The P-selectin Glycoprotein Ligand Functions as a Common Human Leukocyte Ligand for P- and E-selectins

Abstract

P- and E-selectins belong to a family of Ca2+-depend-ent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca2+-dependent manner. This protein has a molecular mass of –120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P-selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E-selectins. P- and E-selectins belong to a family of Ca2+-depend-ent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca2+-dependent manner. This protein has a molecular mass of –120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P-selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E-selectins. P-selectin (CD62P) and E-selectin (CD62E) are members of the selectin family of leukocyte adhesion molecules (1McEver R.P. Thromb. Haemostasis. 1991; 65: 223-228Crossref PubMed Scopus (228) Google Scholar, 2Hynes R.O. Lander A.D. Cell. 1992; 68: 303-332Abstract Full Text PDF PubMed Scopus (761) Google Scholar, 3Lasky L.A. Science. 1992; 258: 964-969Crossref PubMed Scopus (1151) Google Scholar, 4Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7390-7397Crossref PubMed Scopus (952) Google Scholar, 5Bevilacqua M.P. Nelson R.M. J. Clin. Invest. 1993; 91: 379-387Crossref PubMed Scopus (1059) Google Scholar). Both P- and E-selectins mediate leukocyte adhesion to endothelial cells under vascular shear stress in capillary venules. P-selectin also mediates rosetting of activated platelets onto leukocytes (1McEver R.P. Thromb. Haemostasis. 1991; 65: 223-228Crossref PubMed Scopus (228) Google Scholar, 2Hynes R.O. Lander A.D. Cell. 1992; 68: 303-332Abstract Full Text PDF PubMed Scopus (761) Google Scholar, 3Lasky L.A. Science. 1992; 258: 964-969Crossref PubMed Scopus (1151) Google Scholar, 4Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7390-7397Crossref PubMed Scopus (952) Google Scholar, 5Bevilacqua M.P. Nelson R.M. J. Clin. Invest. 1993; 91: 379-387Crossref PubMed Scopus (1059) Google Scholar). P-selectin has been shown to bind to an ∼240-kDa homodimeric sialoglycoprotein (–120 kDa under reducing conditions) from myeloid cells with high affinity (6Moore 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 (421) Google Scholar, 7Norgard K.E. Moore K.L. Diaz S. Stults N.L. Ushiyama S. McEver R.P. Cummings R.D. Varki A. J. Biol. Chem. 1993; 268: 12764-12774Abstract Full Text PDF PubMed Google Scholar, 8Moore K.L. Eaton S.F. Lyons D.E. Lichenstein H.S. Cummings R.D. McEver R.P. J Biol. Chem. 1994; 269: 23318-23327Abstract Full Text PDF PubMed Google Scholar). Recently, a mucin-like transmembrane protein, called PSGL-1, 1The abbreviations used here are: PSGL-1, P-selectin glycoprotein ligand-1; P/ESGL-1, P- and E-selectin glycoprotein ligand-1; Rg, receptor-globulin; mAb, monoclonal antibody; PBS, phosphate-buffered saline at pH 7.4; PAGE, Polyacrylamide gel electrophoresis; IgG, immunoglobulin G; BSA, bovine serum albumin; SLe", Siaa2–3Galβ1-4(Fucal-3)GlcNAc; HBSS, Hank's balanced salt solution with CaCl2 and MgCl2.1The abbreviations used here are: PSGL-1, P-selectin glycoprotein ligand-1; P/ESGL-1, P- and E-selectin glycoprotein ligand-1; Rg, receptor-globulin; mAb, monoclonal antibody; PBS, phosphate-buffered saline at pH 7.4; PAGE, Polyacrylamide gel electrophoresis; IgG, immunoglobulin G; BSA, bovine serum albumin; SLe", Siaa2–3Galβ1-4(Fucal-3)GlcNAc; HBSS, Hank's balanced salt solution with CaCl2 and MgCl2. has been cloned (9Sako D. Chang X.-J. Barone K.M. Vachino G. White H.M. Shaw G. Veldman G.M. Bean K.M. Ahern T.J. Furie B. Cumming D.A. Larsen G.R. Cell. 1993; 75: 1179-1186Abstract Full Text PDF PubMed Scopus (643) Google Scholar). COS cells co-transfected with PSGL-1 and a(l,3/4)fucosyltransferase cDNAs express a homodimer of –240 kDa and support leukocyte adhesion in a P-selectin-dependent manner. We have previously reported the development of a panel of monoclonal antibodies which recognize an ∼240-kDa molecule from human neutrophils and HL-60 cells and inhibit adhesion of these cells to P-selectin (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). These results strongly suggest that the 240-kDa molecule is a leukocyte ligand for P-selectin. A number of leukocyte ligands from various species have been described for P- and E-selectin. E-selectin, but not P-selectin, recognizes an ∼150-kDa monomelic glycoprotein from mouse myeloid cells metabolically labeled with [35S]methionine and [35S]cysteine (11Levinovitz A. Muhlhoff J. Isenmann S. Vestweber D. J. Cell Biol. 1993; 121: 449-459Crossref PubMed Scopus (168) Google Scholar, 12Lenter M. Levinovitz A. Isenmann S. Vestweber D. J. Cell Biol. 1994; 125: 471-481Crossref PubMed Scopus (167) Google Scholar). This binding is Ca2+-dependent and can be blocked by an antibody to mouse E-selectin. In contrast, the mature mouse neutrophils contain glycoproteins of 230 kDa and 130 kDa, which bind to both P- and E-selectins in a Ca2+-dependent, sialidase-sensitive fashion (12Lenter M. Levinovitz A. Isenmann S. Vestweber D. J. Cell Biol. 1994; 125: 471-481Crossref PubMed Scopus (167) Google Scholar). A single glycoprotein (250 kDa under nonreducing conditions and 280 kDa under reducing conditions), isolated from bovine γ/δ T cells, binds to human E-selectin in a Ca2+-dependent, sialidase-sensitive manner (13Walcheck B. Watts G. Jutila M.A. J. Exp. Med. 1993; 178: 853-863Crossref PubMed Scopus (82) Google Scholar). Furthermore, P-selectin, but not E-selectin, recognizes an ∼160-kDa monomelic glycoprotein from mature mouse neutrophils and human HL-60 cells (12Lenter M. Levinovitz A. Isenmann S. Vestweber D. J. Cell Biol. 1994; 125: 471-481Crossref PubMed Scopus (167) Google Scholar). There are some experimental results which suggest that the P-selectin glycoprotein ligand might function as a ligand for E-selectin. First, the conditioned medium from COS cells cotransfected with both the truncated soluble form of PSGL-1 and α(1,3/4)fucosyltransferase cDNAs mediates adhesion to radiolabeled CHO-P-selectin cells and CHO-E-selectin cells. In both cases, the cell adhesion is Ca2+-dependent and inhibitable with respective neutralizing antibodies (9Sako D. Chang X.-J. Barone K.M. Vachino G. White H.M. Shaw G. Veldman G.M. Bean K.M. Ahern T.J. Furie B. Cumming D.A. Larsen G.R. Cell. 1993; 75: 1179-1186Abstract Full Text PDF PubMed Scopus (643) Google Scholar). Second, 125I-PSGL-1 from human neutrophils binds directly to truncated soluble forms of P- and E-selectins in a Ca2+-dependent manner (8Moore K.L. Eaton S.F. Lyons D.E. Lichenstein H.S. Cummings R.D. McEver R.P. J Biol. Chem. 1994; 269: 23318-23327Abstract Full Text PDF PubMed Google Scholar). However, the binding affinity for E-selectin (IC50 ∼25 μΜ) is at least 50-fold weaker than that for P-selectin (IC50 ∼ 0.5 μM). Taken together, these results suggest that E-selectin recognizes several glycoproteins with different molecular masses including ∼130-kDa, ∼150-kDa, and ∼230-kDa monomeric molecules in mouse neutrophils, an ∼250-kDa monomeric molecule from bovine γ/δ T cells, and an ∼240-kDa dimeric molecule (P-selectin glycoprotein ligand) from human neutrophils and HL-60 cells (8Moore K.L. Eaton S.F. Lyons D.E. Lichenstein H.S. Cummings R.D. McEver R.P. J Biol. Chem. 1994; 269: 23318-23327Abstract Full Text PDF PubMed Google Scholar, 11Levinovitz A. Muhlhoff J. Isenmann S. Vestweber D. J. Cell Biol. 1993; 121: 449-459Crossref PubMed Scopus (168) Google Scholar, 12Lenter M. Levinovitz A. Isenmann S. Vestweber D. J. Cell Biol. 1994; 125: 471-481Crossref PubMed Scopus (167) Google Scholar, 13Walcheck B. Watts G. Jutila M.A. J. Exp. Med. 1993; 178: 853-863Crossref PubMed Scopus (82) Google Scholar). However, it is not known whether the P-selectin glycoprotein ligand is the only high affinity ligand recognized by E-selectin in human neutrophils and HL-60 cells. Moreover, the biological importance of the P-selectin glycoprotein ligand on the cell surface of human neutrophils and HL-60 cells as a ligand in E-selectin-mediated leukocyte adhesion remains unclear. In the present study, we show that only one radiolabeled glycoprotein is purified specifically by E-selectin Rg affinity chromatography. The molecular mass and O-linked carbohydrate profile of this molecule are identical with those of the P-selectin glycoprotein ligand isolated by P-selectin affinity chromatography. This molecule is recognized by PL5 mAb, E-selectin Rg, and PSGL-1 antiserum. Furthermore, PL5, a monoclonal IgM antibody raised against the P-selectin ligand (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar), also blocks adhesion of human neutrophils and HL-60 cells to E-selectin. Thus, our results suggest that this glycoprotein is an important human leukocyte ligand for both P- and E-selectins. DIG Glycan Detection Kit and Arthrobacter ureafaeiens sialidase (EC 3.2.1.18) were purchased from Bochringer Mannheim. BBA1 mAb to E-selectin was obtained from R&D Systems. Purified human IgG was purchased from Sigma. Leu-8 mAb to L-selectin was obtained from Breton Dickinson. ImmunoPure IgG Orientation Kit and Micro BCA Kit for measurement of protein concentrations were purchased from Pierce. Affi-Gel 15 Sepharose was obtained from Bio-Rad. Dialysis membrane tubing (12-kDa to 14-kDa molecular mass cut-off) was purchased from Spectra-Pore and used throughout this study. [3H]Glucosamine (20–40 Ci/mmol) was purchased from Amersham or DuPont NEN. SepPak C1H cartridges were obtained from Waters. Supcrdex 200 column and Smart System were purchased from Pharmacia Biotech Inc. All other chemicals were purchased as described previously (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). P-selectin was purified from outdated human platelets as published (11Levinovitz A. Muhlhoff J. Isenmann S. Vestweber D. J. Cell Biol. 1993; 121: 449-459Crossref PubMed Scopus (168) Google Scholar). P-selectin Rg was prepared as described previously.2 E-selectin Rglthe lectin-epidermal growth factor-repeat domains 1 to 6 of E-selectin exoplasmic portion fused with the hinge, CH2, and CH3 domains of human IgGl) was constructed as described (15Walz G. Aruffo A. Kolanus W. Bevilacqua M. Seed B. Science. 1990; 250: 1132-1135Crossref PubMed Scopus (886) Google Scholar).2 The establishment of a stable CHO cell line secreting E-selectin Rg, the serum-free tissue culture of this CHO cell line, and the purification of E-selectin Rg werr exactly carried out as described. 2L. Ma, K.-C. Chou, P. S. Kaytes, B. A. Leone, C. F. Toombs, D. J. Staples, M. S. Babcock, H. D. Fischer, C. W. Smith, and J.-G. Geng, submitted loi- publication. The biotinvlated BBA1 mAb to E-selectin was used (instead of biotinvlated P7 mAb to P-selectin) for sandwich enzyme-linked immunosorbent assay to screen the high secreting clones. The purified E-selectin Rg was dialvzed against PBS, and samples were subject to SDS-PAGE followed by Coomassie Blur staining, as shown in Fig. 1. E-selectin thus prepared mediated a Ca2 '-dependent adhesion of HL-60 cells and human neutrophils (see below). The sample of E-selectin Rg was analyzed by NH.,-terminaI amino acid sequencing with following sequence: WSY.YT-STEAM TYDEASAYXQ QRYTHXVAIQ NK (X: ambiguous residue), fully consistent with the protein sequence of human E-selectin < 16). The EJJi for E-selectin Rg is 15.1 calculated according to the results of acid hydrolysis and compositional analysis of amino acids. Monoclonal IgG antibodies against P-selectin including an adhesion-blocking mAb, P7. an adhesion nonblocking mAb, P23, and a monoclonal IgM antibody against a P-selectin glycoprotein ligand. PL5, were prepared and characterized as described 110), Rabbit antiserum to PSGL-1 was prepared by hyperimmunization of the rabbit with purified recombinant PSGL-1 receptor-globulin. This antiserum was kindly provided by Dr. Dietmar Vestweber (Hans Spermann laboratory at the Max Planck Institute for Immunology. Germany!. CSLEX monoclonal IgM antibody to SLe" was purified from mouse ascites as described previously 110). The purified E-selectin Rg was used as the antigen in primary and booster immunization with BALB/c mice as described 110). Twelve weeks later, the spleen cells of the two mice were fused with myeloma cells. The hybridomas were screened for recognition of E-selectin Rg using an enzyme-linked immunosorbent assay (17Geng J.-G. Moore K.L. Johnson A.E. McEver R.P. J. Biol. Chem. 1991; 266: 22313-22318Abstract Full Text PDF PubMed Google Scholar, 18Geng J.-G. Heavner G.A. McEver R.P. J. Biol. Chem. 1992; 267: 19846-19853Abstract Full Text PDF PubMed Google Scholar). As the immunogen. E-selectin Rg, contained a Kc fragment (without the CH1 domain) of human IgGl, the hybridoma supernatants were preincubated with human IgG at 37 °C for 1 h (0.1 mg/ml final concentration of human IgG) before transferring them into the microtiter wells immobilized with E-selectin Rg to avoid generation of antibodies against the heavy chain of human IgGl. After subcloning twice, we obtained a panel of four hybridomas (El, E2, E3, E4; designated as E for E-selectin). The ascites from individual hybridomas were prepared for purification of antibodies using Protein A chromatography (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). Each of these antibodies recognized E-selectin Rg but did not recognize P-selectin Rg or human IgG in an enzyme-linked immunosorbent assay (data not shown). They also bound to CHO cells expressing full-length E-selectin but did not bind to CHO cells expressing full-length P-selectin or throm-bin-activated platelets using a flow cytometric assay (data not shown). Furthermore, these antibodies did not recognize Jurkat T-lymphocytes whereas Leu-8 mAb to L-selectin did recognize these cells (data not shown). HL-60 cells were metabolically labeled with [3H]glucosamine (50 μCi/ml) in RPMI medium containing 10% fetal calf serum for 48 to 72 h as described previously (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). The P-selectin affinity column was prepared by coupling 10 mg of platelet P-selectin directly onto 10 ml of Affi-Gel 15 Sepharose (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). The E-selectin Rg affinity column was prepared by cross-linking 5 mg of E-selectin Rg onto 2 ml of Protein A resin by dimethyl pimelimidate according to the recommendations of the ImmunoPure IgG Orientation Kit (Pierce). A BSA-Sepharose column (10-ml resin) was used as a guard column for P-selectin and E-selectin Rg affinity columns. For specificity control, the human IgG columns were prepared by cross-linking 10 mg of human IgG onto 2 ml of Protein A resin for each column prepared exactly as the E-selectin Rg column described above. The glycoprotein ligands were purified using an EDTA elution of P-selectin affinity column or E-selectin Rg affinity column as published previously (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). The eluted fractions were pooled together and separately placed into dialysis tubing which had been treated with 0.1% BSA (w/v) to lower nonspecific binding. Samples were dialyzed against H20 at 4 °C overnight. After dialysis, they were frozen in a dry ice/acetone bath and lyophilized in glass tubes to dryness. The lyophilized samples were redissolved in a small volume of PBS, and the radioactivities were counted using a Packard liquid scintillation counter (low level detection). This one-step affinity purification could yield P- and E-selectin ligands radiochemically pure as assessed by SDS-PAGE and autoradiography. To demonstrate cross-depletion of the glycoprotein ligands for P- and E-selectins, the [3H]glucosamine-labeled cell lysates from 2 × 107 cells (labeled with 20 mCi of isotope) were prepared as described above. Two equal aliquote of the cell lysates were applied to the BSA guard column first and then separately applied to P- and E-selectin columns. After washing, the bound materials were eluted from the P-selectin or E-selectin Rg affinity column as described above. The breakthrough from the P-selectin affinity column was reapplied to the E-selectin Rg affinity column, while the breakthrough from the E-selectin Rg affinity column was reapplied to the P-selectin affinity column. After washing, the bound materials were eluted with EDTA as above. Aliquots of each eluted fraction were counted, and radioactivities of these fractions were plotted. The radioactive fractions were pooled, dialyzed, and analyzed by 7% SDS-PAGE and autoradiography as above. The cross-binding properties of the ∼120-kDa molecule on P- and E-selectin affinity columns were demonstrated as follows. The radioactive ligands were purified separately from [3H]glucosamine-labeled HL-60 cells to apparent homogeneity by P- or E-selectin affinity chromatography as above (data not shown I. The purified ligands were dialyzed individually into the buffer containing 1 m.M CaCI., The K-selcctin column-purified ligand (10,000 Cpm) wet reapplied to the P-selectin column, while the P-selectin column-purified ligand (10,000 cpm) was reapplied to the E-selectin column. After washing, the bound ligands were eluted with EDTA from these columns and the radioactivities were counted and plotted. For specificity control, the purified P- and E-selectin ligands (10,000 cpm for each ligand molecule) were separately loaded onto human IgG columns. Following washing, the radioactivities of the EDTA elution fractions were counted. The radiochemically pure glycoprotein ligands isolated by the P- or E-selectin column were precipitated separately with methanol/chloroform (19Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3141) Google Scholar) to remove salts and detergent. Following evaporation to dryness, the O-linked oligosaccharides were released by mild alkaline sodium borohydride treatment (20Carlson D.M. J. Biol. Chem. 1968; 243: 616-626Abstract Full Text PDF PubMed Google Scholar). The released oligosaccharides were dissolved in H2O and washed through SepPak C18 cartridges to remove polypeptide fragments. The released O-linked oligosaccharides were digested with A. ureafaciens sialydase (2 units/ml) in 100 mM acetate buffer, pH 5.0, for 18 h. The desialylated O-linked oligosaccharide "fingerprints" from the molecules isolated on the P- and E-selectin columns were obtained by separating the desialylated oligosaccharides on size exclusion chromatography (GlycoMap Chromatograph, Oxford GlycoSystems). A Bio-Gel P-4 type column (Glycan Sizing Column. Oxford GlycoSystems) and the high resolution program (30 μl/min for 366 min followed by a linear increase to 200 μl/min over 234 mini were used. Two drop fractions (approximately 90 μl) were collected and counted. To obtain biochemical pure P-selectin ligand from unlabeled HL-60 cells, the preparation of membrane lysates from 50 liters of HL-60 cells and the PL5 antibody affinity chromatography were carried out as described previously (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). These materials were dialyzed against PBS, 1 mM CaCl2, 1 mM MgCl2, 0.01% Brij-35 (v/v) and 0.02% N., Na (w/v) (Buffer A) at 4 °C overnight. After a brief centrifugation (14,000 rpm for 10 min at 4 °C), the clean supernatant was loaded onto the P-selectin affinity column as described (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). After washing the P-selectin affinity column with 50 ml of Buffer A, the bound proteins were eluted with PBS, 10 mM EDTA, 0.01% Brij-35 (v/v), and 0.02% NaN:i (w/v) (Buffer B). For the purpose of purification and characterization, the nonradioactive P-selectin ligand was dialyzed against H.¿0 and lyophilized as above. The lyophilized material was then resuspended in 200 μl of PBS, 0.01% Brij-35 (v/v), and 0.02% N.Na (w/v) (Buffer C). Following another brief centrifugation, a 50-μ1 aliquot was injected into a 100-μl sample loop for each run on Superdex 200 size exclusion chromatography, using Buffer C at 40 μl/min at 22 °C and collection as 50-μ1 fractions (Smart System, Pharmacia). The protein peaks were monitored at A280 nm and A260 nm. The pure P-selectin ligand was exactly as above. A aliquot of each P-selectin ligand fraction was counted for The were then by 10 and plotted. Aliquots of the protein from Superdex 200 chromatography were and to a membrane The were separately with E-selectin Rg and PL5 IgM mAb at 1 as as PSGL-1 antiserum of The were also with human IgG and serum of for experimental For the were individually with mAb, IgM and and IgG antibodies at 5 followed by a and a as described previously (10Ma L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). E- and P-selectin and human IgG were in and immobilized onto the microtiter (50 at 4 °C overnight. The cell adhesion assay was exactly as described previously J.-G. Bevilacqua M.P. Moore K.L. G.A. G.A. McEver R.P. 1990; PubMed Scopus Google Scholar). Protein concentrations were using Micro BCA with BSA as (Pierce). ∼120-kDa glycoprotein reducing conditions) can be isolated from HL-60 cells by affinity chromatography using platelet P-selectin or the PL5 mAb (6Moore 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 (421) Google Scholar, 7Norgard K.E. Moore K.L. Diaz S. Stults N.L. Ushiyama S. McEver R.P. Cummings R.D. Varki A. J. Biol. Chem. 1993; 268: 12764-12774Abstract Full Text PDF PubMed Google Scholar, 8Moore K.L. Eaton S.F. Lyons D.E. Lichenstein H.S. Cummings R.D. McEver R.P. J Biol. Chem. 1994; 269: 23318-23327Abstract Full Text PDF PubMed Google Scholar, L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). This molecule from the membrane of human leukocytes and HL-60 cells is also recognized by P-selectin and PL5 mAb by ligand and (6Moore 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 (421) Google Scholar, 7Norgard K.E. Moore K.L. Diaz S. Stults N.L. Ushiyama S. McEver R.P. Cummings R.D. Varki A. J. Biol. Chem. 1993; 268: 12764-12774Abstract Full Text PDF PubMed Google Scholar, 8Moore K.L. Eaton S.F. Lyons D.E. Lichenstein H.S. Cummings R.D. McEver R.P. J Biol. Chem. 1994; 269: 23318-23327Abstract Full Text PDF PubMed Google Scholar, D. Chang X.-J. Barone K.M. Vachino G. White H.M. Shaw G. Veldman G.M. Bean K.M. Ahern T.J. Furie B. Cumming D.A. Larsen G.R. Cell. 1993; 75: 1179-1186Abstract Full Text PDF PubMed Scopus (643) Google Scholar, L. Raycroft L. Asa D. Anderson D.C. Geng J.-G. J. Biol. Chem. 1994; 269: 27739-27746Abstract Full Text PDF PubMed Google Scholar). In this study, we have and purified E-selectin Rg to be used as a for E-selectin. Following the of affinity purification as that for P-selectin ligand we and which can be isolated from [3H]glucosamine-labeled HL-60 cells by E-selectin Rg affinity chromatography. As shown in Fig. E-selectin Rg affinity chromatography specifically isolated only one glycoprotein from labeled human HL-60 cells. this molecule had a molecular mass of –120 kDa under reducing it has a molecular mass identical with that of the P-selectin glycoprotein ligand isolated by P-selectin affinity chromatography The purified ligands from both P- and E-selectin affinity chromatography for than of from the cell lysates which have been applied to the affinity columns (data not shown). As an experimental for the lysates of labeled HL-60 cells were also applied to human IgG affinity column in the of After washing, EDTA elution of this column an of which could not be precipitated by methanol/chloroform (19Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3141) Google Scholar), that it [3H]glucosamine of glycoproteins (data not shown). As P- and E-selectins both bind to the ∼120-kDa we whether these ligands might be the To this cross-depletion were carried Two equal of lysates from [3H]glucosamine-labeled HL-60 cells were applied separately to the P- or E-selectin affinity column in the of After washing, the bound ligands were eluted with EDTA from the P- and E-selectin columns aliquot of each fraction from EDTA elution was counted and for As the bound radioactivities for than of sample only the radioactivities were The radioactivities from the sample and column washing were The collected breakthrough from the E-selectin Rg chromatography was pooled together and reapplied to the P-selectin affinity column while the collected breakthrough from the P-selectin chromatography was pooled together and reapplied to the E-selectin affinity column. After washing, the bound ligands were eluted with EDTA from the P-selectin column and E-selectin column The radioactivities were counted and the fractions were plotted. these demonstrated that the cell lysates through the P-selectin affinity column the of binding to the following E-selectin affinity column A and and C and B). However, it was that the cell lysates through the P- or E-selectin affinity column could not binding to the following E-selectin column or P-selectin column As the of ligand bind