Expression of a polysaccharide capsule is required for the full pathogenicity of many mucosal pathogens such as Streptococcus pneumoniae. Although capsule allows for evasion of opsonization and subsequent phagocytosis during invasive infection, its role during mucosal colonization, the organism's commensal state, remains unknown. Using a mouse model, we demonstrate that unencapsulated mutants remain capable of nasal colonization but at a reduced density and duration compared to those of their encapsulated parent strains. This deficit in colonization was not due to increased susceptibility to opsonophagocytic clearance involving complement, antibody, or the influx of Ly-6G-positive cells, including neutrophils seen during carriage. Rather, unencapsulated mutants remain agglutinated within lumenal mucus and, thus, are less likely to transit to the epithelial surface where stable colonization occurs. Studies of in vitro binding to immobilized human airway mucus confirmed the inhibitory effect of encapsulation. Likewise, pneumococcal variants expressing larger amounts of negatively charged capsule per cell were less likely to adhere to surfaces coated with human mucus and more likely to evade initial clearance in vivo. Removal of negatively charged sialic acid residues by pretreatment of mucus with neuraminidase diminished the antiadhesive effect of encapsulation. This suggests that the inhibitory effect of encapsulation on mucus binding may be mediated by electrostatic repulsion and offers an explanation for the predominance of anionic polysaccharides among the diverse array of unique capsule types. In conclusion, our findings demonstrate that capsule confers an advantage to mucosal pathogens distinct from its role in inhibition of opsonophagocytosis--escape from entrapment in lumenal mucus.

The availability of host and dietary carbohydrates in the gastrointestinal (GI) tract plays a key role in shaping the structure-function of the microbiota. In particular, some gut bacteria have the ability to forage on glycans provided by the mucus layer covering the GI tract. The O-glycan structures present in mucin are diverse and complex, consisting predominantly of core 1-4 mucin-type O-glycans containing α- and β- linked N-acetyl-galactosamine, galactose and N-acetyl-glucosamine. These core structures are further elongated and frequently modified by fucose and sialic acid sugar residues via α1,2/3/4 and α2,3/6 linkages, respectively. The ability to metabolize these mucin O-linked oligosaccharides is likely to be a key factor in determining which bacterial species colonize the mucosal surface. Due to their proximity to the immune system, mucin-degrading bacteria are in a prime location to influence the host response. However, despite the growing number of bacterial genome sequences available from mucin degraders, our knowledge on the structural requirements for mucin degradation by gut bacteria remains fragmented. This is largely due to the limited number of functionally characterized enzymes and the lack of studies correlating the specificity of these enzymes with the ability of the strain to degrade and utilize mucin and mucin glycans. This review focuses on recent findings unraveling the molecular strategies used by mucin-degrading bacteria to utilize host glycans, adapt to the mucosal environment, and influence human health.

The ability of murine tumor cells to metastasize spontaneously from subcutaneous sites is positively correlated with the total sialic acid content of the cells in culture, the degree to which the sialic acid is exposed on the tumor cell surface, and, most strongly, with the degree of sialylation of galactosyl and N-acetylgalactosaminyl residues in cell surface glycoconjugates. These findings suggest that sialic acid on the cell surface may play a role in tumor cell metastasis.

Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30-100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12-1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles' structure. Notably, alpha-2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.

The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to be the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.

Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.

Cell attachment and membrane penetration are functions of the rotavirus outer capsid spike protein, VP4. An activating tryptic cleavage of VP4 produces the N-terminal fragment, VP8*, which is the viral hemagglutinin and an important target of neutralizing antibodies. We have determined, by X-ray crystallography, the atomic structure of the VP8* core bound to sialic acid and, by NMR spectroscopy, the structure of the unliganded VP8* core. The domain has the beta-sandwich fold of the galectins, a family of sugar binding proteins. The surface corresponding to the galectin carbohydrate binding site is blocked, and rotavirus VP8* instead binds sialic acid in a shallow groove between its two beta-sheets. There appears to be a small induced fit on binding. The residues that contact sialic acid are conserved in sialic acid-dependent rotavirus strains. Neutralization escape mutations are widely distributed over the VP8* surface and cluster in four epitopes. From the fit of the VP8* core into the virion spikes, we propose that VP4 arose from the insertion of a host carbohydrate binding domain into a viral membrane interaction protein.

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG Fc N-glycans, which are confined in the space between the two Cγ 2 domains. This also suggests why in contrast to IgG Fc, the IgA1 N-glycans are not undergalactosylated in rheumatoid arthritis.

alpha-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein beta-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve alpha-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the alpha-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the alpha-dystroglycan with laminin.

OBJECTIVE

To test the hypothesis that minor chronic insults such as smoking, chronic bronchitis, and two persistent bacterial infections may be associated with increases in C reactive protein concentration within the normal range and that variations in the C reactive protein concentration in turn may be associated with levels of cardiovascular risk factors and chronic coronary heart disease.

METHODS

Population based cross sectional study.

METHODS

General practices in Merton, Sutton, and Wandsworth.

METHODS

A random sample of 388 men aged 50-69 years from general practice registers. 612 men were invited to attend and 413 attended, of whom 25 non-white men were excluded. The first 303 of the remaining 388 men had full risk factor profiles determined.

METHODS

Measurements of serum C reactive protein concentrations by in house enzyme linked immunosorbent assay (ELISA); other determinations by standard methods. Coronary heart disease was sought by the Rose angina questionnaire and Minnesota coded electrocardiograms.

METHODS

Serum C reactive protein concentrations, cardiovascular risk factor levels, and the presence of coronary heart disease.

RESULTS

Increasing age, smoking, symptoms of chronic bronchitis, Helicobacter pylori and Chlamydia pneumoniae infections, and body mass index were all associated with raised concentrations of C reactive protein. C Reactive protein concentration was associated with raised serum fibrinogen, sialic acid, total cholesterol, triglyceride, glucose, and apolipoprotein B values. C Reactive protein concentration was negatively associated with high density lipoprotein cholesterol concentration. There was a weaker positive relation with low density lipoprotein cholesterol concentration and no relation with apolipoprotein A I value. C Reactive protein concentration was also strongly associated with coronary heart disease.

CONCLUSIONS

The body's response to inflammation may play an important part in influencing the progression of atherosclerosis. The association of C reactive protein concentration with coronary heart disease needs testing in prospective studies.

Many serotype 3 reoviruses bind to two different host cell molecules, sialic acid and an unidentified protein, using discrete receptor-binding domains in viral attachment protein, final sigma1. To determine mechanisms by which these receptor-binding events cooperate to mediate cell attachment, we generated isogenic reovirus strains that differ in the capacity to bind sialic acid. Strain SA+, but not SA-, bound specifically to sialic acid on a biosensor chip with nanomolar avidity. SA+ displayed 5-fold higher avidity for HeLa cells when compared with SA-, although both strains recognized the same proteinaceous receptor. Increased avidity of SA+ binding was mediated by increased k(on). Neuraminidase treatment to remove cell-surface sialic acid decreased the k(on) of SA+ to that of SA-. Increased k(on) of SA+ enhanced an infectious attachment process, since SA+ was 50-100-fold more efficient than SA- at infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of virus to the cell surface where access to a proteinaceous receptor is thermodynamically favored.

Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37 degrees C, suggesting the presence of a receptor-destroying enzyme. Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaxillary mucin as substrate similar to the enzymatic activity found in influenza C viruses. Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact. In addition, hemagglutination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Clostridium perfringens neuraminidase or by addition of sialic acid-containing gangliosides. These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize O-acetylated sialic acid or a similar derivative as cell receptor.

A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).

We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell-surface sialylated glycoproteins while maintaining high cell viability.

The sialic acid-specific leukoagglutinating lectin from the seeds of Maackia amurensis (MAL) has been studied by the techniques of quantitative precipitin formation, hapten inhibition of precipitation, hapten inhibition using an enzyme-linked immunosorbent assay, and lectin affinity chromatography. The ability of the immobilized lectin to fractionate oligosaccharides based on their content of sialic acid has also been investigated. Our results indicate that MAL reacts with greatest affinity with the trisaccharide sequence Neu5Ac/Gc alpha 2,3Gal beta 1,4GlcNAc/Glc. The lectin requires three intact sugar units for binding and does not interact when the beta 1,4-linkage is replaced by a beta 1,3-linkage nor when the "reducing sugar" of the trisaccharide is reduced. Results from enzyme-linked immunosorbent assays show that an N-acetyllactosamine repeating sequence is not required; however, the N-acetyllactosamine repeating sequence does appear to enhance the binding of MAL to a series of glycolipids. In addition, the sialic acid may be substituted with either N-acetyl or N-glycolyl groups without reduction in binding. The C-8 and C-9 hydroxyl groups of sialic acid do not play a role in binding as shown by the strong reaction of periodate-treated glycoproteins. Comparison of the specificity of the three sialic acid-binding lectins indicates that Limax flavus agglutinin binds to Neu5Ac in any linkage and in any position in a glycoconjugate, Sambucus nigra lectin requires a disaccharide of the structure Neu5Ac alpha 2,6Gal/GalNAc, and MAL has a binding site complimentary to the trisaccharide Neu5Ac alpha 2,3Gal beta 1,4GlcNAc/Glc, to which sialic acid contributes less to the total binding affinity than for either S. nigra lectin or L. flavus agglutinin.

When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.

The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). In this study, enzymes were used to selectively degrade glycocalyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI(2)) production were determined. Depletion of HS, HA, and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI(2) production. The results may be interpreted in terms of a glypican-caveolae-eNOS mechanism for shear-induced NO transduction, with PGI(2) being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not.

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. Sialylation is crucial for a variety of cellular functions, such as cell adhesion or signal recognition, and regulates the biological stability of glycoproteins. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. We report that inactivation of the UDP-GlcNAc 2-epimerase by gene targeting causes early embryonic lethality in mice, thereby emphasizing the fundamental role of this bifunctional enzyme and sialylation during development. The need of UDP-GlcNAc 2-epimerase for a defined sialylation process is exemplified with the polysialylation of the neural cell adhesion molecule in embryonic stem cells.

Sialic acids (Sias) are terminal components of many glycoproteins and glycolipids especially of higher animals. In this exposed position they contribute significantly to the structural properties of these molecules, both in solution and on cell surfaces. Therefore, it is not surprising that Sias are important regulators of cellular and molecular interactions, in which they play a dual role. They can either mask recognition sites or serve as recognition determinants. Whereas the role of Sias in masking and in binding of pathogens to host cells has been documented over many years, their role in nonpathological cellular interaction has only been shown recently. The aim of this chapter is to summarize our knowledge about Sias in masking, for example, galactose residues, and to review the progress made during the past few years with respect to Sias as recognition determinants in the adhesion of pathogenic viruses, bacteria, and protozoa, and particularly as binding sites for endogenous cellular interaction molecules. Finally, perspectives for future research on these topics are discussed.

The Nogo-66 receptor (NgR1) is a promiscuous receptor for the myelin inhibitory proteins Nogo/Nogo-66, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). NgR1, an axonal glycoprotein, is the founding member of a protein family composed of the structurally related molecules NgR1, NgR2, and NgR3. Here we show that NgR2 is a novel receptor for MAG and acts selectively to mediate MAG inhibitory responses. MAG binds NgR2 directly and with greater affinity than NgR1. In neurons NgR1 and NgR2 support MAG binding in a sialic acid-dependent Vibrio cholerae neuraminidase-sensitive manner. Forced expression of NgR2 is sufficient to impart MAG inhibition to neonatal sensory neurons. Soluble NgR2 has MAG antagonistic capacity and promotes neuronal growth on MAG and CNS myelin substrate in vitro. Structural studies have revealed that the NgR2 leucine-rich repeat cluster and the NgR2 "unique" domain are necessary for high-affinity MAG binding. Consistent with its role as a neuronal MAG receptor, NgR2 is an axonassociated glycoprotein. In postnatal brain NgR1 and NgR2 are strongly enriched in Triton X-100-insoluble lipid rafts. Neural expression studies of NgR1 and NgR2 have revealed broad and overlapping, yet distinct, distribution in the mature CNS. Taken together, our studies identify NgRs as a family of receptors (or components of receptors) for myelin inhibitors and provide insights into how interactions between MAG and members of the Nogo receptor family function to coordinate myelin inhibitory responses.