Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding. E-selectin binding is unaffected in the presence of the blood group H fucose (alpha 1-2 linked to galactose to form the Le(b) antigen). However, the binding is abolished when in addition alpha 1-3-linked N-acetylgalactosamine to the galactose (blood group A antigen) is present. These results indicate that some E-selectin-mediated adhesive events may be influenced by blood group status.     

Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV and -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.     

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.     

P- and E-selectin use common sites for carbohydrate ligand recognition and cell adhesion

The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P- selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.     

P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.

P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.     

Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001). Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios. The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 [SE]) than the remaining infants (8.5 +/- 0.8; p<0.001). Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042). Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea. We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk.     

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

Selectin/glycoconjugate binding assays for the identification and optimization of selectin antagonists.

In this study we describe ELISA-type P- and L-selectin binding assays for the analysis of selectin antagonists. A biotinylated polyacrylamide-type glycoconjugate containing sialyl Lewis A (sLe(a)-polymer) is utilized as a synthetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sLe(a)-polymer with streptavidin-peroxidase, the complex is added to microtiter plates coated with the recombinant selectins. Binding of sLe(a)-polymer to the immobilized selectins is measured by the peroxidase reaction. SLe(a)-polymer was found to bind to P- and L-selectin in a cation-dependent manner. The interaction of the polymer was blocked by neutralizing anti-P- and anti-L-selectin antibody, respectively. The reference compounds heparin and fucoidan inhibited in both assays. Sialyl Lewis X (sLe(x)) blocked binding to L-selectin by 46% at 3 mM, whereas no inhibition was observed in the P-selectin assay up to 3 mM. Control polymers containing sialic acid or beta-d-glucose instead of sLe(a) weakly bound or failed to bind to the selectins. Both assays are rapid to perform and of low variability. The P-selectin assay was successfully employed to identify and optimize novel carbohydrate-based P-selectin antagonists. The P-, L-, and E-selectin assays were used to determine the fine selectivity of several sLe(x)-related selectin antagonists. These studies together suggest that sLe(a)-polymer-based selectin assays are well suited for primary screening and the characterization of selectin antagonists.     

High affinity binding of the leucocyte adhesion molecule L-selectin to 3'-sulphated-Le(a) and -Le(x) oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides.

The binding of the leucocyte adhesion molecule L-selectin has been investigated toward several structurally defined lipid-linked oligosaccharides immobilized on silica gel chromatograms or plastic wells. In both assay systems the 3'-sulphated Le(a)/Le(x) type tetrasaccharides [formula: see text] were more strongly bound than 3'-sialyl analogues. A considerable binding was observed to the 3'-sulphated oligosaccharide backbone in the absence of fucose but not to a 3'-sialyl analogue or fuco-oligosaccharide analogues lacking sulphate or sialic acid. Affinity for other sulphated saccharides: 3'-sulphoglucuronyl neolactotetraosyl ceramide and glycolipids with sulphate 3'-linked to terminal or sub-terminal galactose or N-acetylgalactosamine was detected in the chromatogram assay only. These studies, together with earlier reports that L-selectin binding to endothelium is inhibited by sulphatide, highlight the relative importance of sulphate in the adhesive specificity of this protein.     

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.     

Carbohydrate microarrays reveal sulphation as a modulator of siglec binding

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.     

Multi-recognition capability of E-selectin in a dynamic flow system, as evidenced by differential effects of sialidases and anti-carbohydrate antibodies on selectin-mediated cell adhesion at low vs. high wall shear stress: a preliminary note.

E-selectin has a "multi-recognition" capability in terms of epitope binding specificity, depending on adhesion conditions (static vs. low- or high-shear stress dynamic systems). Specifically, (i) adhesion based on expression of alpha 2-->3 sialylated Le(x) (SLe(x)) is prominent under static or low shear stress dynamic conditions; (ii) adhesion under high shear stress dynamic conditions does not depend on the known SLe(x) species, but rather on Lex with an adjacent unidentified sialosyl substitution, which shows different susceptibility to sialidases and antibodies compared to known SLe(x).     

Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein.

E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein. This has been achieved by application of the neoglycolipid technology to oligosaccharides released from the glycoprotein by mild alkaline beta-elimination. Oligosaccharides were conjugated to lipid, resolved by thin-layer chromatography, and tested for binding by Chinese hamster ovary cells which had been transfected to express the full-length E-selectin molecule. Several components with strong E-selectin binding activity were revealed among acidic oligosaccharides. The smallest among these was identified by liquid secondary ion mass spectrometric analysis of the neoglycolipid, in conjunction with methylation analysis of the purified oligosaccharide preparation as an equimolar mixture of the Le(a)- and Le(x)/SSEA-1-type fucotetrasaccharides sulfated at position 3 of outer galactose: [formula: see text] To our knowledge this is the first report of a sulfofucooligosaccharide ligand for E-selectin. The binding activity is substantially greater than those of lipid-linked Le(a) and Le(x)/SSEA-1 sequences and is at least equal to that of the 3'-sialyl-Le(x)/SSEA-1 glycolipid analogue.     

New aspects of siglec binding specificities, including the significance of fucosylation and of the sialyl-Tn epitope. Sialic acid-binding immunoglobulin superfamily lectins

The siglecs (sialic acid-binding immunoglobulin superfamily lectins) are immunoglobulin superfamily members recognizing sialylated ligands. Most prior studies of siglec specificities focused on alpha2-3- and alpha2-6-sialyllactos(amin)es and on one or two of the siglecs at a time. Here, we explore several new aspects of specificities of the first six reported siglecs, using sialylated glycans presented in multivalent form, on synthetic polyacrylamide backbones, or on mucin polypeptides. First, we report that binding of siglec-1 (sialoadhesin), siglec-3 (CD33), siglec-4a (myelin-associated glycoprotein), and siglec-5 to alpha2-3 sialyllactosamine is affected markedly by the presence of an alpha1-3-linked fucose. Thus, while siglecs may not interfere with selectin-mediated recognition, fucosylation could negatively regulate siglec binding. Second, in contrast to earlier studies, we find that siglec-3 prefers alpha2-6-sialyllactose. Third, siglec-5 binds alpha2-8-linked sialic acid, making it the siglec least specific for linkage recognition. Fourth, siglecs-2 (CD22), -3, -5, and -6 (obesity-binding protein 1) showed significant binding to sialyl-Tn (Neu5Acalpha2-6-GalNAc), a tumor marker associated with poor prognosis. Fifth, siglec-6 is an exception among siglecs in not requiring the glycerol side chain of sialic acid for recognition. Sixth, all siglecs require the carboxyl group of sialic acid for binding. Finally, the presentation of the sialyl-Tn epitope and/or more extended structures that include this motif may be important for optimal recognition by the siglecs. This was concluded from studies using ovine, bovine, and porcine submaxillary mucins and Chinese hamster ovary cells transfected with ST6GalNAc-I and/or the mucin polypeptide MUC1.     

The contribution of D-mannose, L-fucose, N-acetylglucosamine, and selectin residues on the binding of glycodelin isoforms to human spermatozoa

Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.     

Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1.23:0.36 was isolated from the Pacific brown seaweed Fucus evanescens. The structures of its desulfated and de-O-acetylated derivatives were investigated by 1D and 2D (1)H and (13)C NMR spectroscopy, and the data obtained were confirmed by methylation analysis of the native and desulfated polysaccharides. The fucoidan was shown to contain a linear backbone of alternating 3- and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp(2SO(3)(-))-(1-->4)-alpha-L-Fucp(2SO(3)(-))-(1-->. Additional sulfate occupies position 4 in a part of 3-linked fucose residues, whereas a part of the remaining hydroxyl groups is randomly acetylated.     

Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate-protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Le(a)) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.     

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.     

Receptor-binding specificity of the human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein

The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewis(x) motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.     

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.