Model Glycosulfopeptides from P-selectin Glycoprotein Ligand-1 Require Tyrosine Sulfation and a Core 2-branched O-Glycan to Bind to L-selectin
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m303551200
ISSN1083-351X
AutoresAnne Leppänen, Tadayuki Yago, Vivianne I. Otto, Rodger P. McEver, Richard D. Cummings,
Tópico(s)Immune Response and Inflammation
ResumoL-selectin expressed on leukocytes is involved in lymphocyte homing to secondary lymphoid organs and leukocyte recruitment into inflamed tissue. L-selectin binds to the sulfated sialyl Lewis x (6-sulfo-sLex) epitope present on O-glycans of various glycoproteins in high endothelial venules. In addition, L-selectin interacts with the dimeric mucin P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. PSGL-1 lacks 6-sulfo-sLex but contains sulfated tyrosine residues (Tyr-SO3)at positions 46, 48, and 51 and sLex in a core 2-based O-glycan (C2-O-sLex) on Thr at position 57. The role of tyrosine sulfation and core 2 O-glycans in binding of PSGL-1 to L-selectin is not well defined. Here, we show that L-selectin binds to a glycosulfopeptide (GSP-6) modeled after the extreme N terminus of human PSGL-1, containing three Tyr-SO3 and a nearby Thr modified with C2-O-sLex. Leukocytes roll on immobilized GSP-6 in an L-selectin-dependent manner, and rolling is dependent on Tyr-SO3 and C2-O-sLex on GSP-6. The dissociation constant for binding of L-selectin to GSP-6, as measured by equilibrium gel filtration, is ∼5 μm. Binding is dependent on Tyr-SO3 residues as well as the sialic acid and fucose residues of C2-O-sLex. Binding to an isomeric glycosulfopeptide containing three Tyr-SO3 residues and a core 1-based O-glycan expressing sLex was reduced by ∼90%. All three Tyr-SO3 residues of GSP-6 are required for high affinity binding to L-selectin. Low affinity binding to mono- and disulfated GSPs is largely independent of the position of the Tyr-SO3 residues, except for some binding preference for an isomer sulfated on both Tyr-48 and -51. These results demonstrate that L-selectin binds with high affinity to the N-terminal region of PSGL-1 through cooperative interactions with three sulfated tyrosine residues and an appropriately positioned C2-O-sLex O-glycan. L-selectin expressed on leukocytes is involved in lymphocyte homing to secondary lymphoid organs and leukocyte recruitment into inflamed tissue. L-selectin binds to the sulfated sialyl Lewis x (6-sulfo-sLex) epitope present on O-glycans of various glycoproteins in high endothelial venules. In addition, L-selectin interacts with the dimeric mucin P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. PSGL-1 lacks 6-sulfo-sLex but contains sulfated tyrosine residues (Tyr-SO3)at positions 46, 48, and 51 and sLex in a core 2-based O-glycan (C2-O-sLex) on Thr at position 57. The role of tyrosine sulfation and core 2 O-glycans in binding of PSGL-1 to L-selectin is not well defined. Here, we show that L-selectin binds to a glycosulfopeptide (GSP-6) modeled after the extreme N terminus of human PSGL-1, containing three Tyr-SO3 and a nearby Thr modified with C2-O-sLex. Leukocytes roll on immobilized GSP-6 in an L-selectin-dependent manner, and rolling is dependent on Tyr-SO3 and C2-O-sLex on GSP-6. The dissociation constant for binding of L-selectin to GSP-6, as measured by equilibrium gel filtration, is ∼5 μm. Binding is dependent on Tyr-SO3 residues as well as the sialic acid and fucose residues of C2-O-sLex. Binding to an isomeric glycosulfopeptide containing three Tyr-SO3 residues and a core 1-based O-glycan expressing sLex was reduced by ∼90%. All three Tyr-SO3 residues of GSP-6 are required for high affinity binding to L-selectin. Low affinity binding to mono- and disulfated GSPs is largely independent of the position of the Tyr-SO3 residues, except for some binding preference for an isomer sulfated on both Tyr-48 and -51. These results demonstrate that L-selectin binds with high affinity to the N-terminal region of PSGL-1 through cooperative interactions with three sulfated tyrosine residues and an appropriately positioned C2-O-sLex O-glycan. Selectins are a family of cell adhesion molecules that act in concert with their glycoconjugate ligands to regulate lymphocyte recirculation and leukocyte recruitment into inflammatory sites (1McEver R.P. Moore K.L. Cummings R.D. J. Biol. Chem. 1995; 270: 11025-11028Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar). P-selectin is expressed on activated endothelial cells and activated platelets, E-selectin is expressed on activated endothelial cells, and L-selectin is constitutively expressed on various leukocyte subtypes. L-selectin mediates lymphocyte homing into secondary lymphoid organs and neutrophil recruitment into inflamed tissue (2Butcher E.C. Picker L.J. Science. 1996; 272: 60-66Crossref PubMed Scopus (2519) Google Scholar). High endothelial venules (HEV) 1The abbreviations used are: HEV, high endothelial venules; PSGL-1, P-selectin glycoprotein ligand-1; L-sel-Ig, L-selectin IgG chimera; P-sel-Ig, P-selectin IgG chimera; sLex, sialyl Lewis x (Siaα2–3Galβ1–4(Fucα1–3)GlcNAcβ1-R); 6-sulfo-sLex, Siaα2–3Galβ1–4(Fucα1–3)(6-sulfo)GlcNAcβ1-R; Lex, Lewis x (Galβ1–4(Fucα1–3)GlcNAcβ1-R); GP, glycopeptide; GSP, glycosulfopeptide; PFPL, polyfucosylated polylactosamine; C2-O-sLex, core 2-based O-glycan with sLex; C2-O-Lex-sLex, core 2-based O-glycan with Lex-sLex; C2-O-Lex-Lex-sLex, core 2-based O-glycan with Lex-Lex-sLex; C1-O-sLex, core 1-based O-glycan with sLex; Tyr-SO3- , tyrosine sulfate; MOPS, 4-(N-morpholine)propanesulfonic acid; GlyCAM-1, glycosylation-dependent cell adhesion molecule; mAb, monoclonal antibody; HPLC, high performance liquid chromatography; α1,3-FucTVI, α1,3-fucosyltransferase VI; BSA, bovine serum albumin. of secondary lymphoid organs express various glycoproteins that are bound by L-selectin and may be involved in lymphocyte homing (3Rosen S.D. Am. J. Pathol. 1999; 155: 1013-1020Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These include GlyCAM-1 (4Imai Y. Lasky L.A. Rosen S.D. Nature. 1993; 361: 555-557Crossref PubMed Scopus (332) Google Scholar, 5Imai Y. Rosen S.D. Glycoconj. J. 1993; 10: 34-39Crossref PubMed Scopus (39) Google Scholar), CD34 (6Baumhueter S. Dybdal N. Kyle C. Lasky L.A. Blood. 1994; 84: 2554-2565Crossref PubMed Google Scholar), MadCAM-1 (7Berg E.L. McEvoy L.M. Berlin C. Bargatze R.F. Butcher E.C. Nature. 1993; 366: 695-698Crossref PubMed Scopus (481) Google Scholar), Sgp200 (8Hemmerich S. Butcher E.C. Rosen S.D. J. Exp. Med. 1994; 180: 2219-2226Crossref PubMed Scopus (247) Google Scholar), and podocalyxin (9Sassetti C. Tangemann K. Singer M.S. Kershaw D.B. Rosen S.D. J. Exp. Med. 1998; 187: 1965-1975Crossref PubMed Scopus (220) Google Scholar). L-selectin also binds to PSGL-1 present on the surfaces of other leukocytes, thus mediating leukocyte attachment to already adherent cells, and increasing overall leukocyte recruitment to inflammatory sites (10Walcheck B. Moore K.L. McEver R.P. Kishimoto T.K. J. Clin. Invest. 1996; 98: 1081-1087Crossref PubMed Scopus (299) Google Scholar, 11Guyer D.A. Moore K.L. Lynam E.B. Schammel C.M. Rogelj S. McEver R.P. Sklar L.A. Blood. 1996; 88: 2415-2421Crossref PubMed Google Scholar, 12Spertini O. Cordey A.S. Monai N. Giuffre L. Schapira M. J. Cell Biol. 1996; 135: 523-531Crossref PubMed Scopus (184) Google Scholar, 13Eriksson E.E. Xie X. Werr J. Thoren P. Lindbom L. J. Exp. Med. 2001; 194: 205-218Crossref PubMed Scopus (171) Google Scholar). PSGL-1 is a dimeric, mucin-type glycoprotein ligand originally identified as a ligand for P-selectin (14Moore K.L. Stults N.L. Diaz S. Smith D.F. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (422) Google Scholar), but PSGL-1 also interacts with L- and E-selectin (15McEver R.P. Cummings R.D. J. Clin. Invest. 1997; 100: 485-491Crossref PubMed Google Scholar, 16Cummings R.D. Braz. J. Med. Biol. Res. 1999; 32: 519-528Crossref PubMed Scopus (58) Google Scholar). To date, however, detailed biochemical binding studies have only been carried out for P-selectin and PSGL-1. These studies have shown that P-selectin binds to the extreme N terminus of PSGL-1 by interacting stereospecifically with clustered tyrosine sulfates (Tyr-SO3- ) and a nearby core 2 O-glycan with a sialyl Lewis x (sLex; Siaα2–3Galβ1–4(Fucα1–3)GlcNAcβ1-R) epitope (C2-O-sLex) (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 18Leppanen A. Mehta P. Ouyang Y.B. Ju T. Helin J. Moore K.L. van Die I. Canfield W.M. McEver R.P. Cummings R.D. J. Biol. Chem. 1999; 274: 24838-24848Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 19Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). The use of synthetic glycosulfopeptides modeled after the N-terminal region of PSGL-1 was a key factor in elucidating the molecular requirements for P-selectin binding (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 18Leppanen A. Mehta P. Ouyang Y.B. Ju T. Helin J. Moore K.L. van Die I. Canfield W.M. McEver R.P. Cummings R.D. J. Biol. Chem. 1999; 274: 24838-24848Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 20Leppanen A. Penttila L. Renkonen O. McEver R.P. Cummings R.D. J. Biol. Chem. 2002; 277: 39749-39759Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). By contrast, the interaction between L-selectin and PSGL-1 has been studied less directly with blocking monoclonal antibodies and site-directed mutagenesis of recombinant PSGL-1. Early studies with blocking monoclonal antibodies indicated that L-selectin, like P-selectin, bound to the extreme N terminus of PSGL-1 (10Walcheck B. Moore K.L. McEver R.P. Kishimoto T.K. J. Clin. Invest. 1996; 98: 1081-1087Crossref PubMed Scopus (299) Google Scholar, 11Guyer D.A. Moore K.L. Lynam E.B. Schammel C.M. Rogelj S. McEver R.P. Sklar L.A. Blood. 1996; 88: 2415-2421Crossref PubMed Google Scholar, 12Spertini O. Cordey A.S. Monai N. Giuffre L. Schapira M. J. Cell Biol. 1996; 135: 523-531Crossref PubMed Scopus (184) Google Scholar, 21Tu L. Chen A. Delahunty M.D. Moore K.L. Watson S.R. McEver R.P. Tedder T.F. J. Immunol. 1996; 157: 3995-4004PubMed Google Scholar). This region contains sulfate on tyrosine residues but no sulfate on glycans. However, L-selectin does bind to sulfated carbohydrate ligands on various HEV mucins, such as 6-sulfo-sLex (Siaα2–3Galβ1–4(Fucα1–3)(6-sulfo)GlcNAcβ1-R), an epitope recognized by the mAb MECA-79 (8Hemmerich S. Butcher E.C. Rosen S.D. J. Exp. Med. 1994; 180: 2219-2226Crossref PubMed Scopus (247) Google Scholar, 22Imai Y. Singer M.S. Fennie C. Lasky L.A. Rosen S.D. J. Cell Biol. 1991; 113: 1213-1221Crossref PubMed Scopus (255) Google Scholar, 23Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Surprisingly, a recent study of cells expressing recombinant PSGL-1 suggested that PSGL-1 requires 6-sulfo-sLex determinants rather than tyrosine sulfation to support L-selectin-dependent leukocyte rolling (24Kanamori A. Kojima N. Uchimura K. Muramatsu T. Tamatani T. Berndt M.C. Kansas G.S. Kannagi R. J. Biol. Chem. 2002; 277: 32578-32586Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). This contrasts with earlier observations that L-selectin binds poorly to recombinant PSGL-1 in which the tyrosines are replaced with phenylalanines (25Bernimoulin M.P. Zeng X.L. Abbal C. Giraud S. Martinez M. Michielin O. Schapira M. Spertini O. J. Biol. Chem. 2003; 278: 37-47Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (121) Google Scholar). It has also been proposed that L-selectin recognizes nonsulfated sLex on both core 2 and extended core 1 O-glycans (26Mitoma J. Petryniak B. Hiraoka N. Yeh J.C. Lowe J.B. Fukuda M. J. Biol. Chem. 2003; 278: 9953-9961Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Mutagenesis studies have suggested that Thr-57 is the key O-glycosylation site on PSGL-1 for binding to L-selectin (25Bernimoulin M.P. Zeng X.L. Abbal C. Giraud S. Martinez M. Michielin O. Schapira M. Spertini O. J. Biol. Chem. 2003; 278: 37-47Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (121) Google Scholar). The present study was designed to directly measure the importance of tyrosine sulfation and O-glycosylation at Thr-57 for binding of L-selectin to PSGL-1, utilizing synthetic glyco-(sulfo)peptides (GSPs) modeled after the N terminus of human PSGL-1, and to measure the binding affinity between the GSPs and L-selectin. To this end, we synthesized a set of GSPs containing one, two, three, or no sulfated tyrosine residues and C2-O-sLex at Thr-57. This approach not only allowed us to study the role of tyrosine sulfation but also the stereospecific contribution of individual Tyr-SO3- residues for binding to L-selectin. We also synthesized GSPs containing three Tyr-SO3- residues and a modified O-glycan at Thr-57 to study the role of specific monosaccharide residues of C2-O-sLex, of sialylated polyfucosylated polylactosamine O-glycan and of sialyl Lewis x on extended core 1 O-glycan (C1-O-sLex) for binding to L-selectin. The interaction of L-selectin with GSPs was studied using multiple approaches, including a fluorescence-based solid phase assay, equilibrium gel filtration, and in vitro rolling experiments. Our results demonstrate that L-selectin binds with relatively high affinity to GSPs that contain sulfate on all three tyrosines and that present sLex on a core 2 rather than on an extended core 1 O-glycan. Enzymatic Synthesis of Glycosulfopeptides—Glyco(sulfo)peptide precursors corresponding to amino acid residues 45–61 of human PSGL-1 with a GalNAcα residue at Thr-57 and no, one, two or three Tyr-SO3- residues (Tyr-46, -48, and 51) were synthesized on an automated peptide synthesizer as described (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The peptides were purified on a reversed phase HPLC and characterized by mass spectrometry as described (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The O-glycan at Thr-57 of each peptide was synthesized enzymatically using highly purified or recombinant glycosyltransferases. GP-4, GP-6, GSP-1, GSP-5, GSP-6, GSP(46)-6, GSP(48)-6, GSP(51)-6, GSP(46,48)-6, GSP(46,51)-6, GSP(48,51)-6, and DS-GSP-6 were synthesized and characterized as described (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). GSP-6′ and GSP-6″ were synthesized and characterized as described (20Leppanen A. Penttila L. Renkonen O. McEver R.P. Cummings R.D. J. Biol. Chem. 2002; 277: 39749-39759Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). sLex on extended core 1 in C1-GSP-6 and C1-GP-6 were synthesized as described (18Leppanen A. Mehta P. Ouyang Y.B. Ju T. Helin J. Moore K.L. van Die I. Canfield W.M. McEver R.P. Cummings R.D. J. Biol. Chem. 1999; 274: 24838-24848Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Radiolabeled [3H]GSP-6 was synthesized using nonlabeled GSP-5 as an acceptor and GDP-[3H]Fuc (American Radiolabeled Chemicals Inc., St. Louis, MO) (specific activity 950 or 8480 cpm/pmol) as a donor in a α1,3-FucTVI (Calbiochem) reaction. [3H]GSP-6 was purified from the reaction mixture by reversed phase HPLC. Recombinant Selectin-Ig Chimeras—The vectors that were used to express soluble P- and L-selectin-Ig chimeric proteins were a gift from Dr. Ajit Varki (University of California, San Diego) (28Koenig A. Jain R. Vig R. Norgard-Sumnicht K.E. Matta K.L. Varki A. Glycobiology. 1997; 7: 79-93Crossref PubMed Scopus (94) Google Scholar). P- and L-selectin-Ig chimeric proteins were expressed in 293 cells and purified from the media using Protein A-Sepharose as described (29Moll T. Vestweber D. Methods Mol. Biol. 1999; 96: 77-84PubMed Google Scholar). The purity and homogeneity of the purified selectin-Ig chimeras were analyzed by reducing and non-reducing SDS-PAGE, followed by Coomassie Blue staining and Western blotting. All preparations of P- and L-selectin-Ig chimeras were found to be >90% pure and homogeneously dimeric, showing a molecular weight of ∼190,000. Biotinylation of Glyco(sulfo)peptides and Fluorescence-based Solid Phase Assay—Biotinylation of the C-terminal Cys of each GSP was performed using biotin-HPDP (N-(6-[biotinamido]hexyl)-3′-(2-pyridyldithio)propionamide) (Pierce) as described (20Leppanen A. Penttila L. Renkonen O. McEver R.P. Cummings R.D. J. Biol. Chem. 2002; 277: 39749-39759Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Biotinylated GSPs were dissolved in 20 mm MOPS, pH 7.5, containing 150 mm NaCl, and the concentration of each peptide solution was determined by UV absorbance at 215 nm of a sample subjected to HPLC. Fluorescence-based solid phase assay was performed essentially as described (20Leppanen A. Penttila L. Renkonen O. McEver R.P. Cummings R.D. J. Biol. Chem. 2002; 277: 39749-39759Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, streptavidin-coated black 96-well microtiter plates (Pierce) were washed 3 times with 200 μl of 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 0.02% NaN3 (buffer A) or 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 5 mm EDTA, 0.02% NaN3 (buffer B) and coated for 1.5 h with 1–30 pmol of GSPs in 50 μl of buffer A or B. The wells were then incubated for 1 h with 50 μl of P-sel-Ig chimera (0.5–10 μg/ml), L-sel-Ig chimera (1–30 μg/ml), or anti-PSGL-1 mAb PL1 (2 or 5 μg/ml) (30Li F. Erickson H.P. James J.A. Moore K.L. Cummings R.D. McEver R.P. J. Biol. Chem. 1996; 271: 6342-6348Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) in buffer A or B containing 0.05% Tween 20 and 1% BSA. The wells were subsequently incubated for 1 h with 50 μl of 10–50 μg/ml Alexa Fluor™ 488 goat anti-human IgG (H+L) or with 50 μlof5or10 μg/ml Alexa Fluor™ 488 goat anti-mouse IgG (H+L) (Molecular Probes, Inc., Eugene, OR) in buffer A or B containing 0.05% Tween 20 and 1% BSA. After a final washing, 100 μl of buffer A or B was added to each well and the fluorescence was measured using a Victor2 (Wallac, Turku, Finland) or Tecan Ultra384 (Tecan U.S., Durham, NC) microtiter plate reader with excitation wavelength at 485 nm and emission wavelength at 535 nm. Peptide coating and all incubations were performed at room temperature, and the wells were washed 3 times using buffer A or B containing 0.05% Tween 20. The assays of Figs. 2 and 3 were performed in duplicate, of Figs. 6 and 7 in triplicate, and the results represent averages of two or three determinations, respectively. Background fluorescence reading without peptide coating was subtracted from each sample in each experiment.Fig. 3Binding of L-sel-Ig and P-sel-Ig to different densities of immobilized GSP-6 in a fluorescence-based solid phase assay. Biotinylated GSP-6 was immobilized on streptavidin-coated microtiter wells at different coating densities (see figure). A fixed concentration of L-sel-Ig (A, 10 μg/ml) or P-sel-Ig (B, 5 μg/ml) was incubated with the immobilized GSP-6 in 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 1% BSA, 0.05% Tween 20, and 0.02% NaN3. Fluorescently labeled anti-human IgG (50 μg/ml) was used to detect the bound selectin-Ig chimeras. All assays were performed in duplicate, and the results represent the average of two determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Comparison of binding of L-sel-Ig and P-sel-Ig to different immobilized glyco(sulfo)peptides in a fluorescence-based solid phase assay. Biotinylated glyco(sulfo)peptides were immobilized on streptavidin-coated microtiter wells (A, 10 pmol/well; B, 1 pmol/well). L-sel-Ig (A, 10 μg/ml) or P-sel-Ig (B, 1 μg/ml) was incubated with the immobilized GSPs in either 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 1% BSA, 0.05% Tween 20, and 0.02% NaN3 (light gray bars)orin20mm MOPS, pH 7.5, containing 150 mm NaCl, 5 mm EDTA, 1% BSA, 0.05% Tween 20, and 0.02% NaN3 (dark gray bars). Fluorescently labeled anti-human IgG was used for detection. Monoclonal antibody PL1 was used to confirm that equal amounts of each GSP was immobilized on microtiter wells (not shown). The data of panel A is from one representative experiment of three independent experiments. All assays were performed in triplicate, and the results represent the mean ± S.D. of three determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Binding of L-sel-Ig and P-sel-Ig to immobilized glyco-(sulfo)peptides carrying sLex on extended core 1 O-glycan in a fluorescence-based solid phase assay. Biotinylated GSPs were immobilized on streptavidin-coated microtiter wells (A, 10 pmol/well; B, 1 pmol/well). L-sel-Ig (A, 5 μg/ml) or P-sel-Ig (B, 5 μg/ml) was incubated with immobilized GSPs in 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 1% BSA, 0.05% Tween 20, and 0.02% NaN3. Fluorescently labeled anti-human IgG (10 μg/ml) was used to detect the bound selectin-Ig chimeras. Monoclonal antibody PL1 was used to confirm that equal amounts of each GSP was immobilized on microtiter wells (not shown). All assays were performed in triplicate, and the results represent the mean ± S.D. of three determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Equilibrium Gel Filtration Chromatography—Hummel-Dreyer equilibrium gel filtration experiments (31Hummel J.P. Dreyer W.J. Biochem. Biophys. Acta. 1962; 63: 530-532Crossref PubMed Scopus (934) Google Scholar, 32Akers G.K. Methods Enzymol. 1973; 27: 441-455Crossref PubMed Scopus (36) Google Scholar) with [3H]GSP-6 and L-sel-Ig/P-sel-Ig were conducted in a 2-ml Sephadex G-100 column (0.5 × 10 cm) at physiological salt concentration (150 mm NaCl) or subphysiological salt concentration (50 mm NaCl) in 20 mm MOPS, pH 7.5, containing 2 mm CaCl2, 2 mm MgCl2, 0.02% NaN3 as described (18Leppanen A. Mehta P. Ouyang Y.B. Ju T. Helin J. Moore K.L. van Die I. Canfield W.M. McEver R.P. Cummings R.D. J. Biol. Chem. 1999; 274: 24838-24848Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Various concentrations of L-sel-Ig or P-sel-Ig were applied to the column equilibrated with [3H]GSP-6 (1.3 or 5.6 pmol/ml). Rolling of Neutrophils on Glyco(sulfo)peptides—A 30-μl drop of streptavidin (50 μg/ml) was placed into a demarcated area on a 35-mm tissue culture plate (Corning, Corning, NY) and incubated at 4 °C overnight. The area was washed twice with Hanks' balanced salt solution and then blocked with Hanks' balanced salt solution containing 1% human serum albumin at room temperature for 2 h. Biotinylated GSPs (GSP-6, GSP-1, GP-6, and GP-4) were captured to the adsorbed streptavidin by incubation at 4 °C for 1 h. Site densities of GSPs were determined by binding of radiolabeled anti-PSGL-1 mAb PL1 (33Yago T. Leppanen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar). Human neutrophils were isolated from healthy donors (34Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1985; 76: 2235-2246Crossref PubMed Scopus (278) Google Scholar). Neutrophils (106/ml in Hanks' balanced salt solution containing 0.5% human serum albumin) were perfused over GSPs on 35-mm plates in a parallelplate flow chamber. After 5 min, the accumulated number of rolling cells was measured with a video microscopy system coupled to a digitized image analysis system (Inovision) (27Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (121) Google Scholar, 33Yago T. Leppanen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar). In some experiments, neutrophils were perfused in the presence of 20 μg/ml anti-PSGL-1 mAb PL1 or PL2 (35Moore K.L. Patel K.D. Bruehl R.E. Li F. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (629) Google Scholar), anti-L-selectin mAb DREG-56 or isotype mouse IgG1 mAb. Anti-human L-selectin mAb DREG-56 was purified from hybridomas obtained from American Type Culture Collection (ATCC). An isotype control mouse IgG1 was purchased from BD Pharmingen. Synthesis of Glycosulfopeptides—To study the role of tyrosine sulfation and O-glycosylation of PSGL-1 for binding to L-selectin, we synthesized a series of GSPs corresponding to N-terminal amino acid residues 45–61 of human PSGL-1 (Fig. 1). The GSPs were synthesized chemoenzymatically as described earlier (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 20Leppanen A. Penttila L. Renkonen O. McEver R.P. Cummings R.D. J. Biol. Chem. 2002; 277: 39749-39759Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, the peptide backbones with no Tyr-SO3- or one, two, or three Tyr-SO3- residues and a GalNAc residue at Thr-57 were synthesized on an automated peptide synthesizer. The peptides were cleaved from the solid support, purified by HPLC, and characterized by mass spectrometry as described (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). O-Glycan at Thr-57 was synthesized enzymatically using recombinant or purified glycosyltransferases, and the final products were characterized by mass spectrometry (17Leppanen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 20Leppanen A. Penttila L. Renkonen O. McEver R.P. Cummings R.D. J. Biol. Chem. 2002; 277: 39749-39759Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The GSP-6 series contained one, two, or three Tyr-SO3- residues at Tyr-46, -48, and/or -51 and a simple C2-O-sLex at Thr-57. GSP-6′ and GSP-6″ contained three Tyr-SO3- residues and two (C2-O-Lex-sLex) or three (C2-O-Lex-Lex-sLex) fucosylated lactosamine repeats at Thr-57, respectively. Some control peptides were not sulfated (GP-6 and GP-4). Incompletely glycosylated but fully sulfated GSPs were derivatives of GSP-6 (DS-GSP-6, GSP-5, and GSP-1). Fully sulfated C1-GSP-6 and nonsulfated C1-GP-6 were isomers of GSP-6 and GP-6, respectively, containing the sLex epitope on an extended core 1 branch instead of a core 2 branch. L-selectin Binds to Immobilized GSP-6 and Binding Affinity Is Dependent on the Ligand Density—We first used a newly developed and sensitive fluorescence-based solid phase assay to compare the relative binding affinities of L-selectin and P-selectin to GSP-6. Biotinylated GSP-6 (0.25–10 pmol/well) was first captured quantitatively on streptavidin-coated 96-well plates. Different amounts of L-sel-Ig or P-sel-Ig were incubated in the wells, and bound L-sel-Ig and P-sel-Ig were detected with fluorescently labeled anti-human IgG. At the highest GSP-6 coating density (10 pmol/well), binding of L-sel-Ig to GSP-6 increased linearly with increased concentration of L-sel-Ig, reaching a plateau at 10–30 μg/ml of L-sel-Ig (Fig. 2A). However, at lower GSP-6 densities, no plateau was observed even at the highest concentrations of L-sel-Ig. This shows that the affinity of L-sel-Ig for immobilized GSP-6 is dependent on the ligand density. By contrast, P-sel-Ig generated a saturated binding curve with all GSP-6 coating densities used (Fig. 2B), indicating that the affinity of P-sel-Ig for GSP-6 is less dependent on the density of the immobilized ligand. L-selectin Binds to Immobilized GSP-6 with ∼10-fold Reduced Affinity Compared with P-selectin at Physiological Salt Concentration—Binding affinities of L-sel-Ig and P-sel-Ig for immobilized GSP-6 were first compared using the fluorescence-based solid phase assay. Biotinylated GSP-6 was immobilized on streptavidin-coated plates at different densities, and fixed concentrations of L-sel-Ig (10 μg/ml) and P-sel-Ig (5 μg/ml) were incubated with wells containing varying amounts of GSP-6. Binding of L-sel-Ig to increasing densities of GSP-6 formed a semi-sigmoidal binding curve (Fig. 3A), suggesting that L-sel-Ig binding to immobilized GSP-6 may be cooperative. By contrast, P-sel-Ig bound to GSP-6 forming a typical rectangular hyperbola binding curve (Fig. 3B). Comparison of the GSP-6 densities that give half-maximal binding for L-sel-Ig (∼10 pmol) and P-sel-Ig (∼1 pmol) indicates that L-sel-Ig has ∼10-fold lower affinity for immobilized
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