The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function. Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia. The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. The removal of the desialylated neurites was mediated via the complement receptor-3 (CR3; CD11b/CD18). Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

We investigated the cellular and molecular mechanisms underlying arrhythmias in heart failure. A genetically engineered mouse lacking the expression of the muscle LIM protein (MLP-/-) was used in this study as a model of heart failure. We used electrocardiography and patch clamp techniques to examine the electrophysiological properties of MLP-/- hearts. We found that MLP-/- myocytes had smaller Na+ currents with altered voltage dependencies of activation and inactivation and slower rates of inactivation than control myocytes. These changes in Na+ currents contributed to longer action potentials and to a higher probability of early afterdepolarizations in MLP-/- than in control myocytes. Western blot analysis suggested that the smaller Na+ current in MLP-/- myocytes resulted from a reduction in Na+ channel protein. Interestingly, the blots also revealed that the alpha-subunit of the Na+ channel from the MLP-/- heart had a lower average molecular weight than in the control heart. Treating control myocytes with the sialidase neuraminidase mimicked the changes in voltage dependence and rate of inactivation of Na+ currents observed in MLP-/- myocytes. Neuraminidase had no effect on MLP-/- cells thus suggesting that Na+ channels in these cells were sialic acid-deficient. We conclude that deficient glycosylation of Na+ channel contributes to Na+ current-dependent arrhythmogenesis in heart failure.

A plastic adherent variant line (ESb-M) of a highly invasive and metastatic murine T cell lymphoma (ESb) was found to have lost its metastatic potential while still being tumorigenic in normal syngeneic hosts. The variant retained most of its ESb-derived antigenic and biochemical characteristics but differed at binding sites for certain lectins with specificity for terminal N-acetylgalactosamine residues. Whereas such sites were masked by sialic acid on metastatic ESb cells, they became unmasked on the adherent variant line. Metastatic revertants of ESb-M cells did not express the respective lectin receptor sites because these were again masked by sialic acid. It is suggested that the masking of specific lectin receptors sites on the tumor cell surface is of crucial importance for metastatis. If freely exposed, these sites may change adherence characteristics of the cells possibly not only in vitro (to plastic) but also in vivo.

Newly synthesized class II HLA antigens being transported to the surface of human B-lymphoblastoid cell lines (B-LCL) interact with transferrin-neuraminidase conjugates internalized by means of receptor-mediated endocytosis. Class II antigens, isolated from [35S]methionine-labeled B-LCL after incubation with the conjugates at 37 degrees C, showed extensive desialylation of associated invariant chain and detectable loss of beta-subunit sialic acid on analysis by two-dimensional gel electrophoresis. An equal amount of unconjugated neuraminidase had no effect, and desialylation of class II antigen components was blocked when access of transferrin-neuraminidase conjugates to the B-LCL transferrin receptors was competitively inhibited by the addition of excess iron-saturated transferrin. The conjugates were shown to cycle through the cells in the same way as unconjugated transferrin, being first internalized and then rapidly secreted in an undegraded form. The data suggest that the exocytic pathway taken by class II antigens intersects the route followed by recycling transferrin receptors and that the interaction occurs prior to the dissociation of the invariant chain from the class II antigen complex. Similar intracellular interactions between class II molecules and foreign proteins internalized by antigen-presenting cells may be important in class II antigen-restricted recognition by helper T lymphocytes.

Haemagglutination-inhibition tests (HI) are used to detect increases in influenza antibody in serum. However, they are relatively insensitive for the detection of human antibody responses to avian haemagglutinin, even in the presence of high titres of neutralising antibody after confirmed infection or vaccination. Human influenza viruses bind preferentially sialic acid containing N-acetylneuraminic acid alpha2,6-galactose (SAalpha2,6Gal) linkages while avian and equine viruses bind preferentially those containing N-acetylneuraminic acid alpha2,3-galactose (SAalpha2,3Gal) linkages. Increasing the proportion of SAalpha2,3Gal linkages on the erythrocytes used, by enzymatic modification or change of species, improves the ability of erythrocytes to bind to avian influenza strains and thereby improves the sensitivity of detection of antibody to avian and equine HA in a range of mammalian and human sera using HI tests.

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.

Two H9N2 viruses were isolated, for the first time, from humans in Hong Kong in 1999. Isolation of influenza viruses with a novel subtype of the hemagglutinin (HA) drew attention of health care authorities worldwide from the view of pandemic preparedness. Sequence analysis of the HA genes reveals that HA of A/Hong Kong/1073/99 (H9N2) is most closely related to that of A/quail/HK/G1/97 (H9N2) that contains the internal genes similar to those of Hong Kong/97 (H5N1) viruses. Phylogenetic and antigenic analyses demonstrated the diversity among H9 HA. A/Hong Kong/1073/99 was shown to cause a respiratory infection in Syrian hamsters, suggesting that the virus can replicate efficiently in mammalian hosts. We developed a whole virion test vaccine with a formalin-inactivated egg-grown HK1073. Intraperitoneal administration of the vaccine twice to hamsters conferred a complete protection against challenge infection by the MDCK cell-grown homologous virus. Receptor specificity of HK1073 appeared different from that of other avian influenza viruses of H9 subtype which recognize preferentially alpha-2,3 linked sialic acid. Hemagglutination of HK1073 with guinea pig erythrocytes was inhibited by both alpha-2,3 and alpha-2,6 linked sialic acid containing polymers. These data suggested that HK1073 had acquired a broader host range, including humans. Together with data so far available, the present study suggested that isolation of the H9 influenza viruses from humans requires precaution against the emergence of a novel human influenza.

Comparative study of the oligosaccharide profiles of individual human milk revealed the presence of three different patterns. Four oligosaccharides containing the Fucalpha1-2Gal group were missing in the milk of non-secretor, and three oligosaccharides containing the Fucalpha1-4GlcNAc group were missing in the milk of Lewis negative individuals. Disappearance of some major oligosaccharides in these samples led to the finding of five novel minor oligosaccharides, which were hidden under the missing oligosaccharides. Following these studies, structures of many novel milk oligosaccharides were elucidated. At least 13 core oligosaccharides were found in these oligosaccharides. By adding alpha-fucosyl residues and sialic acid residues to these core oligosaccharides, more than one hundred oligosaccharides were formed. All these oligosaccharides contain lactose at their reducing termini. This evidence, together with the deletion phenomena found in the milk oligosaccharides of non-secretor and Lewis negative individuals, suggested that the oligosaccharides are formed from lactose by the concerted action of glycosyltransferases, which are responsible for elongation and branching of the Galbeta1-4GlcNAc group in the sugar chains of glycoconjugates on the surface of epithelial cells. Therefore, oligosaccharides in human milk could include many structures, starting from the Galbeta1-4GlcNAc group in the sugar chains of various glycoconjugates. Many lines of evidence recently indicated that virulent enteric bacteria and viruses start their infection by binding to particular sugar chains of glycoconjugates on the target cell surfaces. Therefore, milk oligosaccharides could be useful for developing drugs, which inhibit the infection of bacteria and viruses.

Recently, we showed that porcine sialoadhesin (pSn) mediates internalization of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) in alveolar macrophages (Vanderheijden et al., J. Virol. 77:8207-8215, 2003). In rodents and humans, sialoadhesin, or Siglec-1, has been described as a macrophage-restricted molecule and to specifically bind sialic acid moieties. In the current study, we investigated whether pSn is a sialic acid binding protein and, whether so, whether this property is important for its function as a PRRSV receptor. Using untreated and neuraminidase-treated sheep erythrocytes, we showed that pSn binds sialic acid. Furthermore, pSn-specific monoclonal antibody 41D3, which blocks PRRSV infection, inhibited this interaction. PRRSV attachment to and infection of porcine alveolar macrophages (PAM) were both shown to be dependent on the presence of sialic acid on the virus: neuraminidase treatment of virus but not of PAM blocked infection and reduced attachment. Enzymatic removal of all N-linked glycans on the virus with N-glycosidase F reduced PRRSV infection, while exclusive removal of nonsialylated N-linked glycans of the high-mannose type with endoglycosidase H had no significant effect. Free sialyllactose and sialic acid containing (neo)glycoproteins reduced infection, while lactose and (neo)glycoproteins devoid of sialic acids had no significant effect. Studies with linkage-specific neuraminidases and lectins indicated that alpha2-3- and alpha2-6-linked sialic acids on the virion are important for PRRSV infection of PAM. From these results, we conclude that pSn is a sialic acid binding lectin and that interactions between sialic acid on the PRRS virion and pSn are essential for PRRSV infection of PAM.

M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric M, 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia.

Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.

Reovirus induces apoptosis in cultured cells and in vivo. Genetic studies indicate that the efficiency with which reovirus strains induce apoptosis is determined by the viral S1 gene, which encodes attachment protein sigma1. However, the biochemical properties of sigma1 that influence apoptosis induction are unknown. To determine whether the capacity of sigma1 to bind cell surface sialic acid determines the magnitude of the apoptotic response, we used isogenic reovirus mutants that differ in the capacity to engage sialic acid. We found that T3SA+, a virus capable of binding sialic acid, induces high levels of apoptosis in both HeLa cells and L cells. In contrast, non-sialic-acid-binding strain T3SA- induces little or no apoptosis in these cell types. Differences in the capacity of T3SA- and T3SA+ to induce apoptosis are not due to differences in viral protein synthesis or production of viral progeny. Removal of cell surface sialic acid with neuraminidase abolishes the capacity of T3SA+ to induce apoptosis. Similarly, incubation of T3SA+ with sialyllactose, a trisaccharide comprised of lactose and sialic acid, blocks apoptosis. These findings demonstrate that reovirus binding to cell surface sialic acid is a critical requirement for the efficient induction of apoptosis and suggest that virus receptor utilization plays an important role in regulating cell death.

Polysialic acid is a unique carbohydrate composed of a linear homopolymer of alpha2,8-linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) via a typical N-linked glycan in vertebrate neural system. Polysialic acid plays critical roles in neural development by modulating adhesive property of NCAM such as neural cell migration, neurite outgrowth, neural pathfinding, and synaptogenesis. The expression of polysialic acid is temporally and spatially regulated during neural development. Polysialylation of NCAM is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which belong to the family of six genes encoding alpha 2,8-sialyltransferases. ST8Sia II and IV are expressed differentially in tissue-specific and cell-specific manners, and they apparently have distinct roles in development and organogenesis. The presence of polysialic acid is always associated with expression of ST8Sia II and/or IV, suggesting that ST8Sia II and IV are the key enzymes that control the expression of polysialic acid. Both ST8Sia II and IV can transfer multiple alpha 2,8-linked sialic acid residues to an acceptor N-glycan containing a NeuNAc alpha 2-->3 (or 6) Gal beta 1-->4GlcNAc beta 1->>R structure without participation of other enzymes. The two enzymes differently but cooperatively act on NCAM and the amount of polysialic acid synthesized by both enzymes together is greater than that synthesized by either enzyme alone. The polysialyltransferases are thus important regulators in polysialic acid synthesis and contribute to neural development in the vertebrate.

Helicobacterpylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3'-sialyllactose (NeuAc alpha2-3Gal beta1-4Glc; 3'SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC50). Its alpha2-6 isomer (6'SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3'SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (P. G. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. H. Trust, J. Bacteriol. 177:6049-6057, 1995). Pretreatment of the epithelial monolayer with neuraminidase reduced the extent of binding by those bacteria that are sensitive to inhibition by 3'SL. Other potent inhibitors of bacterial binding are the glycoproteins alpha1-acid glycoprotein, fetuin, porcine gastric and bovine submaxillary mucins, and the glycolipid sulfatide, all of which present multivalent sialylated and/or sulfated galactosyl residues under the conditions of the binding assay. Consistent with this pattern, a multivalent neoglycoconjugate containing 20 mol of 3'SL per mol of human serum albumin inhibited bacterial binding with micromolar IC50. The H. pylori isolate most sensitive to inhibition by 3'SL was least sensitive to inhibition by sulfatide, gastric mucin, and other sulfated oligosaccharides. Bacteria that have been allowed to bind epithelial cells are also effectively detached by 3'SL. These results describe a heterogeneous adherence repertoire for these bacteria, but they also confirm the critical role of the 3'SL structure on human gastric epithelial cells as an adherence ligand for recent isolates of H. pylori.

The developmental profiles of the four major brain gangliosides, GM1, GD1a, GD1b, and GT1b, were examined in human frontal lobe covering the period from 10 fetal weeks to 80 years of age. The ganglioside concentration increased approx. 3-fold from the 10th gestational week to the age of about 5 years. Gangliosides GM1 and GD1a increased 12-15-fold during the same period. The most rapid increase of GM1 and GD1a occurred around term, during the period for dendrite arborization, outgrowth of axons and synaptogenesis. GT1b showed a quite different developmental curve. It was the major ganglioside during the 3rd to 5th gestational month, whereafter its concentration dropped rapidly to term, from which time the concentration then increased up to 50 years of age. Similar curves were found for the other gangliosides of the b-series, GD3, GD2, GD1b and GQ1b. Ganglioside 3'-isoLM1 was a characteristic early fetal ganglioside which dropped rapidly to the 5th gestational month, reached a small peak around term and then disappeared during adulthood. The concentration of gangliosides of the neolacto series was larger than that of the lacto series during the whole developmental period. In the beginning of the second trimester, 3'-LM1 constituted 2% and LD1 10% of total ganglioside sialic acid. The new findings demonstrate more dynamic changes of the ganglioside patterns during development than noted in previous studies.

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics.

We used NMR spectroscopy, molecular modeling and infectivity competition assays to investigate the key interactions between the spike protein (VP8(*)) from 'sialidase-insensitive' human Wa and 'sialidase-sensitive' porcine CRW-8 rotaviruses and the glycans of gangliosides G(M1) and G(D1a). Our data provide strong evidence that N-acetylneuraminic acid is a key determinant for binding of these rotaviruses. This is in contrast to the widely accepted paradigm that sialic acids are irrelevant in host cell recognition by sialidase-insensitive rotaviruses.

To identify the rotavirus protein which mediates attachment to cells in culture, viral reassortants between the simian rotavirus strain RRV and the murine strains EHP and EW or between the simian strain SA-11 and the human strain DS-1 were isolated. These parental strains differ in the requirement for sialic acid to bind and infect cells in culture. Infectivity and binding assays with the parental and reassortant rotaviruses indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains. Using ligated intestinal segments of newborn mice and reassortants obtained between the murine strain EW and RRV, we developed an in vivo infectivity assay. In this system, the infectivity of EW was not affected by prior treatment of the enterocytes with neuraminidase, while neuraminidase treatment reduced the infectivity of a reassortant carrying gene 4 from RRV on an EW background more than 80% relative to the controls. Thus, VP4 appears to function as the cell attachment protein in vivo as well as in vitro.

Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.

Mucosally ingested and inhaled antigens are taken up by membranous or microfold cells (M cells) in the follicle-associated epithelium of Peyer's patches or nasopharynx-associated lymphoid tissue. We established a novel M cell-specific monoclonal antibody (mAb NKM 16-2-4) as a carrier for M cell-targeted mucosal vaccine. mAb NKM 16-2-4 also reacted with the recently discovered villous M cells, but not with epithelial cells or goblet cells. Oral administration of tetanus toxoid (TT)- or botulinum toxoid (BT)-conjugated NKM 16-2-4, together with the mucosal adjuvant cholera toxin, induced high-level, antigen-specific serum immunoglobulin (Ig) G and mucosal IgA responses. In addition, an oral vaccine formulation of BT-conjugated NKM 16-2-4 induced protective immunity against lethal challenge with botulinum toxin. An epitope analysis of NKM 16-2-4 revealed specificity to an alpha(1,2)-fucose-containing carbohydrate moiety, and reactivity was enhanced under sialic acid-lacking conditions. This suggests that NKM 16-2-4 distinguishes alpha(1,2)-fucosylated M cells from goblet cells containing abundant sialic acids neighboring the alpha(1,2) fucose moiety and from non-alpha(1,2)-fucosylated epithelial cells. The use of NKM 16-2-4 to target vaccine antigens to the M cell-specific carbohydrate moiety is a new strategy for developing highly effective mucosal vaccines.

The influenza virus subtype H5N1 has raised concerns of a possible human pandemic threat because of its high virulence and mutation rate. Although several approved anti-influenza drugs effectively target the neuraminidase, some strains have already acquired resistance to the currently available anti-influenza drugs. In this study, we present the synergistic application of extended explicit solvent molecular dynamics (MD) and computational solvent mapping (CS-Map) to identify putative 'hot spots' within flexible binding regions of N1 neuraminidase. Using representative conformations of the N1 binding region extracted from a clustering analysis of four concatenated 40-ns MD simulations, CS-Map was utilized to assess the ability of small, solvent-sized molecules to bind within close proximity to the sialic acid binding region. Mapping analyses of the dominant MD conformations reveal the presence of additional hot spot regions in the 150- and 430-loop regions. Our hot spot analysis provides further support for the feasibility of developing high-affinity inhibitors capable of binding these regions, which appear to be unique to the N1 strain.

An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac2 and the N-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound the N-formyl derivative, whereas a N-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.