Siglec-7 is a sialic acid binding receptor with inhibitory potential, expressed on human NK cells and monocytes. It has an unusual binding preference for alpha2,8-linked disialic acids, such as those displayed by ganglioside GD3. Here we have investigated whether siglec-7-GD3 interactions are able to modulate NK cell cytotoxicity. Using synthetic polyacrylamide glycoprobes, siglec-7 was found to be masked at the NK cell surface but it could be unmasked by sialidase treatment of NK cells. GD3 synthase-transfected P815 target cells expressed high levels of GD3 and bound strongly to recombinant siglec-7-Fc protein. Surprisingly, GD3 synthase-transfected P815 cells were killed more effectively by untreated cells in a siglec-7-independent manner. However, following sialidase treatment of NK cells, a siglec-7-dependent inhibition of killing was observed. These findings have important implications for NK cell cytotoxicity against tumor cells like melanoma that express high levels of GD3 ganglioside.

The use of hydrazine to release unreduced N- and O-linked oligosaccharides from glycoproteins has been investigated using several "standard" glycoproteins of previously defined glycosylation. It is shown that hydrazinolysis can be used to release intact N- and O-linked oligosaccharides in an unreduced form. The release of O-linked oligosaccharides occurs with a lower temperature dependence than the release of N-linked oligosaccharides, and the kinetic parameters governing release of oligosaccharides from these standard glycoproteins have been determined. These parameters allow a definition of reaction conditions under which anhydrous hydrazinolysis can be used to selectively release O-linked oligosaccharides (60 degrees C, 5 h) or release both N- and O-linked oligosaccharides (95 degrees C, 4 h) in high yield >> 85%) from all glycoproteins investigated (n = 11). Under these reaction conditions, the recovered N- and O-linked oligosaccharides are structurally intact (as judged by 600-MHz 1H-NMR, laser-desorption mass spectrometry, HPAEC-PAD, gel filtration, and glycosidase digestion), with the possible exception of certain N- and O-acyl substituents of sialic acid. This use of mild hydrazinolysis therefore allows both the simultaneous and sequential chemical release from glycoproteins of O- and N-linked oligosaccharides in their intact unreduced form.

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs.

Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)-its presumptive ancestor-and porcine hemagglutinating encephalomyelitis virus (PHEV). The β1-coronaviruses (β1CoVs) and HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1^A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptor-binding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1^A We then took a comparative structural analysis approach to map the β1CoV S RBS, using the general design of 9-O-Ac-Sia-binding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1^A, and infectivity assays with BCoV-S-pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1^A The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor fine-specificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1^A and that of HKU1 in domain S1^B.

Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcepsilonRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding.

Siglecs are sialic-acid-binding immunoglobulin-like lectins involved in cell-cell interactions and signalling functions in the haemopoietic, immune and nervous systems. Significant advances have been made in our understanding of the link between carbohydrate recognition and signalling for two well-characterised siglecs, CD22 and myelin-associated glycoprotein. Over the past few years, several novel siglecs have been discovered through genomics and functional screens. These "CD33-related" siglecs have molecular features of inhibitory receptors and may be important in regulating leucocyte activation during immune and inflammatory responses.

Mouse lymphocytes incubated on cryostat-cut sections of lymphoid organs (lymph nodes and Peyer's patches) specifically adhere to the endothelium of high endothelial venules (HEV), the specialized blood vessels to which recirculating lymphocytes attach as they migrate from the blood into the parenchyma of the lymphoid organs. Treatment of sections with sialidase eliminated the binding of lymphocytes to peripheral lymph node HEV, had no effect on binding to Peyer's patch HEV, and had an intermediate effect on mesenteric lymph node HEV. These results suggest that sialic acid on endothelial cells may be an organ-specific recognition determinant for lymphocyte attachment.

This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems - such as the human brain - through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous - and sometime quite unexpected - biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the "druggability" of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated "scaffold-dependent" activities, are summarized.

A survey of Haemophilus influenzae strains indicated that around one-third of capsular strains and over two-thirds of non-typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP-Neu5Ac synthetase gene (siaB) resulted in a sialylation-deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non-typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We determine for the first time the site of attachment of sialic acid in the LPS of a non-typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.

Group B Streptococcus (GBS) is an important perinatal pathogen. Because transplacentally acquired maternal antibodies to the GBS capsular polysaccharides (CPS) confer protection, prevention of infant disease may be possible after immunization of women. Unfortunately, the purified CPS of GBS are only variably immunogenic in adults; therefore to enhance immunogenicity we have designed and developed a CPS-protein conjugate vaccine. The lability of a conformationally dependent epitope on the III CPS containing a critical sialic acid residue was important to consider in vaccine design. 100 women were randomized to receive GBS type III CPS-tetanus toxoid conjugate (III-TT) vaccine at one of three doses; unconjugated GBS type III CPS; or saline. Serum samples were obtained before immunization and 2, 4, 8, and 26 wk thereafter, and specific antibody to type III CPS was measured. Vaccines were well tolerated. In sera from recipients of the highest dose of III-TT, CPS-specific IgG levels rose from a geometric mean of 0.09 microg/ml before immunization to 4.53 microg/ml 8 wk later, whereas levels in recipients of unconjugated type III CPS rose from 0.21 microg/ml to 1.41 microg/ml. Lower doses resulted in lower antibody levels. A>> or = 4-fold rise in antibody concentration was achieved in 90% of recipients of III-TT compared with 50% of those that received III CPS (P = 0.0015). Antibodies evoked by the conjugate vaccine recognized a conformationally dependent epitope of the III-CPS, promoted opsonophagocytosis and killing of GBS, and, after maternal immunization, protected neonatal mice from lethal challenge with type III GBS. We conclude that directed coupling of type III GBS polysaccharide to a carrier protein yielded a conjugate vaccine with preserved expression of a highly labile conformational epitope involving sialic acid and enhanced immunogenicity compared with uncoupled CPS.

Sialic acid-containing glycosphingolipids, i.e., gangliosides, constitute a major component of neuronal cells and are thought to be essential for brain function. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyzes the initial step of glycosphingolipid (GSL) biosynthesis. To gain insight into the role of GSLs in brain development and function, a cell-specific disruption of Ugcg was performed as indicated by the absence of virtually all glucosylceramide-based GSLs. Shortly after birth, mice showed dysfunction of cerebellum and peripheral nerves, associated with structural defects. Axon branching of Purkinje cells was significantly reduced. In primary cultures of neurons, dendritic complexity was clearly diminished, and pruning occurred early. Myelin sheaths of peripheral nerves were broadened and focally severely disorganized. GSL deficiency also led to a down-regulation of gene expression sets involved in brain development and homeostasis. Mice died approximately 3 weeks after birth. These results imply that GSLs are essential for brain maturation.

Herpes simplex virus type 1 (HSV-1) contains five glycoproteins, designated gA, gB, gC, gD, and gE. The present studies focused on the synthesis and processing of two of these, gC and gD. By using monoprecipitin antibody to gC, we demonstrated an antigenic and structural relationship between the precursor, pgC(110), and the product, gC(130). Tryptic peptide analysis showed that pgC and gC shared methionine peptides and that these molecules had the same fingerprint pattern as that of gC(130) extracted from the purified virion. These results suggested that post-translational processing of gC involved no major changes in methionine-containing tryptic peptides or in the cleavage sites required to generate those peptides. The syntheses of gC and gD were compared. We found that the glycoproteins were synthesized starting at different times in the infectious cycle; pgD was detected by 2 h postinfection, whereas pgC was first detected at 4 to 6 h postinfection. Both precursor molecules, pgC(110) and pgD(52), are basic glycopolypeptides, and in both cases processing involved changes in molecular weight and charge. These changes were detected by two-dimensional gel electrophoresis. Both glycoproteins exhibited heterogeneity, displayed as a series of spots (6 for gD and 15 to 20 for gC) of increasing negative charge and molecular weight. Neuraminidase treatment decreased the size, number, and acidic charge of the spots, suggesting that processing was due in part, but not entirely, to addition of sialic acid to pgD and pgC.

Our aim was to establish the phylogenetic relation of H9N2 avian viruses in the Middle East to other Asian H9N2 lineages by characterization of 7 viruses isolated from United Arab Emirates (2000-2003). All these viruses had an additional basic amino acid at the hemagglutinin-connecting peptide; 6 contained a mutation associated with increased affinity toward human-like sialic acid substrates. The viruses' surface glycoproteins and most internal genes were >90% similar to those of A/Quail/Hong Kong/G1/97 (H9N2) lineage. The hemadsorbing site of neuraminidase had up to 4 amino acid substitutions, as do human pandemic viruses. M2 sequence analysis revealed amino acid changes at 2 positions, with increasing resistance to amantadine in cell culture. They replicated efficiently in inoculated chickens and were successfully transmitted to contacts. They continue to maintain H5N1-like genes and may augment the spread of H5N1 viruses through regional co-circulation and inapparent infection. These viruses may present as potential pandemic candidates themselves.

We have performed a detailed analysis of all the anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from the whole cell glycoproteins of P388D1 mouse macrophage-like cells labeled for 14 h with [2-3H]mannose. The major anionic species consisted of phosphorylated high mannose-type oligosaccharides containing one or two phosphomonoesters or phosphodiesters in several different positions. In addition we identified hybrid-type molecules containing one, two, or three sialic acid residues. A subset of the latter molecules also contained phosphodiesters or phosphomonoesters on another branch of the same oligosaccharide. Unlike previously reported hybrid-type molecules, these do not have a "bisecting" N-acetylglucosamine residue on the beta-linked mannose. Some of these oligosaccharides contained an unidentified acid-labile group on the core N-acetylglucosamine or the beta-linked mannose. The glycoproteins secreted by these cells were greatly enriched in hybrid oligosaccharides containing one sialic acid and one phosphomonoester. The interaction of the isolated oligosaccharides with bovine liver phosphomannosyl receptor immobilized on Affigel was analyzed. Oligosaccharides with phosphomonoesters were the only species that interacted with high affinity with the receptor, and molecules with two phosphomonoesters showed the best binding. The location of the phosphomonoester on the oligosaccharide influenced the degree of interaction with the receptor. Removal of accessible nonphosphorylated mannose residues improved the binding in some cases. These findings indicate that the generation of the physiological phosphomannosyl ligand on lysosomal enzymes involves removal of the blocking N-acetylglucosamine residues, trimming of certain mannose residues, and correct positioning of the phosphate esters.

Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both alpha(2,3) and alpha(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose.

Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using alpha(1-3,4)-fucosidase, endo-beta-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain alpha2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a>>) GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.

We previously found an inverse relationship between sialidase Neu1 expression and metastatic potential of murine cancer cells. To elucidate the mechanism underlying the cellular events, the human sialidase gene NEU1 was overexpressed or silenced in colon cancer HT-29 cells. When NEU1-overexpressing cells were injected transsplenically into mice, in vivo liver metastasis was significantly reduced. NEU1 suppressed cell migration, invasion and adhesion in vitro, whereas the silencing resulted in the opposite. One of the major molecular changes by NEU1 was decreased sialylation of integrin beta4, assessed by PNA- and MAL-II-lectin blotting of immunoprecipitates with anti-integrin beta4 antibody. The desialylation was accompanied by decreased phosphorylation of the integrin followed by attenuation of focal adhesion kinase and Erk1/2 pathway. Moreover, NEU1 caused downregulation of matrix metalloproteinase-7, overexpression of which is associated with cancer metastasis. Treatment of the cells with GalNAc-alpha-O-benzyl, an inhibitor of O-glycosylation, showed increased PNA-positive integrin beta4 with its decreased phosphorylation, indicating that sialic acid removal from the integrin O-glycans results in the decreased phosphorylation. Biotinylation and immunofluorescence staining exhibited some NEU1 molecules to be at the cell surface accessible to the integrin. These results suggest that NEU1 is important in regulation of integrin beta4-mediated signaling, leading to suppression of metastasis.

Siglecs are sialic acid-binding Ig-like lectins expressed in a highly specific manner, and which are implicated in signaling and adhesive functions. The CD33-related siglecs represent a distinct subgroup that is undergoing rapid evolution within the innate immune system, with the potential to trigger apoptosis and provide inhibitory signals. CD22 is a well-characterised B cell restricted siglec that has been shown to mediate both sialic acid-dependent and -independent signaling functions in B cell regulation. As endocytic receptors, siglecs provide portals of entry for certain viral and bacterial pathogens, as well as therapeutic opportunities for targeting innate immune cells in disease.

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the "two-signal" model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2-like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid-binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid "missing self"-responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

Protein glycosylation is an important post-translational modification associated, among others, with diseases and the efficacy of biopharmaceuticals. Matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS) can be performed to study glycosylation in a high-throughput manner, but is hampered by the instability and ionization bias experienced by sialylated glycan species. Stabilization and neutralization of these sialic acids can be achieved by permethylation or by specific carboxyl group derivatization with the possibility of discrimination between α2,3- and α2,6-linked sialic acids. However, these methods typically require relatively pure glycan samples, show sensitivity to side reactions, and need harsh conditions or long reaction times. We established a rapid, robust and linkage-specific high-throughput method for sialic acid stabilization and MALDI-TOF-MS analysis, to allow direct modification of impure glycan-containing mixtures such as PNGase F-released human plasma N-glycome. Using a combination of carboxylic acid activators in ethanol achieved near-complete ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked variants, in short time using mild conditions. Glycans were recovered by hydrophilic interaction liquid chromatography solid phase extraction and analyzed by MALDI-TOF-MS in reflectron positive mode with 2,5-dihydroxybenzoic acid as the matrix substance. Analysis of the human plasma N-glycome allowed high-throughput detection and relative quantitation of more than 100 distinct N-glycan compositions with varying sialic acid linkages.

Over four decades ago, specific tumor characteristics were ascribed to the increased expression of sialic acid sugars on the surface of cancer cells, and this led to the definition of sialic acids as potential therapeutic targets. Recent advances in glycobiology and cancer research have defined the key processes underlying aberrant expression of sialic acids in cancer, and its consequences, more precisely. These consequences include effects on tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. Collectively, these novel insights provide further rationale for the design and development of therapeutic approaches that interfere with excessively high expression of sialic acids in cancer cells. Strategies to target aberrant sialylation in cancer, however, have evolved comparatively slowly. Here, we review recent findings that emphasize the detrimental effects of hypersialylation on multiple aspects of tumor growth and behavior. We also discuss novel therapeutic strategies.

Sialic acid-binding Ig superfamily lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-11 is a recently identified human-specific CD33-related Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells. Polysialylated neuronal cell adhesion molecule (PSA-NCAM) is a putative ligand of Siglec-11. We observed gene transcription and protein expression of Siglec-11 splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis. Siglec-11 was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec-11 splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells. Stimulation of Siglec-11 by cross-linking suppressed the lipopolysaccharides (LPS)-induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia. Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec-11 transduced microglia. Expression of PSA-NCAM was detected on microglia and neurons by immunohistochemistry and RT-PCR. Coculture of microglia transduced with Siglec-11 and neurons demonstrated neuroprotective function of Siglec-11. The neuroprotective effect of Siglec-11 was dependent on polysialic acid (PSA) residues on neurons, but independent on PSA on microglia. Thus, data demonstrate that human Siglec-11 ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.