A comparative study of the asparagine-linked oligosaccharides on siglec-5, siglec-7 and siglec-8, expressed in a CHO cell line, and their contribution to ligand recognition.

The siglecs (sialic acid-binding immunoglobulin-like lectins) mediate sialic acid-dependent cellular interactions and may in some cases signal through SH2-binding domains. In addition to the previously characterized siglecs, sialoadhesin, CD22, CD33 and myelin-associated glycoprotein, several new ones, siglec-5, siglec-7 and siglec-8, have recently been cloned. Although these novel receptors have generated considerable interest as therapeutic targets because of their expression pattern on immune cells, very little is known about how their lectin activity is regulated. Previous studies with sialoadhesin, CD22 and CD33 have shown that siglec glycosylation has significant effects on binding. To determine any differences in the glycan composition of siglec-5, siglec-7 and siglec-8 that may modify their function, we released and characterized the N-linked oligosaccharide distribution in these three glycoproteins. The glycan pools from siglec-5 and siglec-7 contained a larger proportion of sialylated and core-fucosylated biantennary, triantennary and tetra-antennary oligosaccharides, whereas the carbohydrate mixture released from siglec-8 is noticeably less sialylated and is more abundant in 'high-mannose'-type glycans. In addition, we show that, in contrast with CD22 and CD33, mutating the conserved potentially N-linked glycosylation site in the first domain has no effect on binding mediated by siglec-5 or siglec-7.     

Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell-cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewis(a), which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewis(x), which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewis(a) and sialyl Lewis(x), which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE(2) production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.     

Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake

Sialic acid-binding immunoglobulin-like lectins (siglecs) are expressed predominantly in the haemopoietic and immune systems and exhibit specificities for both the linkage and the nature of sialic acids in N-glycans, O-glycans and glycolipids. Several siglecs, including sialoadhesin (Sn, siglec-1) and siglec-5, bind to NeuAcalpha2,3Gal, a terminal capping structure that can also be displayed on the lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). In the present study, we examined the potential of siglecs expressed on cells of the immune system to function as receptors for sialylated Nm. We used sialylated and non-sialylated LPS derivatives of two serogroups (A and B) of Nm in this study. Using recombinant chimeric soluble receptors, siglec-transfected cell lines and macrophages from wild-type and Sn-deficient mice, we observed that sialylated but not non-sialylated variants of either genetic background were specifically recognized by Sn and siglec-5, whereas other siglecs examined were ineffective. In addition, macrophages expressing Sn, as well as transfectants expressing Sn or siglec-5, bound and phagocytosed sialylated bacteria in a siglec- and sialic acid-dependent manner. This study demonstrates that Nm LPS sialylation can lead to increased bacterial susceptibility to phagocytic uptake, a phenomenon in direct contrast to previously reported protective effects of LPS sialylation.     

A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33.

The siglecs (sialic acid-binding Ig-like lectins) are a distinct subset of the Ig superfamily with adhesion-molecule-like structure. We describe here a novel member of the siglec protein family that shares a similar structure including five Ig-like domains, a transmembrane domain, and a cytoplasmic tail containing two ITIM-signaling motifs. Siglec-10 was identified through database mining of an asthmatic eosinophil EST library. Using the Stanford G3 radiation hybrid panel we were able to localize the genomic sequence of siglec-10 within the cluster of genes on chromosome 19q13.3-4 that encode other siglec family members. We have demonstrated that siglec-10 is an immune system-restricted membrane-bound protein that is highly expressed in peripheral blood leukocytes as demonstrated by Northern, RT-PCR and flow cytometry. Binding assays determined that the extracellular domain of siglec-10 was capable of binding to peripheral blood leukocytes. The cytoplasmic tail of siglec-10 contains four tyrosines, two of which are embedded in ITIM-signaling motifs (Y597 and Y667) and are likely involved in intracellular signaling. The ability of tyrosine kinases to phosphorylate the cytoplasmic tyrosines was evaluated by kinase assay using wild-type siglec-10 cytoplasmic domain and Y-->F mutants. The majority of the phosphorylation could be attributed to Y597 andY667. Further experiments with cell extracts suggest that SHP-1 interacts with Y667 and SHP-2 interacts with Y667 in addition to another tyrosine. This is very similar to CD33, which also binds the phosphatases SHP-1 and SHP-2, therefore siglec-10, as CD33, may be characterized as an inhibitory receptor.     

Carbohydrate microarrays reveal sulphation as a modulator of siglec binding

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.     

Identification and characterization of a novel siglec, siglec-7, expressed by human natural killer cells and monocytes

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8(+) T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of approximately 65 kDa.     

Siglec-7 undergoes a major conformational change when complexed with the alpha(2,8)-disialylganglioside GT1b

The siglecs are a group of mammalian sialic acid binding receptors expressed predominantly in the immune system. The CD33-related siglecs show complex recognition patterns for sialylated glycans. Siglec-7 shows a preference for alpha(2,8)-disialylated ligands and provides a structural template for studying the key interactions that drive this selectivity. We have co-crystallized Siglec-7 with a synthetic oligosaccharide corresponding to the alpha(2,8)-disialylated ganglioside GT1b. The crystal structure of the complex offers a first glimpse into how this important family of lectins binds the structurally diverse gangliosides. The structure reveals that the C-C' loop, a region implicated in previous studies as driving siglec specificity, undergoes a dramatic conformational shift, allowing it to interact with the underlying neutral glycan core of the ganglioside. The structural data in combination with mutagenesis studies show that binding of the ganglioside is driven by extensive hydrophobic contacts together with key polar interactions and that the binding site structure is complementary to preferred solution conformations of GT1b.     

Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Evolution of sialic acid-binding proteins: molecular cloning and expression of fish siglec-4

Siglecs are the largest family of sialic acid-recognizing lectins identified so far with 11 members in the human genome. Most of these siglecs are exclusively expressed by cells of the immune system. Comparison of different mammalian species has revealed differential and complex evolutionary paths for this protein family, even within the primate lineage. To understand the evolution of siglecs, in particular the origin of this family, we investigated the occurrence of corresponding genes in bony fish. Interestingly, only unambiguous orthologs of mammalian siglec-4, a cell adhesion molecule expressed exclusively in the nervous system, could be identified in the genomes of fugu and zebrafish, whereas no obvious orthologs of the other mammalian siglecs were found. As in mammals, fish siglec-4 expression is restricted to nervous tissues as demonstrated by northern blot. Expressed as recombinant protein, fish siglec-4 binds to sialic acids with a specificity similar to the mammalian orthologs. Relatively low sequence similarities in the cytoplasmic tail as well as an additional splice variant found in fish siglec-4 suggest alternative signaling pathways compared to mammalian species. Our observations suggest that this siglec occurs at least in the nervous system of all vertebrates.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Probing sialic acid binding Ig-like lectins (siglecs) with sulfated oligosaccharides

Soluble siglecs-1, -4, -5, -6, -7, -8, -9, and -10 were probed with polyacrylamide glycoconjugates in which: 1) the Neu5Ac residue was substituted by a sulfate group (Su); 2) glycoconjugates contained both Su and Neu5Ac; 3) sialoglycoconjugates contained a tyrosine-O-sulfate residue. It was shown that sulfate derivatives of LacNAc did not bind siglecs-1, -4, -5, -6, -7, -8, -9, and -10; binding of 6'-O-Su-LacNAc to siglec-8 was stronger than binding of 3'SiaLacNAc. The relative affinity of 3'-O-Su-TF binding to siglecs-1, -4, and -8 was similar to that of 3'SiaTF. 3'-O-Su-Le(c) displayed two-fold weaker binding to siglec-1 and siglec-4 than 3'SiaLe(c). The interaction of soluble siglecs with sulfated oligosaccharides containing sialic acid was also studied. It was shown that siglecs-1, -4, -5, -6, -7, -9, and -10 did not interact with these compounds; binding of 6-O-Su-3'SiaLacNAc and 6-O-Su-3'SiaTF to siglec-8 was weaker than that of the corresponding sulfate-free sialoside probes. Siglec-8 displayed affinity to 6'-O-Su-LacNAc and 6'-O-Su-SiaLe(x), and defucosylation of the latter compound led to an increase in the binding. Sialoside probes containing tyrosine-O-sulfate residue did not display increased affinity to siglecs-1 and -5 compared with glycoconjugates containing only sialoside. Cell-bound siglecs-1, -5, -7, and -9 did not interact with 6-O-Su-3'SiaLacNAc, whereas the sulfate-free probe 3'SiaLacNAc demonstrated binding. In contrast, the presence of sulfate in 6-O-Su-6'SiaLacNAc did not affect binding of the sialoside probe to siglecs. 6'-O-Su-SiaLe(x) displayed affinity to cell-bound siglecs-1 and -5; its isomer 6-O-Su-SiaLe(x) bound more strongly to siglecs-1, -5, and -9 than SiaLe(x).     

Identification and molecular characterization of a novel member of the siglec family (SIGLEC9)

Using the positional cloning approach, we have identified siglec-9 (HGMW-approved symbol SIGLEC9) a novel member of the sialic acid-binding Ig-like lectin (Siglec) family, which belongs to the immunoglobulin superfamily (IgSF). We characterized the genomic structure of this gene and determined its chromosomal localization, its homology to other members of the siglec family, and its tissue expression profile. The siglec-9 gene is composed of seven exons, with six intervening introns. The coding region consists of 1392 nucleotides and produces a 463-amino-acid protein. Furthermore, we have localized this gene to 19q13.4, 43.19 kb more telomeric than KLK14 (a member of the kallikrein gene family) through genomic sequencing data and restriction mapping with EcoRI. This novel siglec shows a high degree of homology to many members of the siglec family, including siglec-7 (80%), siglec-8 (72%), siglec-5 (65%), and CD33 (64%). This high degree of homology is also conserved in the extracellular Ig-like domains. Through RT-PCR, we have examined the expression of siglec-9 in a large number of tissues and have found relatively high-level expression in bone marrow, placenta, spleen, and fetal liver. Based on its homology to CD33, we speculate that this gene may also have some utility as a target for immunological antineoplastic therapy.     

Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin-associated glycoprotein

Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids. Analysis has been performed using a novel multivalent platform comprising biotinylated sialosides bound to a streptavidin-alkaline phosphatase conjugate. Each siglec was found to have a unique specificity for binding 16 different sialoside-streptavidin-alkaline phosphatase probes. The relative affinities of monovalent sialosides were assessed for each siglec in competitive inhibition studies. The quantitative data obtained allows a detailed analysis of each siglec for the relative importance of sialic acid and the penultimate oligosaccharide sequence on binding affinity and specificity. Most remarkable was the finding that myelin-associated glycoprotein (Siglec-4) binds with 500-10,000-fold higher affinity to a series of mono- and di-sialylated derivatives of the O-linked T-antigen (Galbeta(1-3)-GalNAc(alpha)OThr) as compared with alpha-methyl-NeuAc.     

CD33/Siglec-3 binding specificity,expression pattern,and consequences of gene deletion in mice

Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to alpha2-3- or alpha2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage.     

Impact of Fc N-glycan sialylation on IgG structure

ABSTRACT

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.     

Ganglioside binding pattern of CD33-related siglecs

Our study deals with the interaction of CD33 related-siglecs-5,-7,-8,-9,-10 with gangliosides GT1b, GQ1b, GD3, GM2, GM3 and GD1a. Siglec-5 bound preferentially to GQ1b, but weakly to GT1b, whereas siglec-10 interacted only with GT1b ganglioside. Siglec-7 and siglec-9 displayed binding to gangliosides GD3, GQ1b and GT1b bearing a disialoside motif, though siglec-7 was more potent; besides, siglec-9 interacted also with GM3. Siglec-8 demonstrated low affinity to the gangliosides tested compared with other siglecs. Despite high structural similarity of CD33 related siglecs, they demonstrated different ganglioside selectivity, in particular to the Neu5Acalpha2-8Neu5Ac motif.     

Induction of sialic acid 9-O-acetylation by diverse gene products: implications for the expression cloning of sialic acid O- acetyltransferases

Sialic acids can be modified by O-acetyl esters at the 7- and/or 9- position, altering recognition by antibodies, lectins and viruses. 9(7)- O-acetylation is mediated by a sialic acid-specific O- acetyltransferase, which has proven difficult to purify. Two groups have recently isolated cDNAs possibly encoding this enzyme, by expression cloning of human melanoma libraries in COS cells expressing the substrate ganglioside GD3. Pursuing a similar approach, we have isolated additional clones that can induce 9-O-acetylation. One clone present in a melanoma library encodes a fusion protein between a bacterial tetracycline resistance gene repressor and a sequence reported to be part of the P3 plasmid. Expression of the open reading frame is necessary for inducing 9-O-acetylation, indicating that this is not a reaction to the introduction of bacterial DNA. Another clone from a rat liver cDNA library induced 9-O-acetylation on COS cells expressing alpha2-6-linked sialic acids, and encodes an open reading frame identical to the Vitamin D binding protein. However, truncation at the 5' end eliminates the amino-terminal hydrophobic signal sequence, predicting cytosolic hyperexpression of a truncated protein. Thus, diverse types of cDNAs can indirectly induce sialic acid 9-O- acetylation in the COS cell system, raising the possibility that the real enzyme may be composed of multiple subunits which would not be amenable to expression cloning. Importantly, the cDNAs we isolated are highly specific in their ability to induce 9-O-acetylation either on alpha2-6-linked sialic acids of glycoproteins (truncated vitamin D binding protein) or on the alpha2-8-linked sialic acids of gangliosides (Tetrfusion protein). These data confirm our prior suggestion that a family of O-acetyltransferases with distinctive substrate specificities exists in mammalian systems.      

Identification and characterization of binding properties of Helicobacter pylori by glycoconjugate arrays

The microaerophilic bacterium Helicobacter pylori is well established for its role in development of different gastric diseases. Bacterial adhesins and corresponding binding sites on the epithelial surface allow H. pylori to colonize the gastric tissue. In this investigation, the adhesion of H. pylori to dot blot arrays of natural glycoproteins and neoglycoproteins was studied. Adhesion was detected by overlay with fluorescence-labeled bacteria on immobilized (neo)glycoproteins. The results confirmed the interaction between the adhesin BabA and the H-1-, Lewis b-, and related fucose-containing antigens. In addition, H. pylori bound to terminal alpha2-3-linked sialic acids as previously described. The use of a sabA mutant and sialidase treatment of glycoconjugate arrays showed that the adherence of H. pylori to laminin is mediated by the sialic acid-binding adhesin, SabA. The adhesion to salivary mucin MUC5B is mainly associated with the BabA adhesin and to a lesser extent with the SabA adhesin. This agrees with reports, that MUC5B carries both fucosylated blood group antigens and alpha2-3-linked sialic acids. The adhesion of H. pylori to fibronectin and lactoferrin persisted in the babA/sabA double mutant. Because binding to these molecules was abolished by denaturation rather than by deglycosylation, it was suggested to depend on the recognition of unknown receptor moieties by an additional unknown bacterial surface component. The results demonstrate that the bacterial overlay method on glycoconjugate arrays is a useful tool for exploration and the characterization of unknown adhesin specificities of H. pylori and other bacteria.