviations BSM bovine submaxillary mucin - CHA I Cepaea hortensis agglutinin I - LFA Limax flavus agglutinin - NeuAc N-acetylneuraminic acid - OSM ovine submaxillary mucin - SNA I Sambucus nigra agglutinin I - THP Tamm-Horsfall protein - TTA Tachypleus tridentatus agglutinin     

Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum

Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the α2,6-sialyl-specific lectin from Sambucus nigra agglutinin (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few α2,6-linked sialic acid residues. Despite their poor α2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the α2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-β2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the α2,6-sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis.     

Lectin analysis of human immunoglobulin G N-glycan sialylation

The lectins Sambucus nigraagglutinin (SNA) and Ricinus communisagglutinin (RCA), specific for 2,6 linked sialylation, and terminal galactose respectively were used to study the occurrence, linkage and distribution of human immunoglobulin G (IgG) sialylation. SNA was shown to bind N-glycan 2,6-linked sialic acid only. Sialidase analysis confirmed that this is the dominant, if not exclusive linkage. Total IgG sialylation was estimated at 1.0[emsp4 ]g SA/mg IgG (or about 0.5 mole per mole) using a biochemical sialic acid assay. SNA displayed strong binding to the IgG Fab fragment in both its native and denatured state. In contrast, SNA failed to bind the IgG Fc fragment in its native form, but displayed strong binding after the Fc was denatured. This allowed the construction of quantitative assays capable of measuring both IgG Fab and Fc 2,6-sialylation without the need for enzymatic peptide digestion.     

Cryptic sialic acid binding lectins on human blood leukocytes can be unmasked by sialidase treatment or cellular activation.

We recently reported that the sialic acid-specific binding sites of CD22 molecules on B cells are masked by endogenous ligands, and can be unmasked by sialidase treatment or cellular activation. Here, we show that many other human blood leukocyte types have endogenous sialic acid binding sites that can be unmasked by sialidase treatment. Truncation of sialic acid side chains on the soluble probes used for detection abolishes all binding, indicating the specificity of the interaction for the details of sialic acid structure. There is limited overlap between alpha2-6- and alpha2-3-sialic acid-specific binding sites, which are unmasked on monocytes, natural killer cells, a minority of mature T cells, neutrophils, and some cultured human leukemic cell lines. Activation with phorbol ester and calcium ionophore causes spontaneous exposure of some of the binding sites, occurring over a period of minutes on neutrophils and several hours on monocytes and U937 leukemia cells. Activation is accompanied by some evidence for desialylation of cell surface molecules. Thus, many human blood cells have specific binding sites for sialic acids, masked by endogenous sialylated ligands. Cellular activation can unmask these sites, possibly by the action of an endogenous sialidase. The nearly universal masking of such sites in unactivated blood cells could explain why many of these sialic acid-binding lectins have not been previously discovered. Similar considerations may apply to sialic acid binding lectins of other cell types and tissues.     

Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors

No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.     

Presence of carnitine acetyltransferase in peroxisomes and in mitochondria of oleic acid-grown Saccharomyces cerevisiae

Abstract Paracoccidiodes brasiliensis , the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N -acetlyneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin ( Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 × 10⁶ residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.