Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAalpha2,3-Gal receptors, we found widespread presence of both SAalpha2,6-Gal and SAalpha2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAalpha2,6-Gal was the dominant receptor type whereas in ducks SAalpha2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAalpha2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAalpha2,3-Gal receptors (both beta(1-4)GlcNAc and beta(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses.

The thiobarbituric acid (TBA) reactivity of human plasma was studied to evaluate its adequacy in quantifying lipid peroxidation as an index of systemic oxidative stress. Two spectrophotometric TBA tests based on the use of either phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochloric acid (pH 0.9, method B) were employed with and without sodium sulfate (SS) to inhibit sialic acid (SA) reactivity with TBA. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Method B gave values of TBA-reactive substances (TBARS) 2-fold higher than those detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standard SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried out in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS formation only in the latter. Protein TBARS may be largely ascribed to SA-containing glycoproteins and, to a minor extent, protein-bound MDA. Indeed, EDTA did not affect protein TBARS assessed in the presence of SS. TBA reactivity of whole plasma and of its lipid fraction was instead inhibited by EDTA, suggesting that lipoperoxides (and possibly monofunctional lipoperoxidation aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostaglandin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipid peroxidation in human plasma. After correction for the interfering effects of SA in the TBA test, 40% of plasma TBARS appears related to in vivo generated prostaglandin endoperoxides and only about 60% to lipoperoxidation products. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma.

The possible presence of gonadotropin receptors in nonpregnant human uterus and human fetoplacental unit was investigated by light microscope immunocytochemistry using a monoclonal antibody to rat luteal hCG/LH receptors. The receptor antibody cross-reacted with human and bovine hCG/LH receptors and appears to be directed against the receptor rather than other proteins, including HLA class I antigens. Uterus and fetoplacental unit contained receptor antibody-binding sites, which indicates the presence of hCG/LH receptors. In the endometrium these receptors were present in glandular and luminal epithelial cells as well as in stromal cells. In the myometrium the receptors were detected in circular and elongated myometrial smooth muscle and vascular smooth muscle. Comparison of immunostaining intensities, which indicates the presence of different amounts of receptors, revealed that luminal and glandular epithelial cells contained more receptors than stromal cells. These cells, in turn, contained more receptors than myometrial and vascular smooth muscle. All cells in secretory phase uterine specimens contained more receptors than corresponding cells from the proliferative phase of the cycle. Midpregnancy placenta, amniotic epithelium, chorionic cytotrophoblasts, and decidual cells contained hCG/LH receptors. At term pregnancy, while receptors in fetal membranes and decidua continue to be detected, placental tissues did not show any detectable receptors unless the tissues were pretreated with neuraminidase. This indicated that term pregnancy placenta contain hCG/LH receptors masked by sialic acid residues. Comparison of immunostaining intensities suggested that syncytiotrophoblasts contained more receptors than cytotrophoblasts at midpregnancy; mesenchymal cells or blood vessels contained no detectable receptors. There were more receptors in decidua than in fetal membranes at mid- and term pregnancy. While the amniotic epithelial receptors decreased, the receptors in chorionic cytotrophoblasts and decidual cells increased from mid- to term pregnancy. In summary, hCG/LH receptors were demonstrated in the nonpregnant human uterus, human placenta, fetal membranes, and decidua. This indicates that hCG/LH may directly regulate functions of these tissues by endocrine, autocrine, or paracrine mechanisms.

Chemoenzymatic synthesis, which combines the flexibility of chemical synthesis and the high selectivity of enzymatic synthesis, is a powerful approach to obtain complex carbohydrates. It is a preferred method for synthesizing sialic acid-containing structures, including those with diverse naturally occurring and non-natural sialic acid forms, different sialyl linkages and different glycans that link to the sialic acid. Starting from N-acetylmannosamine, mannose or their chemically or enzymatically modified derivatives, sialic acid aldolase-catalyzed condensation reaction leads to the formation of sialic acids and their derivatives. These compounds are subsequently activated by a CMP-sialic acid synthetase and transferred to a wide range of suitable acceptors by a suitable sialyltransferase for the formation of sialosides containing natural and non-natural functionalities. The three-enzyme coupled synthesis of sialosides can be carried out in one pot without the isolation of intermediates. The time for synthesis is 4-18 h. Purification and characterization of the product can be completed within 2-3 d.

Previous studies in this laboratory have led to the identification and purification of a chicken cell surface protein named "neural cell adhesion molecule" (N-CAM) that is involved in neural cell-cell and neurite-neurite interactions. In the present investigation, we have found that a similar molecule exists in the mouse and have confirmed that it is also present in rat neural tissue. A monoclonal antibody to chicken N-CAM that crossreacted with mouse and rat brains and an independently derived monoclonal antibody to mouse N-CAM were used to purify an antigen from perinatal mouse and rat brains. The purified neural antigen resembles chicken N-CAM in its ability to neutralize antibodies that inhibit neural cell aggregation and also in its biochemical properties including molecular weight, sialic acid content, amino acid composition, and autoconversion to a smaller polypeptide. Like chicken N-CAM, the murine molecule is found throughout the nervous system and over the entire neuronal cell surface. These results strongly suggest that the molecule is evolutionarily related to chicken N-CAM and prompt the hypothesis that cell adhesion involving N-CAM is a fundamental mechanism existing in nervous systems of different phylogenetic classes of animals.

Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic(ON)). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic(ON). The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.

Monoclonal antibodies directed against nonpolymorphic determinants of HLA-D/DR molecular complex (human Ia antigens) were used to immunoprecipitate HLA-D-region-associated molecules from [35S]methionine internally labeled and 125I surface-labeled B lymphoblastoid B cell lines. Analysis of these by two-dimensional nonequilibrium-pH-gradient electrophoresis reveals a molecular heterogeneity within a 26,000- to 34,000-mol wt range. At least three sets of spots are identified: a very acidic set of 32,000- to 34,000-mol wt, a very intense invariant spot of 31,000 mol wt, and a series of 26,000- to 29,000-mol wt spots of variable charge. Comparison of immunoprecipitates from nine different HTC demonstrates that the polymorphism is localized in this latter set. Pulse-labeling and inhibition of glycosylation by tunicamycin show that the electrophoretic polymorphism is of polypeptide origin, whereas N-linked oligosaccharrides and sialic acid residues contribute to the microheterogeneity of the profile. Two-dimensional gels provide an electrophoretic genotyping procedure and allow analysis of the genetic organization and molecular complexity of the HLA-D/DR molecules.

Humans are genetically incapable of producing the mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), due to an inactivating mutation in the enzyme synthesizing it. Despite this, human cells and tissues appear capable of metabolically incorporating Neu5Gc from exogenous sources, including dietary red meat and dairy products. All normal humans studied are now shown to have circulating Abs against Neu5Gc, with marked differences in isotype levels. The question arises whether such Abs can adversely affect Neu5Gc-expressing human cells or tissues. In this study, we show that although normal human PBMC do not incorporate Neu5Gc during in vitro incubation, activated T cells do. Primary human leukemia cells and human leukemic cell lines are even more efficient at incorporation. Human sera containing naturally high levels of anti-Neu5Gc IgG Abs (hereafter abbreviated GcIg) deposited complement on Neu5Gc-expressing leukemic cells and activated T cells, but not on normal cells. The binding of GcIg resulted in complement-mediated cytotoxicity, which was inhibited by heat inactivation. Low anti-Neu5Gc IgG-containing human sera did not mediate any of these effects. Mixed killing assays confirmed the 15-fold selective killing of leukemic cells over PBMC by GcIg following Neu5Gc feeding. This approach could potentially serve as novel way to target malignant cells for death in vivo using either natural Abs or anti-Neu5Gc Abs prepared for this purpose. Further studies are needed to determine whether deposition of natural GcIg and complement can also target healthy proliferating immune cells for death in vivo following incorporation of dietary Neu5Gc.

Chinese hamster ovary (CHO) cells cluster in the presence of pertussis toxin, a response that is correlated with the ADP-ribosylation of a Mr = 41,000 membrane protein by the toxin. A ricin-resistant line of CHO cells (CHO-15B) which specifically lacks the terminal NeuAc----Gal beta 4GlcNAc oligosaccharide sequence on glycoproteins did not cluster in response to pertussis toxin. These cells do contain the Mr = 41,000 protein substrate for the enzymatic activity of the toxin which suggests that pertussis toxin, like certain plant lectins, does not bind to or is not internalized by the CHO-15B cells. There was no evidence of pertussis toxin binding to gangliosides or neutral glycolipids isolated from CHO cells but the toxin bound to a Mr = 165,000 component in N-octyglucoside extracts of CHO cells that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Plant lectins from Ricinus communis and Erythina cristagalli detected a similar size band in CHO cells and also did not react with CHO-15B cells. Unlike pertussis toxin, these plant lectins recognized two other major bands in CHO cell extracts and reacted best after sialidase treatment of nitrocellulose transfers containing CHO cell extracts. Conversely, sialidase treatment abolished binding a pertussis toxin and wheat germ agglutinin, a plant lectin that reacts with multivalent sialic acid residues on glycoproteins, to the Mr = 165,000 band. Purified B oligomer of pertussis toxin also uniquely detected a Mr = 165,000 component in CHO cell extracts while the A subunit of pertussis toxin was unreactive. These results indicate that pertussis toxin binds to a CHO cell glycoprotein with N-linked oligosaccharides and that sialic acid contributes to the complementary receptor site for the toxin. In addition, they suggest that a glycoprotein may serve as a cell surface receptor for pertussis toxin and that this interaction is mediated by a lectin-like binding site located on the B oligomer.

In this study the sugar composition of podocalyxin was determined in puromycin aminonucleoside-treated (PAN) rats and controls. Podocalyxin from both control and PAN rats bound 125I-WGA and 125I-peanut lectin (the latter only after neuraminidase treatment) on nitrocellulose transfers. Purified podocalyxin from both control and PAN rats was found to contain sialic acid, Gal, GlcNac, and Man but lacked Fuc and GalNac by gas-liquid chromatography. In PAN rats the sialic acid content of podocalyxin was reduced from 4.5% to 1.5%, whereas the concentration of the other sugars (with the possible exception of Gal) was similar to that of controls. The density of podocalyxin on the epithelial cell surface was estimated after immunogold labeling with anti-podocalyxin IgG, and no differences were found between PAN rats and controls. These data indicate that the reduced total glomerular sialic acid content found in PAN is due to the combined effects of the decreased podocyte plasmalemmal surface area and the reduced sialic acid content of podocalyxin.

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.

Initial studies with the monoclonal antibody F8-11-13 described in this paper showed that it reacted strongly with B lymphocytes, did not react at all with granulocytes, and reacted only weakly with a small subpopulation of thymocytes and peripheral T lymphocytes. This picture was entirely different from that seen with monoclonal antibodies to the leukocyte common (LC) antigen, where 100% of all the above-mentioned leukocyte populations were positive. Biochemical studies using detergent solubilized membranes labeled with 3H at the sialic acid residues showed that the molecule bearing the F8-11-13 determinant was a glycoprotein of 215,000 mol wt, and that the peak depleted by F8-11-13 monoclonal antibody affinity columns corresponded to the high molecular weight region of a broad peak previously shown to be completely depleted by monoclonal antibody (F10-89-4) affinity columns directed at the LC antigen. Proof that the F8-11-13 determinant was expressed on some LC molecules was established by cross-inhibition studies with affinity-column-purified and depleted material. This finding of a serologically identifiable conformational or other structural change selectively expressed on the LC molecule of a functionally discrete population of lymphocytes has interesting implications for the structure and function of the LC molecule, and might be relevant to functional consideration of other membrane molecules.

A program (HOOK) is described for generating potential ligands that satisfy the chemical and steric requirements of the binding region of a macromolecule. Functional group sites with defined positions and orientations are derived from known ligand structures or the multicopy simulation search (MCSS) method (Miranker, A., Karplus, M. Proteins 11:29-34, 1991). HOOK places molecular "skeletons" from a database into the protein binding region by making bonds between sites ("hooks") on the skeleton and functional groups. The nonpolar interactions with the binding region of candidate molecules are assessed by use of a simplified van der Waals potential. The method is illustrated by constructing ligands for the sialic acid binding site of the hemagglutinin from the influenza A virus and the active site of chloramphenicol acetyltransferase. Aspects of the HOOK program that lead to a highly efficient search of 10(5) or more skeletons for binding to 10(2) or more functional group minima are outlined.

Sialome sweet sialome: As sialic acids are involved in many host-pathogen recognition events and are markers of embryonic and malignant tissues, there is great interest in methods for the enrichment and identification of sialylated glycoproteins from complex tissues. Now N-(4-pentynoyl)mannosamine can be used to metabolically label sialylated glycoproteins in living animals, enabling future identification of new biomarkers.

Sialic acids are negatively charged acidic sugars, and sialylglycoconjugates often play important roles in various biological phenomena. Sialyltransferases are involved in the synthesis of sialylglycoconjugates, and 20 members of the mammalian sialyltransferase family have been identified to date. These sialyltransferases are grouped into four families according to the carbohydrate linkages they synthesize: beta-galactoside alpha2,3-sialyltransferases (ST3Gal I-VI), beta-galactoside alpha2,6-sialyltransferases (ST6Gal I and II), GalNAc alpha2,6-sialyltransferases (ST6GalNAc I-VI), and alpha2,8-sialyltransferases (ST8Sia I-VI). Analysis of the amino acid sequence similarities, substrate specificities, and gene structures of mouse sialyltransferases has revealed that they can be further divided into seven subfamilies. The genomic structural resemblance of members of the same subfamily suggests that they arose from a common ancestral gene through gene duplication events. These multiple sialyltransferase genes are needed for fine control of the expression of sialylglycoconjugates, resulting in a variety of developmental stage- and tissue-specific glycosylation patterns.

The type III polysaccharide of -roup B Streptococcus has been isolated and purified by a method that employs washing of intact cells at neutral pH. That the polysaccharide prepared by this procedure is the "native" type III antigen is suggested by its molecular size in excess of 10(6) daltons, its degradation by acid and heat treatment to a fragment with immunologic characteristics of the classical HCl antigen, and its type-specific serologic activity. The type III polysaccharide in native form contains sialic acid, galactose, glucose, glucosamine, heptose, and mannose. It is acidic in nature, is resistant to neuramindiase degradation, contains no O-acetyl groups, and does not share antigenic determinants with capsular type K1 antigen of Escherichia coli or Group B polysaccharide antigen of Neiserria meningitidis.

The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO(2)) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO(2) toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO(2) resin. By combining TiO(2) enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO(2)/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.

Vascular adhesion protein-1 (VAP-1) is a dimeric 170-kDa endothelial transmembrane molecule that under normal conditions is most strongly expressed on the high endothelial venules of peripheral lymph nodes and on hepatic endothelia. It is a glycoprotein that mediates tissue-selective lymphocyte adhesion in a sialic acid-dependent manner. In this study, we report the detection of a soluble form of VAP-1 in circulation. We developed a quantitative sandwich ELISA using novel anti-VAP-1 mAbs and used it to determine the levels of soluble VAP-1 (sVAP-1) in the serum of healthy individuals and in patients with inflammatory diseases. In healthy persons, circulating sVAP-1 concentrations were 49 to 138 ng/ml. Immunoblotting studies revealed that the apparent molecular mass of dimeric sVAP-1 is slightly (approximately 10 kDa) higher than that of transmembrane VAP-1 under nonreducing conditions. In contrast, the electrophoretic mobilities of monomeric sVAP-1 and transmembrane VAP-1 were similar after reduction and boiling. Adhesion assays showed that the circulating sVAP-1 modulates lymphocyte binding to endothelial cells. Inflammation can cause an elevation of serum sVAP-1 levels, because sVAP-1 concentrations in patients with certain liver diseases were two- to fourfold higher than those in normal individuals. In contrast, rheumatoid arthritis and inflammatory bowel diseases were not associated with elevated levels of sVAP-1. These findings indicate that there is a functionally active, soluble form of VAP-1 in circulation and suggest that the serum level of sVAP-1 might be a useful marker of disease activity in inflammatory liver diseases.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are sialic acid-binding proteins, which are expressed on many cell types of the immune system. B cells express two members of the Siglec family, CD22 (Siglec-2) and Siglec-G, both of which have been shown to inhibit B-cell signaling. CD22 recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and inhibits B-cell receptor (BCR)-induced Ca2+ signaling on normal B cells. CD22 interacts specifically with ligands carrying alpha2-6-linked sialic acids. Interaction with these ligands in cis regulates the association of CD22 with the BCR and thereby modulates the inhibitory function of CD22. Interaction of CD22 to ligands in trans can regulate both B-cell migration as well as the BCR signaling threshold. Siglec-G is a recently identified protein with an inhibitory function restricted to a B-cell subset, the B1 cells. Siglec-G inhibits Ca2+ signaling specifically in these cells. In addition, it controls the cellular expansion and antibody secretion of B1 cells. Thus, both Siglecs modulate BCR signaling on different B-cell populations in a mutually exclusive fashion.

Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phylogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of approximately 1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the "unique" Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars.

Intravenous immunoglobulin (IVIg) therapy is used to treat a wide range of autoimmune conditions and consists of pooled immunoglobulin G (IgG) from healthy donors. The immunosuppressive effects of IVIg are, in part, attributed to terminal α2,6-linked sialic acid residues on the N-linked glycans of the IgG Fc (fragment crystallizable) domain. This α2,6-sialylated Fc (sFc) has been reported to bind to the carbohydrate recognition domain (CRD) of the cell-surface lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and its murine orthologue SIGN-R1 (specific intracellular adhesion molecule-grabbing non-integrin R1) and, via this interaction, to signal the downstream expression of immunosuppressive cytokines and receptors. Consistent with this model, the antiinflammatory effect of IVIg treatment is abolished in a murine knock-out of SIGN-R1 and can be restored by a knock-in with human DC-SIGN. In contrast, however, existing glycan array and X-ray crystallographic studies indicate that the CRDs of both SIGN-R1 and DC-SIGN bind to a restricted set of primarily oligomannose-type glycans that does not include the glycans found on sFc. We attempted to reconcile these immunological and biophysical observations. We first generated hypersialylated, desialylated, deglycosylated and untreated serum IgG and found that the affinity for the complete extracellular region of the DC-SIGN tetramer was similar for all antibody glycoforms. Moreover, the binding could be attributed to cross-reactive, polyclonal Fab (fragment antigen-binding) specificities in serum as neither recombinant Fc nor sFc bound to DC-SIGN. In addition, serum IgG exhibited no competition against known ligands of the DC-SIGN CRD. These findings lead us to suggest that IVIg therapy does not involve binding of IgG Fc to DC-SIGN and that alternative cell-surface lectins are required for the antiinflammatory activity of sFc.

Several properties of 10 cell lines derived from the polyoma-induced PW20 Wistar-Furth rat renal sarcoma have been examined, including the ability of the tumor cells to metastasize spontaneously from subcutaneous sites in syngeneic hosts, the platelet-aggregating activity of material extracted by urea from the surface of cultured cells, the sialic acid content of the platelet-aggregating material, and the degree of sialylation of cell surface glycoconjugates in cultured cells. A correlation has been observed among all of these parameters. The results suggest a possible link between the degree of cell surface sialylation of tumor cells, their ability to aggregate platelets, and their ability to metastasize.

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity--5'5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody--inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.