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• W2004432012 abstract "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 (Kd ∼ 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 (Kd∼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 (Kd ∼ 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 (Kd ∼ 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. 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 (Kd ∼ 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 (Kd∼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 (Kd ∼ 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 (Kd ∼ 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. sialyl Lewis x core-2-based O-glycan with sLex P-selectin glycoprotein ligand-1 soluble P-selectin glycopeptide glycosulfopeptide tyrosine sulfate high performance liquid chromatography association constant dissociation constant 4-[N-morpholine]propanesulfonic acid adenosine 3′-phosphate 5′-phosphosulfate N-(9-fluorenyl)methoxycarbonyl fucose Selectins and their glycoconjugate ligands promote the tethering and rolling of circulating leukocytes on blood vessel endothelial cells, platelets, and other leukocytes. L-, E-, and P-selectin weakly bind to the sialyl Lewis x (sLex)1tetrasaccharide determinant NeuAcα2–3Galβ1–4(Fucα1–3)-GlcNAc-R, but each selectin binds with higher affinity to specific sialylated and fucosylated macromolecular ligands (1McEver R.P. Moore K.L. Cummings R.D. J. Biol. Chem. 1995; 270: 11025-11028Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 2McEver R.P. Glycoconj. J. 1997; 14: 585-591Crossref PubMed Scopus (240) Google Scholar, 3Cummings R.D. Lowe J.B. Varki A. Cummings R.D. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology (1999). Cold Spring Harbor Laboratory Press, Boston, MA1999: 391-416Google Scholar). The best characterized ligand for selectins is P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin present on the surface of all leukocytes (4McEver R.P. Cummings R.D. J. Clin. Invest. 1997; 100: 485-492Crossref PubMed Google Scholar, 5Moore K.L. Leuk. Lymphoma. 1998; 29: 1-15Crossref PubMed Scopus (94) Google Scholar, 6Cummings R.D. Braz. J. Med. Biol. Res. 1999; 32: 519-528Crossref PubMed Scopus (58) Google Scholar, 7Yang J. Furie B.C. Furie B. Thromb. Haemostasis. 1999; 81: 1-7Crossref PubMed Scopus (220) Google Scholar). PSGL-1 binds with relatively high affinity (Kd ∼ 300 nm) to P-selectin expressed on the surface of activated endothelial cells and platelets (8Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Antibody blockade studies (9Borges E. Eytner R. Moll T. Steegmaier M. Campbell M.A. Ley K. Mossmann H. Vestweber D. Blood. 1997; 90: 1934-1942Crossref PubMed Google Scholar) and targeted disruption of the gene for PSGL-1 in mice (10Yang J. Hirata T. Croce K. Merrill-Skoloff G. Tchernychev B. Williams E. Flaumenhaft R. Furie B.C. Furie B. J. Exp. Med. 1999; 190: 1769-1782Crossref PubMed Scopus (291) Google Scholar) confirm that PSGL-1 is the primary ligand for P-selectin on leukocytes. The N terminus of mature human PSGL-1 begins at residue 42, after removal of the signal peptide from residues 1–18 and a propeptide from residues 19–41 (11Sako 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 (647) Google Scholar). Sulfation of tyrosine residues andO-glycosylation in the mature N-terminal region of PSGL-1 appear necessary for high affinity binding of PSGL-1 to P-selectin (4McEver R.P. Cummings R.D. J. Clin. Invest. 1997; 100: 485-492Crossref PubMed Google Scholar), but the importance of modifications at specific amino acid residues is not well understood. Studies employing site-directed mutagenesis of recombinant PSGL-1, which was co-expressed in Chinese hamster ovary cells or COS cells with specific glycosyltransferases, suggested that a core-2 based O-glycan with the sialyl Lewis x (C2-O-sLex) antigen at Thr-57 and at least one of the Tyr residues at Tyr-46, -48 or -51 are required for measurable binding of PSGL-1 to P-selectin (12Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 13Pouyani T. Seed B. Cell. 1995; 83: 333-343Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 14Liu W. Ramachandran V. Kang J. Kishimoto T.K. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 7078-7087Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Whether the individual Tyr residues make distinct contributions to P-selectin recognition has not been examined. Mutation of Thr-44 to alanine did not inhibit binding of recombinant PSGL-1, suggesting that an O-glycan at this position does not contribute to binding. However, this residue might beO-glycosylated in hematopoietic cells but not in transfected Chinese hamster ovary cells. To obtain more precise information about the interaction of P-selectin with O-glycans and TyrSO3 residues on PSGL-1, we recently used purified and recombinant glycosyltransferases and a tyrosyl-protein sulfotransferase to generate synthetic glycosulfopeptides (GSPs) modeled after the mature N-terminal region of PSGL-1. A glycosulfopeptide-6 (GSP-6) was synthesized to contain three TyrSO3 residues at Tyr-46, -48, or -51 and a nearby C2-O-sLex at Thr-57 (16Leppänen 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 (186) Google Scholar). GSP-6 binds to P-selectin with an affinity (Kd ∼ 350 nm) close to that of native, neutrophil-derived PSGL-1 (Kd ∼ 300 nm) (8Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 16Leppänen 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 (186) Google Scholar). Consistent with this finding, monomeric forms of full-length PSGL-1 and a trypsin-derived glycosulfopeptide from the N terminus of HL-60 cell-derived PSGL-1 also bind with high affinity to P-selectin (17Epperson T.K. Patel K.D. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 7839-7853Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Very little is known about the stereospecific contributions of tyrosine sulfation and O-glycosylation to binding of PSGL-1 to P-selectin. Substitution of any two of the three tyrosines with phenylalanines in recombinant PSGL-1 impairs the mechanical properties of its bonds with P-selectin in shear flow, but these substitutions could affect peptide structure as well as prevent sulfation (18Ramachandran 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). Synthetic conjugates that express sLex on different types of O-glycans bind differentially to P-selectin (16Leppänen 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 (186) Google Scholar, 19Jain R.K. Piskorz C.F. Huang B.G. Locke R.D. Han H.L. Koenig A. Varki A. Matta K.L. Glycobiology. 1998; 8: 707-717Crossref PubMed Scopus (28) Google Scholar). Notably, a GSP modeled after the N terminus of PSGL-1 that contains sLex expressed on a core-2 based O-glycan binds much better to P-selectin than an isomeric GSP that contains sLex expressed on an extended core-1 basedO-glycan (16Leppänen 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 (186) Google Scholar). It is not clear whether the position of theO-glycan relative to TyrSO3 residues,i.e. at Thr-44 versus Thr-57, contributes to binding. The interpretation of the effects of amino acid substitutions on post-translational modifications of PSGL-1 has significant limitations because it is difficult to structurally define specific post-translational modifications on recombinant glycoproteins. There is usually microheterogeneity of glycosylation that complicates the interpretation of binding affinities. The use of synthetic, homogeneously glycosylated glycosulfopeptides offers marked advantages, but the tyrosyl-protein sulfotransferase adds sulfate from PAPS donor to all available Tyr residues within the consensus sequence. Thus, enzymatic sulfation does not readily allow sulfation of individual Tyr residues within a peptide that contains multiple Tyr residues (16Leppänen 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 (186) Google Scholar). To gain more insight into the contributions of individual TyrSO3 residues, the peptide backbone, specific sugar residues, and their site-specific contributions, we generated synthetic GSPs modeled after the mature N terminus of human PSGL-1 that contain TyrSO3 residues and O-glycans at specific sites. Our results demonstrate that P-selectin differentially recognizes GSPs containing one, two, or three TyrSO3 residues and that the relative contributions of each TyrSO3 residue differ. P-selectin binds weakly to non-sulfated anionic peptides containing C2-O-sLex, but the O-glycan must be placed at Thr-57 rather than at the alternative site at Thr-44. These studies further extend our knowledge about the roles of peptide, sulfate, and carbohydrate determinants in binding of PSGL-1 to P-selectin. Glyco(sulfo)peptides were synthesized on an Applied Biosystems 433A peptide synthesizer using Fmoc chemistry. The preloaded Wang resin (p-hydroxymethylphenoxymethyl resin) and liquid reagents and solvents were purchased from Applied Biosystems, Warrington, Great Britain. Tri-O-acetyl-GalNAc and tyrosine sulfates were incorporated into the peptide during the solid phase peptide synthesis using tri-O-acetyl-GalNAcα-Fmoc-Thr (Glycotech, Rockville, MD) and Fmoc-Tyr(-SO3H)-H sodium salt (Bachem California Inc., Torrance, CA). Other Fmoc amino acid derivatives were from Calbiochem-Novabiochem. Sulfated peptides 2-GSP(46)-1, 2-GSP(48)-1, 2-GSP(51)-1, 2-GSP(46,48)-1, 2-GSP(46,51)-1, 2-GSP(48,51)-1, and 2-GSP-1 were cleaved from the resin as described (20Kitagawa K. Futaki S. Yagami T. Sumi S. Inoue K. Int. J. Pept. Protein Res. 1994; 43: 190-200Crossref PubMed Scopus (18) Google Scholar). Briefly, 70–100 mg of resin containing peptide was cleaved in 700 μl of 90% aqueous trifluoroacetic acid containing 2.5% (v/v) m-cresol for 5 h at 4 °C under continuous stirring. Nonsulfated 2-GP-1 was cleaved from the resin in 700 μl of 90% aqueous trifluoroacetic acid containing 5% (v/v) thioanisole and 2.5% (v/v) ethanedithiol for 15 min on ice, and incubation was continued for 1 h 45 min at room temperature under stirring. The cleaved peptides were then precipitated and washed with tert-butyl methyl ether. The dried peptides were dissolved in 30% aqueous acetonitrile to a concentration of 2 mg/ml. An equal volume of methanol was added, and the free sulfhydryl of the C-terminal cysteine was converted into S-S-CH3 by incubating the peptides in 4 mm methyl methanethiosulfonate (Pierce) for 1 h at room temperature. The complete derivatization was confirmed by reversed phase HPLC. Tri-O-acetyl-GalNAc was deacetylated using 18 mm sodium methylate as described (16Leppänen 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 (186) Google Scholar). The deacetylated peptides were purified by reversed phase HPLC and analyzed by electrospray mass spectrometry (see Table I).Figure 5Equilibrium binding affinities of glycosulfopeptides for sPS at physiological salt concentration.Equilibrium gel filtration experiments were carried out by loading 50 pmol–3.2 nmol of sPS into gel filtration columns equilibrated with the indicated [3H]glycosulfopeptides in buffer (specific activity, 1000 cpm/pmol and 8000 cpm/ml in 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 2 mmCaCl2, 2 mm MgCl2, 0.02% NaN3). The bound GSP and free sPS concentrations were calculated from the equilibrium gel filtration data by dividing the molar amounts of GSPs and sPS by the peak volume of GSP-sPS complex (3 fractions, 420–435 μl). Representative data is shown for 2-GSP-6 (panel A) and 2-GSP(46,48)-6 (panel C). The calculated binding data for each of the glycosulfopeptides is shown inpanels B and D–G. Dissociation constants for indicated glycosulfopeptides were calculated using a rectangular hyperbola equation to derive the nonlinear curve-fitting. Theinset in panel A indicates the elution positions of sPS and 2-GSP-6 in the gel filtration column. H, correlation between elution positions (Δ) of glyco(sulfo)peptides on immobilized sPS and association constants (Ka) derived from equilibrium gel filtration data (Ka = 1/Kd). OD, optical density.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Enzymatic synthesis of glycosulfopeptide 2-GSP-6. Glyco(sulfo)peptides containing tyrosine sulfate at specified position(s) and GalNAcα at Thr-57 were acceptors for glycosyltransferase reactions. The synthesis of each peptide in Fig. 1was performed as indicated for 2-GSP-6. β1,3-GalT, core-1 β1,3-galactosyltransferase; β1,6-GlcNAcT, core-2 β1,6-N-acetylglucosaminyltransferase; β1,4-GalT, β1,4-galactosyltransferase; α2,3-(N)-sialylT, α2,3-(N)-sialyltransferase; α1,3-FucT, α1,3-fucosyltransferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IElectrospray mass spectrometric analysis of HPLC-purified glyco(sulfo)peptide samplesGlyco(sulfo)peptideCalculated massObserved massDa2-GP-12501.72501.02-GSP(46)-12581.72580.82-GSP(48)-12581.72581.12-GSP(51)-12581.72581.02-GSP(46,48)-12661.72661.02-GSP(46,51)-12661.72661.32-GSP(48,51)-12661.72661.22-GSP-12741.72740.82-GP-53320.43319.82-GSP(46)-53400.43400.12-GSP(48)-53400.43400.52-GSP(51)-53400.43400.02-GSP(46,48)-53480.43480.32-GSP(46,51)-53480.43480.02-GSP(48,51)-53480.43480.22-GSP-53560.53559.92-GP-63466.53466.02-GSP(46)-63546.53546.32-GSP(48)-63546.53546.22-GSP(51)-63546.53546.22-GSP(46,48)-63626.53626.42-GSP(46,51)-63626.53626.72-GSP(48,51)-63626.53626.32-GSP-63706.63706.33-GSP-63878.83878.33-GSP-6′3878.83878.4Shown are calculated and observed masses for the indicated glyco(sulfo)peptides (for structures, see Fig. 1). In some cases minor signals (peptide mass minus 80 mass units) that represent partially desulfated peptides were present (not shown). Open table in a new tab Shown are calculated and observed masses for the indicated glyco(sulfo)peptides (for structures, see Fig. 1). In some cases minor signals (peptide mass minus 80 mass units) that represent partially desulfated peptides were present (not shown). HPLC-purified peptides (2-GP-1, 2-GSP(46)-1, 2-GSP(48)-1, 2-GSP(51)-1, 2-GSP(46,48)-1, 2-GSP(46,51)-1, 2-GSP(48,51)-1, and 2-GSP-1) were acceptors for glycosyltransferase reactions (see Fig. 2). Sialyl Lewis x on a core-2-based O-glycan was synthesized at Thr-57 in each peptide using recombinant or highly purified glycosyltransferases essentially as described (16Leppänen 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 (186) Google Scholar). The reactions were accomplished by adding one glycosyltransferase and one donor at a time; after the reaction was >95% complete a new glycosyltransferase and a donor were added. At each step of the synthesis, a small aliquot of the reaction mixture was analyzed by HPLC. The completeness of each reaction was easily monitored by HPLC, because the addition of each monosaccharide reduced the retention time for the peptides (data not shown). At the end of the reaction sequence the reaction mixtures were deproteinated by chloroform-methanol (2:1) extraction, and samples were further purified by HPLC. The masses of the final products (2-GSP-6 series) and of peptide products after sialyltransferase reaction (2-GSP-5 series) were verified by electrospray mass spectrometry (see Table I). The sialyltransferase products served as acceptors for the synthesis of radiolabeled peptides. Radiolabeled glycosulfopeptides (2-GP-6, 2-GSP(46)-6, 2-GSP(48)-6, 2-GSP(51)-6, 2-GSP(46,48)-6, 2-GSP(46,51)-6, 2-GSP(48,51)-6, and 2-GSP-6) were synthesized using sialyltransferase products (2-GSP-5 series) as acceptors and either GDP-[3H]Fuc (1000 cpm/pmol) (American Radiolabeled Chemicals Inc., St. Louis, MO) or GDP-[14C]Fuc (96,000 cpm/nmol) (Amersham Pharmacia Biotech) as a donor in the α1,3-fucosyltransferase VI (Calbiochem) reaction. [3H]2-GSP-2 was synthesized using 2-GSP-1 as an acceptor and UDP-[3H]Gal (1000 cpm/pmol) (Amersham Pharmacia Biotech) as a donor in the core-1 β1,3-galactosyltransferase reaction. [3H]2-GSP-5 was synthesized using 2-GSP-4 as an acceptor and CMP-[3H]NeuAc (1000 cpm/pmol) (NEN Life Science Products, Inc.) as a donor in the α2,3-(N)-sialyltransferase (Calbiochem) reaction. Radiolabeled products were purified by HPLC. Isomeric glycopeptides 3-GP-6 and 3-GP-6′ were synthesized as described (16Leppänen 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 (186) Google Scholar). Matrix-assisted laser desorption ionization/time of flight mass spectrometric analysis confirmed the masses of the HPLC-purified products (observedm/z 3637.3 for 3-GP-6 and observedm/z 3636.6 for 3-GP-6′; calculatedm/z 3637.7 for both peptides). Glycopeptides 3-GP-6 and 3-GP-6′ were sulfated enzymatically using human tyrosyl-protein sulfotransferase-1 and either [35S]PAPS (544 000 cpm/nmol) (NEN Life Science Products, Inc.) or nonlabeled PAPS (Sigma) as a sulfate donor as described (16Leppänen 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 (186) Google Scholar). The fully sulfated products 3-GSP-6 and 3-GSP-6′ were purified using HPLC, and the masses of the final products were verified using electrospray mass spectrometry (Table I). Glyco(sulfo)peptide samples were filtered on a Spin-X membrane (Corning Costar, Cambridge, MA) and analyzed in a reversed phase C-18 HPLC column (Vydac, Hesperia, CA) on a Beckman System Gold HPLC. The following solvent systems were used at a flow rate of 1 ml/min: 1–10 min, isocratic 20% aqueous acetonitrile containing 0.1% trifluoroacetic acid; 10–70 min, linear gradient 20–45% aqueous acetonitrile containing 0.1% trifluoroacetic acid (non-, mono-, and disulfated peptides); 1–10 min, isocratic 15% aqueous acetonitrile containing 0.1% trifluoroacetic acid; 10–70 min, linear gradient 15–40% aqueous acetonitrile containing 0.1% trifluoroacetic acid (trisulfated peptides). The UV absorbance at 215 nm was followed, and/or the radioactivity of the collected fractions was measured. To prevent desulfation, the glycosulfopeptide products eluted from HPLC were rapidly cooled on ice and dried under vacuum. Hummel-Dreyer equilibrium gel filtration experiments were conducted in 2 ml of Sephadex G-100 columns (0.5 × 10 cm) as described (16Leppänen 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 (186) Google Scholar).3H-Labeled peptides (specific activity 1000 cpm/pmol) were used in the buffer at a concentration of 8000 cpm/ml. Two different concentrations of NaCl were used: 150 mm or 50 mm NaCl in 20 mm MOPS, pH 7.5, containing 2 mm CaCl2, 2 mm MgCl2, and 0.02% NaN3. Inhibition experiments with EDTA were conducted using 1 mm EDTA with 150 mm or 50 mm NaCl in 20 mm MOPS, pH 7.5, containing 0.02% NaN3. The amount of soluble P-selectin (sPS) (21Ushiyama S. Laue T.M. Moore K.L. Erickson H.P. McEver R.P. J. Biol. Chem. 1993; 268: 15229-15237Abstract Full Text PDF PubMed Google Scholar) used was 500 pmol in high salt buffer or 50 pmol in low salt buffer. Inhibition experiments with anti-P-selectin monoclonal antibody G1 (22Geng J.G. Bevilacqua M.P. Moore K.L. McIntyre T.M. Prescott S.M. Kim J.M. Bliss G.A. Zimmerman G.A. McEver R.P. Nature. 1990; 343: 757-760Crossref PubMed Scopus (802) Google Scholar) were performed using the same molar amount of G1 and sPS (500 pmol or 50 pmol) in buffers containing Ca2+. sPS was coupled to Ultralink Biosupport Medium (Pierce) at a density of 6.5 mg/ml. An sPS column (0.9 ml, 0.5 × 4.5 cm) was equilibrated with 20 ml of 20 mm MOPS, pH 7.5, containing 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 0.02% NaN3. Radiolabeled peptides dissolved in 200 μl of equilibration buffer (1000–2000 cpm, 1–10 pmol) were chromatographed in the P-selectin column by collecting 0.5-ml fractions at a flow rate of 250 μl/min. Bound peptides were eluted using 10 mmEDTA instead of divalent cations in buffer. In some experiments, 50 mm NaCl was used instead of 150 mm NaCl in buffers containing Ca2+ or EDTA (1 mm). To derive the Δ value for each bound peptide, the elution volume of unbound peptide (2 ml) was subtracted from the elution volume of bound peptide. [3H]2-GSP-6 (506,000 cpm, 0.5 nmol) was desialylated by adding 13 milliunits ofArthrobacter ureafaciens neuraminidase (Sigma) in 400 μl of 0.2 m sodium acetate, pH 5.5, for 14 h at 37 °C. The reaction mixture was deproteinated by chloroform-methanol (2:1) extraction and purified by HPLC. [3H]2-GP-6 (10,000 cpm, 10 pmol) was digested with Streptomyces griseusPronase E (2 mg/ml) (Sigma) in 100 μl of 0.1 m Tris, pH 8.0, containing 1 mm CaCl2 for 19 h at 60 °C. Pronase was inactivated by boiling for 30 min and was removed by chloroform-methanol (2:1) extraction. The aqueous phase was dried under vacuum and dissolved by 100 μl of 20 mmMOPS, pH 7.5, containing 50 mm NaCl and 0.02% NaN3. An aliquot of Pronase-digested [3H]2-GP-6 (1, 300 cpm) was chromatographed on a P-selectin column using 20 mm MOPS, pH 7.5, containing 50 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 0.02% NaN3. Electrospray mass spectra were collected in the negative ion mode using an API365 triple quadrupole mass spectrometer (PerkinElmer Sciex Instruments, Thornhill, Ontario, Canada). Samples were dissolved in 1% triethylamine in 50% aqueous methanol to a concentration of 5 pmol/μl and injected into the mass spectrometer with a nanoelectrospray ion source (MDS Protana A/S, Odense M, Denmark). We explored the possibility that only one or two TyrSO3 residues together with a nearbyO-glycan can support high affinity binding to P-selectin. To this end we synthesized a series of glyco(sulfo)peptides corresponding to the extreme N terminus of human PSGL-1 (residues 45–61) with one, two, or three TyrSO3 residues at defined positions or without TyrSO3 residues (Fig. 1). Each glyco(sulfo)peptide contained a C-terminal Cys residue for future coupling of the peptide to artificial supports. Each glyco(sulfo)peptide was subsequently modified by purified or recombinant glycosyltransferases to generate a core-2-basedO-glycan expressing the sLex antigen at Thr-57 (Fig. 2). To compare the roles of sialic acid and fucose for binding to P-selectin, peptides with three TyrSO3 residues but having incomplete glycosylation were also synthesized (Fig. 1). Peptides with one, two, or three TyrSO3 residues (Tyr-46, -48, and/or -51) and an α-linked GalNAc residue at Thr-57 were synthesized chemically using Fmoc derivatives of TyrSO3 and tri-O-acetyl-GalNAcα-Thr during the solid phase peptide synthesis. The sulfated peptides were cleaved from the solid support under mild conditions because of the acid lability of TyrSO3 residues (20Kitagawa K. Futaki S. Yagami T. Sumi S. Inoue K. Int. J. Pept. Protein Res. 1994; 43: 190-200Crossref PubMed Scopus (18) Google Scholar). After peptide cleavage, the C-terminal Cys of each peptide was protected by converting the free sulfhydryl into S-S-CH3 to prevent oxidation and dimerization of the peptide. After deacetylation of tri-O-acetyl-GalNAc, the peptides were purified by reversed phase HPLC, characterized by mass spectrometry, and used as acceptors for enzymatic glycosylation. sLex on a core-2-basedO-glycan was synthesized on each peptide at Thr-57 using recombinant or highly purified glycosyltransferases (Fig. 2). The completeness of each glycosyltransferase reaction was easily monitored by HPLC, because the addition of each monosaccharide reduced the retention time for the peptides by ∼0.5–2 min (data not shown). Electrospray mass spectrometry was used to analyze the masses of the final products. The mass of each glyco(sulfo)peptide matched the calculated mass (Table I). Radiolabeled peptides were synthesized using either GDP-[3H]Fuc or GDP-[14C]Fuc in the reactions with α1,3-fucosyltransferase, which is the final step in the synthesis. In addition, radiolabeled glycosulfopeptides with three TyrSO3 residues but with incomplete O-glycan structure were synthesized. These glycosulfopeptides are 2-GSP-2 (core-1 O-glycan), desialylated 2-GSP-6, and 2-GSP-5, which lacks a fucose residue (Fig. 1). We previously showed that a core-2-based O-glycan containing sLex at Thr-57 together with three TyrSO3residues supported high affinity binding of a glycosulfopeptide (GSP-6) to P-selectin (16Leppänen 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 (186) Google Scholar). 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