Impaired emotional response to stress in mice lacking galectin-1 or galectin-3.

Galectin-1 (Gal-1) and galectin-3 (Gal-3) are multifunctional glycan-binding proteins, expressed in the brain and in its limbic structures that are involved in behavioral control. Gal-1 induces the expression of the brain-derived neurotrophic factor (BDNF) and promotes adult neural stem cells proliferation, biological events impaired in stress-related psychiatric disorders, such as depression and anxiety. Despite that, there is no evidence regarding galectin involvement in emotional control during stressful situations. Thus, we analyzed the behavioral phenotype of Gal-1 or Gal-3 knock-out mice (Gal-1 KO or Gal-3 KO) in different experimental models predictive of depressive and compulsive-like behaviors. METHODS: C57BL-6 Gal-1 KO, Gal-3 KO, and wild-type mice (WT) were analyzed under the open field test (OFT) and, 6 h later, under the forced swim test (FST). Additionally, independent groups of male mice, lacking galectins or not, were exposed to the tail suspension test (TST) or to the marble burying test (MBT). The hippocampus and prefrontal cortex (PFC) of the mice submitted to MBT were dissected to access BDNF levels. RESULTS: Both Gal-1 and Gal-3 KO mice showed increased time of immobility in the FST and in the TST compared to WT animals, thus reflecting an impaired stress-coping behavior. Additionally, Gal-1 and Gal-3 KO female mice presented increased compulsive-like behavior in the MBT, without significant changes in the locomotor activity. BDNF levels were found to be decreased in the PFC of Gal-1 KO mice. DISCUSSION: Our results demonstrate that the absence of either endogenous Gal-1 and Gal-3 impairs stress-coping and increases compulsive-like behavior, suggesting that Gal-1 and Gal-3 are involved in the neurobiology of depression and obsessive-compulsive-like disorder.

Trichuris suis soluble products induce Rab7b expression and limit TLR4 responses in human dendritic cells.

Inflammatory immune disorders such as inflammatory bowel disease and multiple sclerosis are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases; however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on Toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide-stimulated DCs with or without T. suis treatment showed that co-stimulation with T. suis SPs resulted in a downregulation of both the myeloid differentiation primary response gene 88-dependent and the TIR-domain-containing adaptor-inducing interferon-ß-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.

Presentation Modality of Glycoconjugates Modulates Dendritic Cell Phenotype.

The comparative dendritic cell (DC) response to glycoconjugates presented in soluble, phagocytosable, or non-phagocytosable display modalities is poorly understood. This is particularly problematic, as the probing of immobilized glycans presented on the surface of microarrays is a common screen for potential candidates for glycan-based therapeutics. However, the assumption that carbohydrate-protein interactions on a flat surface can be translatable to development of efficacious therapies, such as vaccines, which are delivered in soluble or phagocytosable particles, has not been validated. Thus, a preliminary investigation was performed in which mannose or glucose was conjugated to cationized bovine serum albumin and presented to DCs in soluble, phagocytosable, or non-phagocytosable display modalities. The functional DC response to the glycoconjugates was assessed via a high throughput assay. Dendritic cell phenotypic outcomes were placed into a multivariate, general linear model (GLM) and shown to be statistically different amongst display modalities when comparing similar surface areas. The GLM showed that glycoconjugates that were adsorbed to wells were the most pro-inflammatory while soluble conjugates were the least. DC interactions with mannose conjugates were found to be calcium dependent and could be inhibited via anti-DC-SIGN antibodies. The results of this study aim to resolve conflicts in reports from multiple laboratories showing differential DC profiles in response to similar, if not identical, ligands delivered via different modalities. Additionally, this study begins to bridge the gap between microarray binding data and functional cell responses by highlighting the phenotypes induced from adsorbed glycoconjugates as compared to those in solution or displayed on microparticles.

Selectin ligands promote ultrasound contrast agent adhesion under shear flow.

Contrast-enhanced ultrasound imaging has shown promise in the field of molecular imaging. This technique relies upon the adhesion of ultrasound contrast agent (UCA) to targeted molecular markers of disease. This is accomplished by coating the surface of the contrast agent with a ligand that specifically binds to the intended molecular marker. Most UCA particles remain in the blood space, and their retention is influenced by the forces imposed by blood flow. For a UCA bound to a molecular target on the vascular endothelium, blood flow imposes a dislodging force that counteracts retention. Additionally, contrast agent adhesion to the molecular marker requires rapid binding kinetics, especially in rapid blood flow. The ability of a ligand:target bond complex to mediate fast adhesion and withstand dislodging force is necessary for efficient ultrasound-based molecular imaging. In the current study, we describe a flow-based adhesion assay which, combined with a novel automated tracking algorithm, enables quick determination of the ability of a targeting ligand to mediate effective contrast agent adhesion. This system was used to explore the adhesion of UCA targeted to the proinflammatory endothelial protein P-selectin via four targeting ligands, which revealed several interesting adhesive behaviors. Contrast agents targeted with glycoconjugate ligands modeled on P-selectin glycoprotein ligand 1 exhibited primarily unstable or transient adhesion, while UCA targeted with an anti-P-selectin monoclonal antibody exhibited primarily firm adhesion, although the efficiency with which these agents were recruited to the target surface was relatively low.

Dimerization of a selectin and its ligand stabilizes cell rolling and enhances tether strength in shear flow.

Selectins mediate rolling of leukocytes by rapid formation and dissociation of selectin-ligand bonds, which are assumed to require high mechanical strength to prevent premature dissociation by the forces applied in shear flow. This assumption is based largely on the observation that increasing wall shear stress increases only modestly the dissociation of transient leukocyte tethers on very low selectin densities. P-selectin binds to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. Both PSGL-1 and P-selectin are extended homodimers. We perfused transfected cells expressing wild-type dimeric PSGL-1 or a chimeric monomeric form of PSGL-1 on immobilized dimeric or monomeric forms of P-selectin. Cells expressing dimeric or monomeric PSGL-1 tethered to P-selectin at equivalent rates. However, cells expressing dimeric PSGL-1 established more stable rolling adhesions, which were more shear resistant and exhibited less fluctuation in rolling velocities. On low densities of dimeric P-selectin, increasing wall shear stress more rapidly increased transient tether dissociation of cells expressing monomeric PSGL-1 than dimeric PSGL-1. Tether dissociation on low densities of monomeric P-selectin was even more shear sensitive. We conclude that dimerization of both PSGL-1 and P-selectin stabilizes tethering and rolling, probably by increasing rebinding within a bond cluster. Because transient tethers may have more than one bond, the mechanical strength of selectin-ligand bonds is likely to be lower than initially estimated. Tether strength may rely more on bond clusters to distribute applied force.

Antibodies to glycans dominate the host response to schistosome larvae and eggs: is their role protective or subversive?

Multiple exposures of chimpanzees to the radiation-attenuated schistosome vaccine provoked a strong parasite-specific cellular and humoral immune response. Specific IgM and IgG were directed mainly against glycans on antigens released by cercariae; these were also cross-reactive with soluble antigens from larvae, adult worms, and eggs. Egg deposition was the major antigenic stimulus after challenge of vaccinated and control chimpanzees with normal parasites, eliciting strong antiglycan responses to egg secretions. Glycan epitopes recognized included LacdiNAc, fucosylated LacdiNAc, Lewis(X) (weakly), and those on keyhole limpet hemocyanin. Antibodies to peptide epitopes became prominent only during the chronic phase of infection, as glycan-specific IgM and IgG decreased. Because of their intensity and cross-reactivity, the antiglycan responses resulting from infection could be a smoke screen to subvert the immune system away from more vulnerable larval peptide epitopes. Their occurrence in humans might explain the long time required for antischistosome immunity to build up after infection.

Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin on leukocytes that binds to selectins. P-selectin binds to an N-terminal region of PSGL-1 that requires sulfation of at least one of three clustered tyrosines (TyrSO(3)) and an adjacent core-2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). We synthesized glycosulfopeptides (GSPs) modeled after this region of PSGL-1 to explore the roles of individual TyrSO(3) residues, the placement of C2-O-sLe(x) relative to TyrSO(3), the relative contributions of fucose and sialic acid on C2-O-sLe(x), and the function of the peptide sequence for binding to P-selectin. Binding of GSPs to P-selectin was measured by affinity chromatography and equilibrium gel filtration. 2-GSP-6, which has C2-O-sLe(x) at Thr-57 and TyrSO(3) at residues 46, 48, and 51, bound to P-selectin with high affinity (K(d) approximately 650 nm), whereas an isomeric trisulfated GSP containing C2-O-sLe(x) at Thr-44 bound much less well. Non-sulfated glycopeptide (2-GP-6) containing C2-O-sLe(x) at Thr-57 bound to P-selectin with approximately 40-fold lower affinity (K(d) approximately 25 microm). Proteolysis of 2-GP-6 abolished detectable binding of the residual C2-O-sLe(x)-Thr to P-selectin, demonstrating that the peptide backbone contributes to binding. Monosulfated and disulfated GSPs bound significantly better than non-sulfated 2-GP-6, but sulfation of Tyr-48 enhanced affinity (K(d) approximately 6 microm) more than sulfation of Tyr-46 or Tyr-51. 2-GSP-6 lacking sialic acid bound to P-selectin at approximately 10% that of the level of the parent 2-GSP-6, whereas 2-GSP-6 lacking fucose did not detectably bind; thus, fucose contributes more than sialic acid to binding. Reducing NaCl from 150 to 50 mm markedly enhanced binding of 2-GSP-6 to P-selectin (K(d) approximately 75 nm), demonstrating the charge dependence of the interaction. These results reveal a stereospecific interaction of P-selectin with PSGL-1 that includes distinct contributions of each of the three TyrSO(3) residues, adjacent peptide determinants, and fucose/sialic acid on an optimally positioned core-2 O-glycan.

Influenza virus infection of desialylated cells.

Sialic acid has long been considered to be the sole receptor for influenza virus. The viral hemagglutinin (HA) is known to bind cell surface sialic acid, and sialic acids on viral glyco-proteins are cleaved by the viral neuraminidase (NA) to promote efficient release of progeny virus particles. However, NWS-Mvi, a mutant virus completely lacking NA, grows well in MDCK cells continuously treated with exogenous neuraminidase (sialidase). Exogenous sialidase quantitatively releases all sialic acids from purified glycoproteins and glycolipids of MDCK cells and efficiently removes surface sialic acid from intact cells. Binding of NWS-Mvi and parent influenza viruses to MDCK cells is indistinguishable, and is only partially reduced by sialidase treatment of the cells. Both mutant and wild-type viruses enter enzymatically desialylated cells and initiate transcription. The ability of influenza A reassortant viruses to infect desialylated cells is shared by recent H3N2 clinical isolates, suggesting that this may be a general property of influenza A viruses. We propose that influenza virus infection can result from sialic acid-independent receptors, either directly or in a multistage process. When sialic acid is present, it may act to enhance virus binding to the cell surface to increase interaction with secondary receptors to mediate entry. Understanding virus entry will be critical to further efforts in infection control and prevention.

Expression cloning of a new member of the ABO blood group glycosyltransferases, iGb3 synthase, that directs the synthesis of isoglobo-glycosphingolipids.

The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: beta-d-galactosyl-1,4-glucosylceramide alpha-1, 3-galactosyltransferase (iGb(3) synthase) from a rat placental cDNA expression library. iGb(3) synthase acts on lactosylceramide, LacCer (Galbeta1,4Glcbeta1Cer) to form iGb(3) (Galalpha1,3Galbeta1, 4Glcbeta1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40-50% identity) to members of the ABO gene family that includes: murine alpha1, 3-galactosyltransferase, Forssman (Gb(5)) synthase, and the ABO glycosyltransferases. In contrast to the murine alpha1, 3-galactosyltransferase, iGb(3) synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb(3) synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb(3) functions, including the globo- and isoglobo-series of glycolipids.

Cloning of Gb3 synthase, the key enzyme in globo-series glycosphingolipid synthesis, predicts a family of alpha 1, 4-glycosyltransferases conserved in plants, insects, and mammals.

We have cloned Gb(3) synthase, the key alpha1, 4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb(3) synthase, the alpha1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Galbeta1,4Glcbeta1Cer (LacCer), to produce Gb(3) (Galalpha1,4LacCer) or iGb(3) (Galalpha1, 3LacCer), respectively. GalNAc can be added sequentially to either Gb(3) or iGb(3) yielding globoside and Forssman from Gb(3), and isogloboside and isoForssman from iGb(3). Gb(3) synthase is not homologous to iGb(3) synthase but shows 43% identity to a human alpha1,4GlcNAc transferase that transfers a UDP-sugar in an alpha1, 4-linkage to a beta-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb(3) synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an alpha1,4-glycosyltransferase, possibly Gb(3) synthase, throughout evolution. The isolated Gb(3) synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb(3) synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb(3) glycolipid, also called P(k) antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb(3) and are resistant to verotoxin become susceptible to the toxin following transfection with Gb(3) synthase cDNA.

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains.

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.

Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-selectin. To further address the ability of P-selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-selectin recognition.

Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes.

Infections of animals with parasitic worms, such as Schistosoma mansoni, induce humoral immune responses to carbohydrate antigens, raising the possibility that such antigens might be useful targets for the development of vaccines and new diagnostic approaches. Here we describe the identification of fucosylated LacdiNAc (LDNF) [GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc-R] as a new carbohydrate antigen in S. mansoni that induces humoral immune responses in infected mice. The presence of antibodies was determined by ELISA using a neoglycoconjugate synthesized to express LDNF sequences. Sera from S. mansoni-infected, but not uninfected, mice contain IgM, IgG, IgA, and IgE antibodies to LDNF. The IgG antibodies are primarily of the IgG1 and IgG3 subclasses, with no detectable levels of the complement-fixing IgG2a and IgG2b isotypes. An IgM monoclonal antibody, designated SMLDNF1, was generated from the spleens of S. mansoni-infected mice, and the antibody exhibits specific recognition of LDNF sequences, but not other fucosylated glycans tested. Immunocytochemical analysis demonstrates that LDNF antigens are localized on the tegumental surface of adult S. mansoni. Western blot analysis indicates that LDNF sequences are expressed on numerous high-molecular-weight glycoproteins from the three major human schistosome species, as well as the bird schistosome Trichobilharzia ocellata. The identification of LDNF antigen on the tegumental glycoproteins of schistosomes and the ability to synthesize LDNF conjugates should aid in the development of glycan-based vaccines and immunodiagnostic tests for schistosomiasis and in determining the role(s) of the glycans in worm development and pathogenesis.

Core alpha1-->3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep.

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

Schistosome glysoconjugates.

Schistosomes are trematodes known as blood flukes that cause schistosomiasis in people and animals. The male and female worms reside mainly in intestinal veins where they lay eggs that result in a wide-ranging pathology in infected individuals. A growing body of evidence indicates that carbohydrates on glycoproteins, glycolipids and glycosaminoglycans synthesized by the parasite are targets of humoral immunity and may play a role in modulating host immune responses. Carbohydrate antigens may provide protective immunity against infection. In addition, recent evidence indicates that glycoconjugates and carbohydrate-binding proteins from the parasites and their hosts participate in egg adhesion and granuloma formation involved in disease pathology. This review will highlight our current knowledge of the glycoconjugates synthesized by the parasites and their immunological and biological properties. There is increasing anticipation in the field that information about the glycobiology of these parasites may lead to carbohydrate-based vaccines and diagnostics for the disease and perhaps new therapies for treating infected individuals.

Tyrosine replacement in P-selectin glycoprotein ligand-1 affects distinct kinetic and mechanical properties of bonds with P- and L-selectin.

Selectins are adhesion molecules that initiate tethering and rolling of leukocytes on the vessel wall. Rolling requires rapid formation and breakage of selectin-ligand bonds that must have mechanical strength to resist premature dissociation by the forces applied in shear flow. P- and L-selectin bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. To define determinants on PSGL-1 that contribute to the kinetic and mechanical properties of bonds with selectins, we compared rolling of transfected preB cells expressing P- or L-selectin on transfected cell monolayers expressing wild-type PSGL-1 or PSGL-1 constructs with substitutions in targeted N-terminal residues. Rolling through P- or L-selectin required a Thr or Ser at a specific position on PSGL-1, the attachment site for an essential O-glycan, but required only one of three nearby Tyr residues, which are sites for Tyr-SO(3) formation. The adhesive strengths and numbers of cells rolling through P- or L-selectin were similar on wild-type PSGL-1 and on each of the three PSGL-1 constructs containing only a single Tyr. However, the cells rolled more irregularly on the single-Tyr forms of PSGL-1. Analysis of the lifetimes of transient tethers on limiting densities of PSGL-1 revealed that L-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at all shears examined. In sharp contrast, P-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at higher shear but not at lower shear. Thus, tyrosine replacements in PSGL-1 affect distinct kinetic and mechanical properties of bonds with P- and L-selectin.

Mice infected with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAc beta 1-->4GlcNAc) determinants.

Schistosoma mansoni is a parasitic trematode infecting humans and animals. We reported previously that adult S. mansoni synthesizes complex type biantennary N-glycans bearing the terminal sequence GalNAc beta 1-->4GlcNAc-R (lacdiNAc or LDN). We now report that mice infected with S. mansoni generate antibodies to LDN, as assessed by ELISA using a synthetic neoglycoconjugate containing LDN sequences. Sera of infected mice, but not uninfected mice, contained primarily IgM and low levels of IgG toward LDN. Interestingly, these antibodies also recognize bovine milk glycoproteins, which are known to express LDN sequences. The anti-LDN in sera of infected mice were affinity purified on immobilized bovine milk glycoproteins and shown to specifically bind LDN. An IgM monoclonal antibody (SMLDN1.1) was derived from the spleens of S. mansoni infected mice and shown to specifically bind LDN determinants. Immunoblots with affinity purified anti-LDN and SMLDN1.1 demonstrate that LDN sequences occur primarily on N-glycans of numerous glycoproteins of adult S. mansoni. LDN sequences are also expressed in many glycoproteins from S. japonicum and S. haematobium. The availability of antibody to LDN determinants should aid in defining the roles of these glycans in helminth and vertebrate biology.

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.

Structure and function of the selectin ligand PSGL-1.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->R). In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

Pig xenogeneic antigen modification with green coffee bean alpha-galactosidase.

Green coffee bean alpha-galactosidase can cleave the terminal alpha-galactose (alphaGal) on oligosaccharides that form the major antigen on pig endothelial cells recognized by primate-specific antibodies. Studies have been made of the conditions under which it is functional (e.g. temperature, pH) and of its biochemical and immunologic effects. Pig-to-rhesus monkey vein transplants were studied to identify the efficiency of the enzyme in delaying hyperacute rejection. When a graft became occluded, biopsies were taken for light microscopy (hematoxylin and eosin), scanning electron microscopy (SEM) and immunostaining with Griffonia simplicifolia IB4 lectin (GSIB4), and for IgM, IgG and C3. alpha-Galactosidase was stable for 72-96 h and was effective at 4 degrees C and pH 6.9 (conditions of human liver graft storage), although better function was obtained at 20 degrees C and pH 6.5. Using the porcine PK15 cell assay, the cytotoxicity of human serum was reduced after treatment of the pig cells with the enzyme. In vitro studies demonstrated that porcine veins treated with alpha-galactosidase lost endothelial expression of the Gal epitope within 30 min. SEM, however, demonstrated endothelial damage beginning within 2 h, probably caused by the alpha-galactosidase, as no damage was found in phosphate-buffered saline-treated veins, where the Gal epitope was preserved for >3 h. No change was found in either group on light microscopy. In vivo studies demonstrated that patency of the alpha-galactosidase-treated veins (mean 2.5 h) was longer than that of untreated veins (0.23 h) (P < 0.01). Biopsies showed no GSIB4 lectin staining for alpha-Gal epitopes and much less IgM and C3 deposition in the treated group. Light microscopy and SEM demonstrated more severe endothelial damage, hemorrhage, and fibrin formation in the untreated group. Galactosidase is effective in removing the terminal alphaGal and delays the onset of hyperacute rejection of pig veins transplanted into monkeys. However, its effect is temporary and, on its own, its use is unlikely to prolong survival of pig organs transplanted into primates sufficiently to be of clinical value.