Publications

Core 2 O-glycans terminated with sialyl-Lewis x (sLe(X)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLe(X) extended from a core 2 branch (C2-O-sLe(X)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLe(X) was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(X) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(X)-related glycans. Unlike anti-sLe(X) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both.

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.

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.

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.

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.

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.

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.

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.

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.

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.