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• W2149194080 abstract "Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2-/- mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2-/- males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2-/- mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2-/- males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. Protein tyrosine O-sulfation is a post-translational modification that occurs in most eukaryotes (1Huttner W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (178) Google Scholar, 3Huttner W.B. Baeuerle P.A. Mol. Cell. Biol. 1988; 6: 97-140Google Scholar). In mouse and man, tyrosine sulfation is mediated by one of only two tyrosylprotein sulfotransferases (EC 2.8.2.20), TPST1 and TPST2 (4Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (142) Google Scholar, 6Beisswanger R. Corbeil D. Vannier C. Thiele C. Dohrmann U. Kellner R. Ashman K. Niehrs C. Huttner W.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11134-11139Crossref PubMed Scopus (117) Google Scholar). These enzymes catalyze the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate, the universal sulfate donor, to tyrosine residues in polypeptides (7Lee R.W. Huttner W.B. J. Biol. Chem. 1983; 258: 11326-11334Abstract Full Text PDF PubMed Google Scholar). TPST enzymes are type II transmembrane proteins that reside in the trans-Golgi network and have luminally oriented catalytic domains (4Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (142) Google Scholar, 6Beisswanger R. Corbeil D. Vannier C. Thiele C. Dohrmann U. Kellner R. Ashman K. Niehrs C. Huttner W.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11134-11139Crossref PubMed Scopus (117) Google Scholar, 8Baeuerle P.A. Huttner W.B. J. Cell Biol. 1987; 105: 2655-2664Crossref PubMed Scopus (192) Google Scholar). Thus, tyrosine sulfation occurs only on soluble and transmembrane proteins that transit the Golgi en route to either secretion or incorporation into the plasma membrane. Accordingly, all of the native tyrosine-sulfated proteins described to date fall into one of these two categories (2Moore K.L. J. Biol. Chem. 2003; 278: 24243-24246Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Consensus features for tyrosine sulfation have been proposed based on the amino acid sequences flanking known sulfation sites coupled with in vitro studies on the sulfation of various synthetic peptides (PROSITE accession number PS00003). In addition, a software tool for prediction of tyrosine sulfation sites in proteins called Sulfinator has been developed (9Monigatti F. Gasteiger E. Bairoch A. Jung E. Bioinformatics (Oxf.). 2002; 15: 769-770Crossref Scopus (224) Google Scholar). However, the positive predictive value of these features and the Sulfinator tool is not known. Some known tyrosine sulfation sites do not fulfill proposed consensus features, and some are not predicted by Sulfinator. Thus, unlike some other post-translational modifications, there is no way to reliably predict sites of sulfation. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. For example, tyrosine sulfation of P-selectin glycoprotein ligand-1 (PSGL-1 2The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; HCII, heparin cofactor II; mAb, monoclonal antibody; pY, phosphotyrosine; sY, sulfotyrosine; scFv, single chain Fv; MOPS, 3-(N-morpholino)propanesulfonic acid; MBS, MOPS-buffered saline; TBS, Tris-buffered saline; NRK, normal rat kidney; LC-MS/MS, liquid chromatography-tandem mass spectrometry; FTICR, Fourier transform ion cyclotron resonance; PVDF, polyvinylidene difluoride. 2The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; HCII, heparin cofactor II; mAb, monoclonal antibody; pY, phosphotyrosine; sY, sulfotyrosine; scFv, single chain Fv; MOPS, 3-(N-morpholino)propanesulfonic acid; MBS, MOPS-buffered saline; TBS, Tris-buffered saline; NRK, normal rat kidney; LC-MS/MS, liquid chromatography-tandem mass spectrometry; FTICR, Fourier transform ion cyclotron resonance; PVDF, polyvinylidene difluoride.; CD162) expressed on leukocytes is required for cell-cell interactions mediated by P- and L-selectins in the vasculature (10Wilkins P.P. Moore K.L. McEver R.P. Cummings R.D. J. Biol. Chem. 1995; 270: 22677-22680Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 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 (392) Google Scholar). In the co-crystal of the lectin/epidermal growth factor domain of P-selectin and a recombinant glycosulfopeptide mimetic of the N-terminal domain of PSGL-1, the sulfate groups at Tyr48 and Tyr51 are involved in direct protein-protein contacts with P-selectin (13Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). A great deal of recent interest has focused on the role of tyrosine sulfation in G-protein-coupled receptor function after Farzan et al. (14Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar) showed that CCR5, a major human immunodeficiency virus coreceptor, is tyrosine-sulfated. Sulfation of 1 or more tyrosine residues in the N-terminal extracellular domain of CCR5 is required for optimal binding of CCL3, CCL4, and CCL5 and for optimal human immunodeficiency virus coreceptor function. Similar studies indicated that tyrosine sulfation of the N-terminal domains of other chemokine receptors (CXCR4, CCR2B, CX3CR1, CCR8, CXCR3) as well as other G-protein-coupled receptors (C5a; C3a; SIP1; and the follicle-stimulating, luteinizing, and thyroid-stimulating hormone receptors), is required for optimal binding of their cognate ligands (2Moore K.L. J. Biol. Chem. 2003; 278: 24243-24246Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 15Gutierrez J. Kremer L. Zaballos A. Goya I. Martinez A.C. Marquez G. J. Biol. Chem. 2004; 279: 14726-14733Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Colvin R.A. Campanella G.S. Manice L.A. Luster A.D. Mol. Cell. Biol. 2006; 26: 5838-5849Crossref PubMed Scopus (100) Google Scholar). Likewise, tyrosine sulfation is required for optimal proteolytic processing of progastrin (18Bundgaard J.R. Vuust J. Rehfeld J.F. EMBO J. 1995; 14: 3073-3079Crossref PubMed Scopus (56) Google Scholar), proteolytic activation of coagulation factors V and VIII by thrombin (19Pittman D.D. Tomkinson K.N. Michnick D. Selighsohn U. Kaufman R.J. Biochemistry. 1994; 33: 6952-6959Crossref PubMed Scopus (68) Google Scholar, 21Michnick D.A. Pittman D.D. Wise R.J. Kaufman R.J. J. Biol. Chem. 1994; 269: 20095-20102Abstract Full Text PDF PubMed Google Scholar), proteolysis of the complement C4 α-chain by C1s (22Hortin G.L. Farries T.C. Graham J.P. Atkinson J.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1338-1342Crossref PubMed Scopus (66) Google Scholar), binding of glycoprotein Ibα to thrombin (23Celikel R. McClintock R.A. Roberts J.R. Mendolicchio G.L. Ware J. Varughese K.I. Ruggeri Z.M. Science. 2003; 301: 177-179Crossref PubMed Scopus (166) Google Scholar), binding of glycoprotein Ibα (24Dong J.F. Li C.Q. Lopez J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (106) Google Scholar, 25Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and factor VIII (26Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar) to von Willebrand factor (26Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar), binding of cholecystokinin to the cholecystokinin A receptor (27Jensen R.T. Lemp G.F. Gardner J.D. J. Biol. Chem. 1982; 257: 5554-5559Abstract Full Text PDF PubMed Google Scholar), and optimal binding of hirudin to thrombin (28Stone S.R. Hofsteenge J. Biochemistry. 1986; 25: 4622-4628Crossref PubMed Scopus (511) Google Scholar). However, for many of the known tyrosine-sulfated proteins, there is no information on the role of the sulfotyrosine residue(s) in protein function. Many more tyrosine-sulfated proteins are likely to exist and await description. However, the pace of discovery has been very slow. One the major barriers to developing a full understanding of the importance of tyrosine sulfation in biological systems has been a lack of a facile means to identify additional tyrosinesulfated proteins and probes to explore the role of tyrosine sulfation in protein function. An antibody reagent able to detect sulfotyrosine residues would be highly desirable. We attempted to generate anti-sulfotyrosine monoclonal antibodies using the strategy employed by Glenney et al. (29Glenney Jr., J.R. Zokas L. Kamps M.P. J. Immunol. Methods. 1988; 109: 277-285Crossref PubMed Scopus (171) Google Scholar) to generate anti-phosphotyrosine monoclonal antibodies. However, we failed to generate detectable antibody responses to sulfotyrosine in mice. Here, we describe the identification and characterization of a novel anti-sulfotyrosine antibody generated using phage display technology. We show that this antibody binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of the sequence context. Furthermore, we show that it can detect tyrosine-sulfated proteins in complex biological samples, and we demonstrate its utility in affinity purification of tyrosine-sulfated proteins from crude tissue samples. Materials—Human neutrophil PSGL-1 and human platelet P-selectin were purified as described previously (30Moore 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, 31Moore K.L. J. Tissue Cult. Methods. 1994; 16: 255-257Crossref Scopus (11) Google Scholar). Purified bovine factors X1 and X2 were provided by Charles Esmon (Oklahoma Medical Research Foundation), and human heparin cofactor II (HCII) expressed in Escherichia coli (32Han J.H. Tollefsen D.M. Biochemistry. 1998; 37: 3203-3209Crossref PubMed Scopus (12) Google Scholar) was a gift from Douglas Tollefsen (Washington University, St. Louis, MO). Purified human plasma C4 was purchased from Advanced Research Technology (San Diego, CA), and human plasma HCII and mouse fibrinogen were from Hematologic Technologies, Inc. (Essex Junction, VT). Phosphotyrosine (pY) and sulfotyrosine (sY) were purchased from Sigma and Bachem, respectively. The pentapeptides LDYDF, LD(sY)DF, and LD(pY)DF were synthesized and high pressure liquid chromatography-purified (>95% purity) by Biosynthesis Inc. (Lewisville, TX). Anti-phosphotyrosine monoclonal antibody (mAb) PY20 was purchased from MP Biomedicals. Identification of Antibody Binding to Sulfotyrosine—A single chain Fv (scFv) phagemid library, an expanded version of the 1.38 × 1010 library (33Vaughan T.J. Williams A.J. Pritchard K. Osbourn J.K. Pope A.R. Earnshaw J.C. McCafferty J. Hodits R.A. Wilton J. Johnson K.S. Nat. Biotechnol. 1996; 14: 309-314Crossref PubMed Scopus (855) Google Scholar), was used to select antibodies that bind to a protein containing sulfated tyrosines. The immobilized target protein was the purified dimeric 19ek.Fc recombinant protein (13Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar), which contains three sulfated tyrosines within the N-terminal 19 amino acids of human PSGL-1 (QATE(sY)E-(sY)LD(sY)DFLPETEPP) and is fused to human IgG1 Fc via an enterokinase cleavage site. Several scFv clones were isolated following multiple rounds of a panning procedure (33Vaughan T.J. Williams A.J. Pritchard K. Osbourn J.K. Pope A.R. Earnshaw J.C. McCafferty J. Hodits R.A. Wilton J. Johnson K.S. Nat. Biotechnol. 1996; 14: 309-314Crossref PubMed Scopus (855) Google Scholar). The purified PSG2 scFv clone was identified as an scFv fragment whose binding to 19.ek.Fc was inhibited in the presence of increasing concentrations of the murine PSGL-1/Fc fusion protein, which contains sulfated tyrosines within a different sequence context (34Yang J. Galipeau J. Kozak C.A. Furie B.C. Furie B. Blood. 1996; 87: 4176-4186Crossref PubMed Google Scholar). The PSG2 scFv fragment was converted to a full-length intact IgG4λ antibody (designated PSG2) and expressed in mammalian Chinese hamster ovary cells as described (35Thompson J.E. Vaughan T.J. Williams A.J. Wilton J. Johnson K.S. Bacon L. Green J.A. Field R. Ruddock S. Martins M. Pope A.R. Tempest P.R. Jackson R.H. J. Immunol. Methods. 1999; 227: 17-29Crossref PubMed Scopus (47) Google Scholar). PSG2 Purification—PSG2-expressing Chinese hamster ovary cells were expanded from a single vial of frozen cells in α-minimal essential medium (Mediatech, Inc.) containing 10% heatinactivated dialyzed fetal bovine serum (Sigma), 2 mml-glutamine, 100 nm methotrexate, 1 mg/ml G418, 100 units/ml penicillin, and 100 μg/ml streptomycin into 850-cm2 roller bottles. At ≈90% confluence, the serum-containing medium was removed; the monolayer washed with warm phosphate-buffered saline; and the medium was replaced with α-minimal essential medium, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. The conditioned medium was harvested and replaced with fresh medium twice each week until senescence. The conditioned medium was clarified by centrifugation, followed by addition of benzamidine toa 5 mm final concentration, sterilization by passage through a 0.2-μm filter, and freezing at -20 °C. To purify PSG2, the conditioned medium was batch-adsorbed to protein G-Sepharose overnight at room temperature; the resin was packed into a column and washed extensively with MOPS-buffered saline (MBS; 0.1 m NaCl and 20 mm MOPS, pH 7.5); and bound mAb was eluted with ImmunoPure® Gentle Ag/Ab Elution Buffer (Pierce). Eluted mAb was exhaustively dialyzed against MBS, clarified by centrifugation (48,000 × g, 30 min), and stored at -80 °C. The purified IgG was >95% pure as assessed by SDS-PAGE. The purified material co-eluted with an IgG standard on a Tosoh Bioscience G3000 SWXL size exclusion column (7.8 × 300 mm) using 150 mm NaCl and 20 mm sodium phosphate, pH 6.7, as the mobile phase. Epitope Mapping—Peptide spot synthesis was performed as described previously (36Bridges K.G. Chopra R. Lin L. Svenson K. Tam A. Jin G. Cowling R. Lovering F. Akopian T.N. DiBlasio-Smith E. Annis-Freeman B. Marvell T.H. LaVallie E.R. Zollner R.S. Bard J. Somers W.S. Stahl M.L. Kriz R.W. Peptides (N.Y.). 2006; 27: 1877-1885Crossref PubMed Scopus (15) Google Scholar). The arrays were defined on the membranes by coupling a β-alanine spacer, and peptides were synthesized using standard diisopropyl carbodiimide/hydroxybenzotriazole coupling chemistry as described previously (37Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (913) Google Scholar, 38Molina F. Laune D. Gougat C. Pau B. Granier C. Pept. Res. 1996; 9: 151-155PubMed Google Scholar). Activated amino acids were spotted using an Abimed ASP 222 robot, and the washing and Fmoc (N-(9-fluorenyl)methoxycarbonyl) deprotection steps were done manually. Following the final synthesis cycle, the membranes were treated with acetic anhydride, and side chains were deprotected, resulting in an array of peptides that were N-terminally acetylated and attached via the C terminus. Membranes were washed with methanol, blocked with 1% casein in Tris-buffered saline (TBS; 100 mm NaCl and 20 mm Tris-HCl, pH 6.9), and then incubated with 1 μg/ml PSG2 in TBS for 1 h with gentle shaking. The membranes were washed four times for 2 min with TBS and probed with horseradish peroxidase-conjugated anti-Fc antibody in TBS and 1% casein. After washing with TBS, bound protein was visualized using Super Signal West reagent (Pierce) and a digital camera. SDS-PAGE and Western Blotting—All samples were electrophoresed on 4-15% Tris-HCl/SDS-polyacrylamide gels (Bio-Rad). For Coomassie Blue staining, 1 μg of purified proteins was loaded, whereas for Western blotting, 10-20 ng of proteins was loaded. For Western blotting, the PSG2 or control IgG4λ mAb was used as primary antibody at 30 ng/ml, followed by 200 ng/ml horseradish peroxidase-conjugated anti-human IgG as secondary antibody. Bound secondary antibody was detected using ECL Plus (Amersham Biosciences), followed by either autoradiography or imaging on a Storm 860 scanner (GE Healthcare). Enzyme-linked Immunosorbent Assays—Microtiter plates (Immulon 1B, Dynex) were coated with bovine factor X1 or X2 (5 μg/ml, 100 μl/well) in sodium carbonate, pH 9.6, overnight at 4 °C. Plates were washed three times with TBS containing 0.05% Tween 20, blocked with TBS and 1% bovine serum albumin for 2 h, and washed twice with TBS containing 0.05% Tween 20. Tyrosine sulfate, tyrosine phosphate, or various peptides in MBS were added (50 μl/well) to triplicate wells. PSG2 (60 ng/ml, 50 μl/well) in MBS was then added to all wells. After 1 h, plates were washed five times with TBS containing 0.05% Tween 20, and a 1:5000 dilution of horseradish peroxidase-conjugated anti-human IgG in the same buffer was added (100 μl/well) and incubated for 1 h. Plates were washed five times with TBS containing 0.05% Tween 20, and then 2,2′-azinodi(3-ethylbenzthiazoline 6-sulfonate) peroxidase substrate (Kirkegaard & Perry Laboratories, Inc.) was added (100 μl/well). Plates were read at 405 nm using a VersaMax microplate reader (Molecular Devices Corp.). Pervanadate Treatment of Normal Rat Kidney (NRK) Cells—NRK cells (ATCC CRL-6509) were grown to confluence in Dulbecco's modified Eagle's medium with 2 mml-glutamine and 10% fetal bovine serum at 37 °C and 5% CO2. A fresh stock solution of pervanadate ions (30 mm) was prepared by combining 100 μl of 100 mm Na3VO4, 10 μl of 30% H2O2, and 223 μl of phosphate-buffered saline, followed by incubation in the dark for 10 min at room temperature. NRK cells were then incubated for 30 min at 37 °C and 5% CO2 in complete medium alone or containing 100 μm pervanadate. After 30 min, the medium was removed, and the cells were detached and collected by centrifugation (90 × g, 5 min). Cells were washed once with phosphate-buffered saline and collected by centrifugation, and cell pellets were lysed with 1% Triton X-100 in 100 mm NaCl, 20 mm HEPES, pH 7.2, 10% glycerol, 1 mm phenylmethanesulfonyl fluoride, 10 μg/ml aprotinin, 160 μg/ml benzamidine, 10 μg/ml antipain, 10 μg/ml leupeptin, and 1 mm Na3VO4. Lysates were incubated in the dark for 20 min at 4 °C and then clarified by centrifugation (14,000 × g, 10 min, 4 °C). Protein content was determined using the BCA protein assay (Pierce), and lysates were snap-frozen in liquid N2 and stored at -80 °C until used. Glycan Array Screening—The PSG2 mAb was used to screen a glycan microarray (Glycan Array version 3.5) at the Protein-Glycan Interaction Core of the Consortium for Functional Glycomics (Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center) as described previously (39Bochner B.S. Alvarez R.A. Mehta P. Bovin N.V. Blixt O. White J.R. Schnaar R.L. J. Biol. Chem. 2005; 280: 4307-4312Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In total, 206 unique glycans are represented on the array. Briefly, biotinylated glycosides (40Korchagina E. Bovina N.V. Bioorg. Khim. 1992; 18: 283-298PubMed Google Scholar) were bound to streptavidin-coated microtiter plates in replicates of four. Purified bovine factors X1 and X2 were biotinylated using sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce) and included on the array (30 pmol/well) as negative and positive controls, respectively. Precoated plates were washed three times with 100 μl of wash buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, and 0.05% Tween 20). The PSG2 mAb (10 μg/ml) in wash buffer containing 1% bovine serum albumin was added to each well and incubated for 1 h. The plates were washed, and 25 μl of fluorescein isothiocyanate-conjugated anti-human IgG (2 μg/ml) in wash buffer was added and incubated for 1 h. The wells were washed three times; 25 μl of wash buffer was added to each well; and bound secondary antibody was detected using a VICTOR2™ 1420 multilabel counter (PerkinElmer Life Sciences) at excitation and emission wavelengths of 485 and 535 nm, respectively. Generation of Tpst Double Knock-out Mice—Tpst1-null (Tpst1tm1Klm, MGI:2183366) and Tpst2-null (Tpst2tm1Klm, MGI:3512111) mice were generated and characterized as described previously (41Ouyang Y.B. Crawley J.T.B. Aston C.E. Moore K.L. J. Biol. Chem. 2002; 277: 23781-23787Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 42Borghei A. Ouyang Y.B. Westmuckett A.D. Marcello M.R. Landel C.P. Evans J.P. Moore K.L. J. Biol. Chem. 2006; 281: 9423-9431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The Tpst1 and Tpst2 genes reside on chromosome 5, and the physical distance between the two is ≈18.4 megabases. To generate the Tpst double knock-out mice, Tpst1-/- males were mated with Tpst2-/- females to generate obligate in trans compound heterozygotes. Male and female offspring from this cross were mated, and their offspring were screened by PCR for the presence of the wild-type and mutant alleles at both loci as described (41Ouyang Y.B. Crawley J.T.B. Aston C.E. Moore K.L. J. Biol. Chem. 2002; 277: 23781-23787Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 42Borghei A. Ouyang Y.B. Westmuckett A.D. Marcello M.R. Landel C.P. Evans J.P. Moore K.L. J. Biol. Chem. 2006; 281: 9423-9431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Male and female Tpst1-/-,Tpst2+/- offspring were then interbred to generate double homozygotes. Animals were mated, housed, and fed as described previously (41Ouyang Y.B. Crawley J.T.B. Aston C.E. Moore K.L. J. Biol. Chem. 2002; 277: 23781-23787Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Adhesion of Neutrophils to P-selection under Flow Conditions—Adhesion assays were performed as described previously (43Patel K.D. Moore K.L. Nollert M.U. McEver R.P. J. Clin. Investig. 1995; 96: 1887-1896Crossref PubMed Scopus (172) Google Scholar). Briefly, 2 ml of 1 μg/ml human platelet P-selectin in Hanks' balanced salt solution was coated on 35-mm tissue culture plates overnight at 4 °C and blocked with 1% human serum albumin in Hank's balanced salt solution for 2 h at room temperature. Human neutrophils were isolated as described previously (44Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Investig. 1985; 76: 2235-2246Crossref PubMed Scopus (274) Google Scholar), resuspended at 4 × 106 cells/ml in Hank's balanced salt solution and 1% human serum albumin, and perfused over the P-selection surface at a wall shear stress of 1 dyne/cm2. Neutrophil adhesion was allowed to equilibrate for 4 min, and then non-adherent cells were flushed from the system with Hank's balanced salt solution and 1% human serum albumin. After an additional 4 min of perfusion, the control human IgG4λ (50 μg/ml) or PSG2 (50 μg/ml) mAb was added to the perfusate. Neutrophil adhesion was visualized by phase-contrast video microscopy, and the number of rolling neutrophils was determined every 20 s. PSG2 Affinity Chromatography and Peptide Sequencing—Epididymides were collected from 10 sexually mature wild-type mice and homogenized in cold MBS containing a mixture of protease inhibitors (Complete Mini, Roche Applied Science) using a Dounce homogenizer. A post-nuclear supernatant was obtained (800 × g, 10 min), which was then subjected to centrifugation (100,000 × g, 60 min) to prepare a soluble protein fraction. The soluble fraction (8.5 ml, ≈28 mg of total protein) was applied to a PSG2-Affi-Gel-10 column (4 mg of mAb/ml of resin, 0.9 × 5 cm) at 50 μl/min. The column was washed extensively with MBS and then with 100 mm NH4OAc, and the column was eluted with the sulfated peptide LD(sY)DF (4 mm) in 100 mm NH4OAc at 30 μl/min. Flow-through and elution fractions were monitored by SDS-PAGE, followed by PSG2 Western blotting or silver staining. Sperm/epididymal proteins eluted from the PSG2 column were concentrated and electrophoresed on 4-15% Tris-HCl/SDS-polyacrylamide gels under nonreducing conditions, and separated proteins were visualized by colloidal Coomassie Blue staining. Gels were washed with H2O, and bands were excised, washed again with H2O and then with 60% acetonitrile in H2O, and dried in a SpeedVac. The gel pieces were reduced in 10 mm dithiothreitol in 50 mm NH4HCO3 for 1 h at 56 °C and alkylated in 55 mm iodoacetamide in 50 mm NH4HCO3 for 45 min at 22 °C in the dark. After washing with 50 mm NH4HCO3, the gel pieces were dehydrated with 60% acetonitrile in H2O and dried. Gel pieces were re-swollen with trypsin in 50 mm NH4HCO3 and incubated overnight at 37 °C. Peptides were eluted from the gel slices by repeated extraction (three times, 60 min) with 60% acetonitrile and 0.1% trifluoroacetic acid in H2O. Eluates were pooled, dried down, and redissolved in 2% acetonitrile in 0.1% trifluoroacetic acid prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Mass Spectrometry Analysis—Nano-LC-MS/MS experiments were performed on a Thermo Electron LTQ FT hybrid linear ion trap/7-tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a Thermo Electron nanospray ion source, Surveyor MS pump, and microautosampler. The tryptic peptide mixture was separated on a PicoFrit column (12 cm, inner diameter, × 50 μm) packed with C18 reversed-phase resin (Magic C18AQ, 100-Å pore size, 3-μm particle size, Michrom Bioresources, Inc.). The column was equilibrated before sample injection for 10 min at 2% solvent B (0.1% (v/v) formic acid in acetonitrile) and 98% solvent A (0.1% (v/v) formic acid in H2O) at a flow rate of 140 nl/min. The column was developed with a linear gradient of 2-50% solvent B over 30 min at a flow rate of 320 nl/min. The LTQ FT mass spectrometer was operated in the data-dependent acquisition mode using the TOP10 method: a full-scan mass spectrum acquired in the FTICR mass spectrometer, followed by 10 MS/MS experiments performed with the LTQ FT mass spectrometer on the 10 most abundant ions detected in the full-scan mass spectrum. All tandem mass spectra from each LC-MS run were searched against the NCBI Protein Database using the Mascot search engine. Searches were performed with tryptic specificity allowing four missed cleavages and a tolerance on the mass measurement of 10 ppm in MS mode and 0.5 Da for MS/MS ions. Possible structure modifications allowe" @default.
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• W2149194080 date "2006-12-01" @default.
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• W2149194080 title "Detection and Purification of Tyrosine-sulfated Proteins Using a Novel Anti-sulfotyrosine Monoclonal Antibody" @default.
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• W2149194080 doi "https://doi.org/10.1074/jbc.m609398200" @default.
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