Publications

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.

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.

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.

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.

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.

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.

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.

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize alpha1,3-fucosylated GlcNAc and terminal beta-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal beta-Gal, and the glycoconjugates lack the Lewis x (Le(x)) antigen or other related fucose-containing antigens, such as sialylated Le(x), Le(a), Le(b) Le(y), or H-type 1. Direct assays of parasite extracts demonstrate the presence of an alpha1,3-fucosyltransferase (alpha1,3FT) and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT), but not beta1,4-galactosyltransferase. The H. contortus alpha1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galalpha1-->4GlcNAcbeta1-->3Galbeta1-->4Glc to form lacto-N-fucopentaose III Galbeta1-->4[Fuca1-->3]GlcNAc beta1-->3Galbeta1-4GIc, which contains the Le(x) antigen, and the acceptor lacdiNAc (LDN) GalNAcbeta1-->4GlcNAc to form GalNAc beta1-->4[Fualpha1-->3]GlcNAc. The alpha1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 microM and a Vmax of 10.8 nmol-mg(-1) h(-1). The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The alpha1,3FT acts best with type-2 glycan acceptors (Galbeta1-->4GlcNAcbeta1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus alpha1,3FT can synthesize the Le(x) antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-->R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.