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.     

L-selectin interactions with novel mono- and multisulfated Lewisx sequences in comparison with the potent ligand 3'-sulfated Lewisa.

The cell adhesion molecule L-selectin binds to 3'-sialyl-Lewis (Le)x and -Lea and to 3'-sulfo-Lex and -Lea sequences. The binding to 3'-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on the N-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6'-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3'-sulfo-Lex, the 6-sulfation enhances and 6'-sulfation suppresses L-selectin binding. The 6'-sulfation without 3'-sialyl or 3'-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3'-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and inner N-acetylglucosamine do not influence the binding. Although binding to the 6,3'-sulfo-Lex and 6-sulfo, 3'-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3'-sulfo-Lex series are more potent than the corresponding 3'-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3'-sulfo-Lex (and also the 3'-sulfo-Lea) would seem most appropriate.     

Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems

This communication is concerned with the binding specficityof the leukocyte-adhesion molecule L-selectin (leukocyte homingreceptor) towards structurally defined sulphated oligosaccharidesof the blood group Le^a and Le^x series, and of the glycolsaminoglycanseries heparin, chondroitin sulphate and keratan sulphate. Therecombinant soluble form of the rat L-selectin (L-selectin-IgGFc chimera) investigated here was shown previously to bind tolipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated atgalactose, such as sulphatides and a mixture of 3-sulphatedLe^a/Le^x type tetrasaccharides isolated from ovarian cystadenoma,as well as to the HNK-1 glycolipid with 3-O sulphated glucuronicacid. In the present study, the L-selectin investigated in bothchromatogram binding and plastic microwell binding experimentsusing neoglycolipids was found to bind to the individual 3-sulphatedLe^a and Le^x sequences (penta-, tetra- and trisaccharides), andwith somewhat lower intensities to their non-fucosylated analogues.Glycosaminoglycan disaccharides of keratan sulphate, heparinand chondroitin sulphate types were also bound by L-selectinin one or both assay systems, leading to the conclusion thatclustered glycosaminoglycan oligosaccharides with 6-O sulphationof N-acetylgalactctosamine, N-acetylglucosamine or glucosamine,4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronicacid, N-sulphation of glucosamine and, to a lesser extent, thenon-sulphated uronic acid-contahing disaccharides, can supportL-selectin adhesion. As inflammatory chemokines (short-rangestimulators of lymphocyte migration which trigger integrin activation)are known to bind to endothelial glycosaminoglycans, we proposethat the binding of the lymphocyte membrane L-selectin to endothelialglycosaminoglycans may provide a link between the selectin-mediatedand integrin-mediated adhesion systems in leukocyte extravasationcascades. The posibility is also raised that lymphocyte L-selectininteractions with glycosaminoglycans may contribute to pathologiesof glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoidarthritis and inflammatory lesions of the cornea. glycosaminoglycans leukocyte adhesion cascades neoglycolipids oligosaccharide presentation sulphated oligosaccharides     

Comparison of L-selectin and E-selectin ligand specificities: the L-selectin can bind the E-selectin ligands sialyl Le(x) and sialyl Le(a).

The L- and E-selectins are leukocyte and endothelial cell surface molecules which mediate leukocyte-endothelial cell adhesion by interacting with carbohydrate ligands. In the present study we find that L-selectin, like E-selectin, can interact with synthetic neoglycoproteins containing Sialyl Le(x) (Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta-R), or Sialyl Le(a) (Neu5Ac-alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta-R). Additionally, both the E-selectin and L-selectin can bind the peripheral lymph node addressin, a high endothelial venule ligand for L-selectin. Despite overlapping interactions, the L- and E-selectins discriminate between their native ligands. The peripheral lymph node addressin is a preferential ligand for L-selectin; and furthermore, L-selectin expressing cells do not interact detectably with the cutaneous lymphocyte antigen, a native glycoprotein ligand for E-selectin found on a subset of lymphocytes associated with the skin.     

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.     

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.     

Synthesis of a lipid conjugate of SO3Le(a) and its enhancement on liposomal binding to activated platelets

3'-O-Sulfated Le(a) (SO3Le(a)) is one of the most potent natural oligosaccharide ligands of selectins. The specific interactions between SO3Le(a) and E-/P-selectins are critical in the inflammation process. This paper described an efficient synthesis of a lipid conjugate of SO3Le(a) and its combination with phospholipid and cholesterol to form SO3Le(a)-coated liposomes by the freeze-thaw and extrusion method. The size (D = 78 nm) and stability of the resultant glycoliposomes were comparable to that of liposomes without the glycoconjugate. It was further observed that the incorporation of SO3Le(a) into liposomes could significantly enhance their adhesion to activated platelets as a result of the specific binding between SO3Le(a) on the glycoliposome and the P-selectin on activated platelets. The glycoliposome constructs may be useful for antiinflammation or for targeted delivery of drugs to endothelial cells that express E- and P-selectins.     

1,3-beta-D glucan binding protein (BGBP) from the white shrimp,Penaeus vannamei,is also a heparin binding protein

Shrimp BGBP was purified as a 100 kDa glycoprotein by affinity chromatography using immobilised heparin. BGBP bound simple carbohydrates, glycosaminoglycans like heparin sulphate and glycoproteins, but it was unable to agglutinate erythrocytes. Using an ELISA-based microplate assay, it was shown that simple carbohydrates such as n-glucose and D-mannose are competitive inhibitors of heparin sulphate binding to BGBP. Based on these properties BGBP is considered as a new type of heparin binding protein.     

Monomeric and Multimeric Blockers of Selectins: Comparison of <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> Activity

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.     

Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis(a) and ABH blood group glycotopes

Pseudomonas aeruginosa Fuc > Man specific lectin, PA-IIL, is an important microbial agglutinin that might be involved in P. aeruginosa infections in humans. In order to delineate the structures of these lectin receptors, its detailed carbohydrate recognition profile was studied both by microtiter plate biotin/avidin-mediated enzyme-lectin-glycan binding assay (ELLSA) and by inhibition of the lectin-glycan interaction. Among 40 glycans tested for binding, PA-IIL reacted well with all human blood group ABH and Le(a)/Le(b) active glycoproteins (gps), but weakly or not at all with their precursor gps and N-linked gps. Among the sugar ligands tested by the inhibition assay, the Le(a) pentasaccharide lacto-N-fucopentaose II (LNFP II, Galbeta1-3[Fucalpha1-4]GlcNAcbeta1-3Galbeta1-4Glc) was the most potent one, being 10 and 38 times more active than the Le(x) pentasaccharide (LNFP III, Galbeta1-4 [Fucalpha1-3]GlcNAcbeta1-3Galbeta1-4Glc) and sialyl Le(x) (Neu5Acalpha2-3Galbeta1-4[Fucalpha1-3] GlcNAc), respectively. It was 120 times more active than Man, while Gal and GalNAc were inactive. The decreasing order of PA-IIL affinity for the oligosaccharides tested was: Le(a) pentaose > or = sialyl Le(a) tetraose > methyl alphaFuc > Fuc and Fucalpha1-2Gal (H disaccharide)>2'-fucosyllactose (H trisaccharide), Le(x) pentaose, Le(b) hexaose (LNDFH I) and gluco-analogue of Le(y) tetraose (LDFT)>H type I determinant (LNFP I)>Le(x) trisaccharide (Galbeta1-4[Fucalpha1-3]GlcNAc) > sialyl Le(x) trisaccharide > Man > Gal, GalNAc, and Glc (inactive). The results presented here, in accordance with the crystal 3D structural data, imply that the combining site of PA-IIL is a small cavity-type best fitting Fucalpha1- with a specific shallow groove subsite for the remainder part of the Le(a) saccharides, and that polyvalent glycotopes enhance the reactivity. The Fuc > Man Ralstonia solanacearum lectin RSL, which resembles PA-IIL in sugar specificity, differs from it in it's better fit to the B and A followed by H oligosaccharides vs. Fuc, whereas, the second R. solanacearum lectin RS-IIL (the structural homologue of PA-IIL) binds Man > Fuc. These results provide a valuable information on PA-IIL interactions with mammalian glycoforms and the possible spectrum of attachment sites for the homing of this aggressive bacterium onto the target molecules. Such information might be useful for the antiadhesive therapy of P. aeruginosa infections.     

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.     

Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.     

Probing sialic acid binding Ig-like lectins (siglecs) with sulfated oligosaccharides

Soluble siglecs-1, -4, -5, -6, -7, -8, -9, and -10 were probed with polyacrylamide glycoconjugates in which: 1) the Neu5Ac residue was substituted by a sulfate group (Su); 2) glycoconjugates contained both Su and Neu5Ac; 3) sialoglycoconjugates contained a tyrosine-O-sulfate residue. It was shown that sulfate derivatives of LacNAc did not bind siglecs-1, -4, -5, -6, -7, -8, -9, and -10; binding of 6'-O-Su-LacNAc to siglec-8 was stronger than binding of 3'SiaLacNAc. The relative affinity of 3'-O-Su-TF binding to siglecs-1, -4, and -8 was similar to that of 3'SiaTF. 3'-O-Su-Le(c) displayed two-fold weaker binding to siglec-1 and siglec-4 than 3'SiaLe(c). The interaction of soluble siglecs with sulfated oligosaccharides containing sialic acid was also studied. It was shown that siglecs-1, -4, -5, -6, -7, -9, and -10 did not interact with these compounds; binding of 6-O-Su-3'SiaLacNAc and 6-O-Su-3'SiaTF to siglec-8 was weaker than that of the corresponding sulfate-free sialoside probes. Siglec-8 displayed affinity to 6'-O-Su-LacNAc and 6'-O-Su-SiaLe(x), and defucosylation of the latter compound led to an increase in the binding. Sialoside probes containing tyrosine-O-sulfate residue did not display increased affinity to siglecs-1 and -5 compared with glycoconjugates containing only sialoside. Cell-bound siglecs-1, -5, -7, and -9 did not interact with 6-O-Su-3'SiaLacNAc, whereas the sulfate-free probe 3'SiaLacNAc demonstrated binding. In contrast, the presence of sulfate in 6-O-Su-6'SiaLacNAc did not affect binding of the sialoside probe to siglecs. 6'-O-Su-SiaLe(x) displayed affinity to cell-bound siglecs-1 and -5; its isomer 6-O-Su-SiaLe(x) bound more strongly to siglecs-1, -5, and -9 than SiaLe(x).     

Antibody binding to a conformation-dependent epitope induces L-selectin association with the detergent-resistant cytoskeleton

L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37 degrees C, at 4 degrees C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.     

The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse

The high-molecular-mass salivary mucin MG1, one of two major mucins produced by human salivary glands, plays an important role in oral health by coating the tooth surface and by acting as a bacterial receptor. Here this mucin was purified from the submandibular/sublingual saliva of a blood group O individual. The presence of MUC5B as the major mucin in this preparation was confirmed by amino acid analysis and its reactivity with the monoclonal antibody PAN H2. To structurally characterize MG1 carbohydrates the O-glycans were released by reductive beta-elimination. Nuclear magnetic resonance spectroscopy of the nonfractionated mixture showed that (1) fucose was present in blood group H, Le(a), Le(x), Le(b), and Le(y) epitopes; (2) NeuAc was mainly linked alpha 2-3 to Gal or alpha 2-6 to GalNAcol; and (3) the major internal structures were core 1 and core 2 sequences. After this preliminary analysis the released oligosaccharides were separated into neutral (56%), sialylated (26%), and sulfated (19%) fractions, with an average length of 13, 17, and 41 sugar residues, respectively. Gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of mixtures of neutral and sialylated oligosaccharides revealed at least 62 neutral and 25 sialylated oligosaccharides consisting of up to 20 monosaccharide residues. These results showed that the MG1-derived oligosaccharides were much longer than those of MG2, and only a few species were found on both molecules. Thus, these two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body.     

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.     

Biosynthesis of L-selectin ligands: sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases

The leukocyte adhesion molecule L-selectin mediates lymphocyte homing to secondary lymphoid organs and to certain sites of inflammation. The cognate ligands for L-selectin possess the unusual sulfated tetrasaccharide epitope 6-sulfo sialyl Lewis x (Siaalpha2-->3Galbeta1-->4[Fucalpha1-->3][SO(3)-->6]GlcNAc). Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Four recently discovered GlcNAc-6-sulfotransferases are the first candidate contributors to the biosynthesis of 6-sulfo sLex in the context of L-selectin ligands. Here we report the in vitro activity of the four GlcNAc-6-sulfotransferases on a panel of synthetic oligosaccharide substrates that comprise structural motifs derived from sialyl Lewis x. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. On the basis of these results, we propose possible pathways for 6-sulfo sialyl Lewis x biosynthesis and suggest that sulfation may be an early committed step.     

Characterization of heparin oligosaccharides binding specifically to antithrombin III using mass spectrometry

Heparan sulfate (HS) is a sulfated glycosaminoglycan attached to a core protein on the cell surface. Protein binding to cell surface HS is a key regulatory event for many cellular processes such as blood coagulation, cell proliferation, and migration. The concept whereby protein binding to HS is not random but requires a limited number of sulfation patterns is becoming clear. Here we describe a hydrophobic trapping assay for screening a library of heparin hexasaccharides for binders to antithrombin III (ATIII). The hexasaccharide compositions are defined with their building block content in the following format: (DeltaHexA:HexA:GlcN:SO 3:Ac). Of five initial compositions present in the library, (1:2:3:6:1), (1:2:3:7:1), (1:2:3:7:0), (1:2:3:8:0), and (1:2:3:9:0), only two are shown to bind ATIII, namely, (1:2:3:8:0) and (1:2:3:9:0). The use of amide hydrophilic interaction (HILIC) liquid chromatography-mass spectrometry permitted reproducible quantitative analysis of the composition of the initial library as well as that of the binding fraction. The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII-mediated inhibition of Factor Xa in vitro.     

Mutagenesis of the ezrin-radixin-moesin binding domain of L-selectin tail affects shedding, microvillar positioning, and leukocyte tethering

L-selectin is a cell adhesion molecule that mediates the initial capture (tethering) and subsequent rolling of leukocytes along ligands expressed on endothelial cells. We have previously identified ezrin and moesin as novel binding partners of the 17-amino acid L-selectin tail, but the biological role of this interaction is not known. Here we identify two basic amino acid residues within the L-selectin tail that are required for binding to ezrin-radixinmoesin (ERM) proteins: arginine 357 and lysine 362. L-selectin mutants defective for ERM binding show reduced localization to microvilli and decreased phorbol 12-myristate 13-acetate-induced shedding of the L-selectin ectodomain. Cells expressing these L-selectin mutants have reduced tethering to the L-selectin ligand P-selectin glycoprotein ligand-1, but rolling velocity on P-selectin glycoprotein ligand-1 is not affected. These results suggest that ERM proteins are required for microvillar positioning of L-selectin and that this is important both for leukocyte tethering and L-selectin shedding.     

Cutting edge: evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils

Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca(2+) concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca(2+) mobilization, whereas mAbs against the beta(2) integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca(2+) signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.