Anne Leppänen

A Novel Glycosulfopeptide Binds to P-selectin and Inhibits Leukocyte Adhesion to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO3) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLex) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO3 or C2-O-sLex do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6′, which contains sLex 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 aK d of ∼350 nm. GSP-6 (<5 μm) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLex (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.

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 (TyrSO3) and an adjacent core-2-based O-glycan expressing sialyl Lewis x (C2-O-sLex). We synthesized glycosulfopeptides (GSPs) modeled after this region of PSGL-1 to explore the roles of individual TyrSO3 residues, the placement of C2-O-sLexrelative to TyrSO3, the relative contributions of fucose and sialic acid on C2-O-sLex, 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-sLex at Thr-57 and TyrSO3 at residues 46, 48, and 51, bound to P-selectin with high affinity (K d ∼ 650 nm), whereas an isomeric trisulfated GSP containing C2-O-sLex at Thr-44 bound much less well. Non-sulfated glycopeptide (2-GP-6) containing C2-O-sLex at Thr-57 bound to P-selectin with ∼40-fold lower affinity (K d ∼25 μm). Proteolysis of 2-GP-6 abolished detectable binding of the residual C2-O-sLex-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 ∼ 6 μm) more than sulfation of Tyr-46 or Tyr-51. 2-GSP-6 lacking sialic acid bound to P-selectin at ∼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 ∼ 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 TyrSO3residues, adjacent peptide determinants, and fucose/sialic acid on an optimally positioned core-2 O-glycan.

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow.

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin–binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-β-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin–ligand interactions.

Structurally Distinct Requirements for Binding of P-selectin Glycoprotein Ligand-1 and Sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and α1–3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of α1–3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.

Replacing a Lectin Domain Residue in L-selectin Enhances Binding to P-selectin Glycoprotein Ligand-1 but Not to 6-Sulfo-sialyl Lewis x

Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.

The linear tetrasaccharide, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc, isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAcβ1-6Galβ1-4GlcNAc, resisted the action of endo-β-galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc were cleaved in the expected manner.

Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.

Biosynthesis of Branched Polylactosaminoglycans EMBRYONAL CARCINOMA CELLS EXPRESS MIDCHAIN β1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE ACTIVITY THAT GENERATES BRANCHES TO PREFORMED LINEAR BACKBONES

Two types of β1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcβ1–3Galβ1–4GlcNAcβ1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcβ1–3)Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc from the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc and Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc from the linear hexasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo-β-galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml−1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAcβ1-6Galβ1-4GlcNAc, or those of the tetrasaccharides Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc and Galβ1-4GlcNAcβ1-6Galβ1-4Glc. The isomeric glycans which contained the GlcNAcβ1-3Galβ1-4GlcNAc/Glc sequence were readily cleaved.

Glycosulfopeptides modeled on P-selectin glycoprotein ligand 1 inhibit P-selectin-dependent leukocyte rolling in vivo

Leukocytic inflammation can be limited by inhibiting selectin‐dependent leukocyte rolling. In spite of intensive efforts to develop small molecule selectin inhibitors with defined structure‐activity profiles, inhibition of P‐selectin‐dependent leukocyte rolling in vivo by such a compound has yet to be described. We recently reported that glycosulfopeptides (GSP), modeled on the high affinity selectin ligand PSGL‐1, inhibit leukocyte binding to P‐selectin in vitro. Here, we have used intravital microscopy to investigate whether GSP can inhibit P‐selectin‐dependent leukocyte rolling in vivo. Surgical preparation of the mouse cremaster muscle for intravital microscopy induced P‐selectin‐dependent leukocyte rolling. Baseline rolling was recorded for 1 min followed by i.v. injection of GSP. 2‐GSP‐6 and 4‐GSP‐6 substantially reversed P‐selectindependent leukocyte rolling, whereas control GSP, which are not fully glycosylated, did not. Inhibition of leukocyte rolling by 2‐ and 4‐GSP‐6 lasted 2–4 min. Clearance studies with 125Ilabeled 4‐GSP‐6 demonstrated rapid reduction in its circulating levels concurrent with accumulation in urine. These data represent the first demonstration that a precisely defined structure based on a natural P‐selectin ligand can inhibit P‐selectin‐dependent leukocyte rolling in vivo.