Post-translational Modifications of Recombinant P-selectin Glycoprotein Ligand-1 Required for Binding to P- and E-selectin
1996; Elsevier BV; Volume: 271; Issue: 6 Linguagem: Inglês
10.1074/jbc.271.6.3255
ISSN1083-351X
AutoresFugang Li, Patricia P. Wilkins, Suzanne Crawley, Jasminder Weinstein, Richard D. Cummings, Rodger P. McEver,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoP-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galβ1-3GalNAcα1-Ser/Thr); they lack core 2 O-linked glycans (Galβ1-3(Galβ1-4GlcNAcβ1-6)GalNAcα1-Ser/Thr) because they do not express the core 2 β1-6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack α1-3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an α1-3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of β-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galβ1-3GalNAcα1-Ser/Thr); they lack core 2 O-linked glycans (Galβ1-3(Galβ1-4GlcNAcβ1-6)GalNAcα1-Ser/Thr) because they do not express the core 2 β1-6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack α1-3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an α1-3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of β-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin. INTRODUCTIONThe selectins are a group of three Ca2+-dependent lectins that mediate rolling adhesion of leukocytes on the vessel wall during inflammation (reviewed in McEver et al.(1.McEver R.P. Moore K.L. Cummings R.D. J. Biol. Chem. 1995; 270: 11025-11028Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar)). L-selectin, expressed on leukocytes, binds to constitutively or inducibly expressed carbohydrate ligands on endothelial cells. E-selectin, expressed on cytokine-activated endothelium, and P-selectin, expressed on thrombin-activated endothelial cells and platelets, bind to carbohydrate ligands on myeloid cells and subsets of lymphocytes. Leukocyte rolling requires that selectins rapidly associate and then dissociate from their ligands in a manner largely independent of shear stress(2.Alon R. Hammer D.A. Springer T.A. Nature. 1995; 374: 539-542Crossref PubMed Scopus (600) Google Scholar). The selectins interact weakly with sialylated and fucosylated oligosaccharides such as sialyl Lewis x (sLex), ( 1The abbreviations used are: sLexsialyl Lewis xCHOChinese hamster ovaryC2GnTcore 2 β1-6-N-acetylglucosaminyltransferaseDHFRdihydrofolate reductaseFuc-TfucosyltransferaseHBSSHanks' balanced salt solutionPSGL-1P-selectin glycoprotein ligand-1mAbmonoclonal antibodyPAGEpolyacrylamide gel electrophoresis.) but bind with higher affinity/avidity to only a few glycoproteins(1.McEver R.P. Moore K.L. Cummings R.D. J. Biol. Chem. 1995; 270: 11025-11028Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Studies with mAbs support a physiologic role for one of these glycoproteins, P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 accounts for all the high affinity binding sites for P-selectin on human leukocytes(3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar). PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin under physiologic shear forces(3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar, 4.Norman K.E. Moore K.L. McEver R.P. Ley L. Blood. 1995; 86: 4417-4421Crossref PubMed Google Scholar), and it also contributes to the rolling of neutrophils on E-selectin (5.Patel K.D. Moore K.L. Nollert M.U. McEver R.P. J. Clin. Invest. 1995; 96: 1887-1896Crossref PubMed Scopus (172) Google Scholar).PSGL-1 is a membrane glycoprotein with two identical disulfide-linked subunits(6.Moore K.L. Stults N.L. Diaz S. Smith D.L. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (421) Google Scholar). Each subunit has at most three N-linked glycans, but has many clustered, sialylated O-linked glycans(6.Moore K.L. Stults N.L. Diaz S. Smith D.L. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (421) Google Scholar, 7.Norgard 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), some with polylactosamine terminating in sLex(8.Moore 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). PSGL-1 is a type I membrane protein with an extracellular domain that is rich in serines, threonines, and prolines(9.Sako 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). PSGL-1 binds both E-selectin and P-selectin(8.Moore 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, 9.Sako 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, 10.Lenter M. Levinovitz A. Isenmann S. Vestweber D. J. Cell Biol. 1994; 125: 471-481Crossref PubMed Scopus (167) Google Scholar, 11.Asa D. Raycroft L. Ma L. Aeed P.A. Kaytes P.S. Elhammer Å.P. Geng J.-G. J. Biol. Chem. 1995; 270: 11662-11670Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The structural requirements for binding are not well characterized, although there is evidence that PSGL-1 does not interact identically with P- and E-selectin(5.Patel K.D. Moore K.L. Nollert M.U. McEver R.P. J. Clin. Invest. 1995; 96: 1887-1896Crossref PubMed Scopus (172) Google Scholar, 8.Moore 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). PSGL-1 must be sialylated and fucosylated to bind both molecules(6.Moore K.L. Stults N.L. Diaz S. Smith D.L. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (421) Google Scholar, 9.Sako 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, 10.Lenter M. Levinovitz A. Isenmann S. Vestweber D. J. Cell Biol. 1994; 125: 471-481Crossref PubMed Scopus (167) Google Scholar). Enzymatic removal of N-linked glycans from human neutrophil PSGL-1 does not affect binding to P-selectin, suggesting that O-linked glycans are required for binding(6.Moore K.L. Stults N.L. Diaz S. Smith D.L. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (421) Google Scholar). It is not known if O-linked glycans are required for binding to E-selectin. Fab fragments of PL1, an IgG mAb to PSGL-1, block binding of PSGL-1 to P-selectin but only partially inhibit binding to E-selectin(3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar, 5.Patel K.D. Moore K.L. Nollert M.U. McEver R.P. J. Clin. Invest. 1995; 96: 1887-1896Crossref PubMed Scopus (172) Google Scholar). The PL1 epitope is located near the N terminus (3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar), ( 2F. Li, H. P. Erickson, K. L. Moore, J. A. James, R. D. Cummings, and R. P. McEver, manuscript in preparation.) near three clustered tyrosines within a consensus motif for tyrosine sulfation(9.Sako 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, 12.Huttner W.B. Baeuerle P.A. Mod. Cell Biol. 1988; 6: 97-140Google Scholar). PSGL-1 is sulfated on tyrosine rather than on carbohydrate, and enzymatic removal of sulfate from tyrosine eliminates binding of PSGL-1 to P-selectin(13.Wilkins 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). These data demonstrate that tyrosine sulfate functions in conjunction with sialylated and fucosylated glycans to mediate high affinity binding of PSGL-1 to P-selectin. It is not known if tyrosine sulfation is required for PSGL-1 to bind E-selectin.To further characterize the post-translational modifications that confer binding of PSGL-1 to P-selectin and E-selectin, we transfected CHO cells, which have well characterized oligosaccharide structures, with cDNAs for PSGL-1 and various glycosyltransferases. PSGL-1 was co-expressed with a branching enzyme for O-linked glycans (core 2 β1-6-N-acetylglucosaminyltransferase (C2GnT)), and/or an α1-3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Recombinant PSGL-1 co-expressed in CHO cells with the appropriate glycosyltransferases bound avidly to both P- and E-selectin.EXPERIMENTAL PROCEDURESMaterialsCarrier-free [35S]sulfate (1.1-1.6 Ci/mmol) and [3H]glucosamine (20-45 Ci/mmol) were purchased from DuPont NEN. [3H]Thymidine (5 Ci/mmol) was obtained from Amersham Corp. Sialidase from Arthrobacter ureafaciens and Galβ1-3GalNAc were obtained from Sigma. Endo-α-N-acetylgalactosaminidase was obtained from Boehringer Mannheim. α1-3/4-L-fucosidase was obtained from Takara Biochemicals. Phycoerythrin-streptavidin was purchased from Molecular Probes, Inc. Lipofectamine(™) and all cell culture reagents were purchased from Life Technologies, Inc. Other chemicals were ACS grade or better; unless stated otherwise, they were obtained from Fisher.AntibodiesThe anti-human P-selectin mAbs S12 and G1 were prepared as described previously(14.McEver R.P. Martin M.N. J. Biol. Chem. 1984; 259: 9799-9804Abstract Full Text PDF PubMed Google Scholar, 15.Geng 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 (798) Google Scholar). G1, but not S12, blocks binding of P-selectin to leukocytes. The anti-human E-selectin mAbs ES1 and ES2 were prepared as described previously(5.Patel K.D. Moore K.L. Nollert M.U. McEver R.P. J. Clin. Invest. 1995; 96: 1887-1896Crossref PubMed Scopus (172) Google Scholar). ES1, but not ES2, blocks binding of E-selectin to leukocytes. The anti-human PSGL-1 mAbs PL1 and PL2 were prepared as described previously(3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar). PL1, but not PL2, blocks binding of PSGL-1 to P-selectin. These mAbs are all of the IgG1 subclass. The hybridoma secreting the IgM anti-sLex mAb CSLEX-1 (16.Fukushima K. Hirota M. Terasaki P.I. Wakisaka A. Togashi H. Chia D. Suyama N. Fukushi Y. Nudelman E. Hakomori S. Cancer Res. 1984; 44: 5279-5285PubMed Google Scholar) was obtained from the ATCC. Rabbit antiserum to a peptide corresponding to residues 42-56 of the cDNA-derived amino acid sequence of human PSGL-1 was prepared as described previously(8.Moore 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). MOPC21 (IgG1) was purchased from Cappel-Organon Technika. Fluorescein isothiocyanate-conjugated goat anti-mouse IgG/IgM was purchased from Caltag Laboratories.ProteinsHuman platelet P-selectin and human neutrophil PSGL-1 were purified as described previously(8.Moore 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, 17.Moore K.L. J. Tissue Cult. Methods. 1994; 16: 255-257Crossref Scopus (11) Google Scholar). Human neutrophil PSGL-1 was radiolabeled with 125I as described previously (8.Moore 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). A recombinant soluble form of human P-selectin (sPS, formerly called tPS) was purified as described previously(18.Ushiyama 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). A recombinant soluble form of human E-selectin (sES, formerly called tES) was a gift from Dr. David Lyons(8.Moore 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).cDNAsThe PSGL-1 cDNA was amplified by the polymerase chain reaction from human leukocyte cDNAs as described previously(3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar). We reported that this cDNA encoded an additional decamer repeat between amino acids 127 and 128 of the original published sequence (9.Sako 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) and changed Ala128 to Pro. On reexamination, the decamer sequence was found to be inserted between amino acids 128 and 129, which did not change the original sequence. The additional decamer sequence is TEAQTTQPVP. This additional decamer has been observed in most PSGL-1 cDNAs(19.Veldman G.M. Bean K.M. Cumming D.A. Eddy R.L. Sait S.N.J. Shows T.B. J. Biol. Chem. 1995; 270: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The cDNA insert was excised from the pBK-EF vector with ScaI and KpnI and ligated into the mammalian expression vector pZeoSV (Invitrogen). The pZeoSV plasmid allows selection for resistance to the antibiotic Zeocin(™) in both Escherichia coli and mammalian cells.A mutant PSGL-1 cDNA was created in which the codons encoding the tyrosines at residues 46, 48, and 51 were replaced with codons encoding phenylalanines. Mutagenesis was performed with a polymerase chain reaction protocol(20.Disdier M. Morrissey J.H. Fugate R.D. Bainton D.F. McEver R.P. Mol. Biol. Cell. 1992; 3: 309-321Crossref PubMed Scopus (125) Google Scholar). The PSGL-1 cDNA template was inserted into Bluescript (Stratagene) at the XbaI and KpnI sites. The outside primers for the polymerase chain reaction included a restriction site for XbaI on the 5′ end in the Bluescript vector and for a unique StuI site in the coding region of the PSGL-1 cDNA. The amplified product was sequenced to confirm that it contained the expected substitutions. It was then excised with XbaI and StuI and used to replace the corresponding XbaI-StuI fragment of wild-type PSGL-1 in Bluescript. The entire PSGL-1 mutant cDNA was then excised from Bluescript with XbaI and KpnI and inserted into pZeoSV.The C2GnT cDNA was amplified by the reverse transcriptase polymerase chain reaction from total RNA from human testis (Clontech). The two oligonucleotides used for polymerase chain reaction (5′-GCG GCG GCG TCT AGA CCA CCA TGC TGA GGA CGT TGC TGC GAA G-3′ and 5′-CGC GCG CGT CGA CTC ACA GTC AGT GTT TTA ATG TCT CCA AAG C-3′) were designed to match the 5′ and 3′ ends of the published sequence of the human C2GnT cDNA(21.Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 89: 9326-9330Crossref Scopus (279) Google Scholar). The primers also included additional non-complementary sequence that included an XbaI site 5′ to the initiating methionine and a SalI site 3′ to the stop codon. The amplified product was ligated into the PCRII vector using the TA cloning kit (Invitrogen). The entire insert was sequenced to confirm its identity with the published sequence. The insert was then excised with EcoRI and ligated into the expression vector pcDNA3 (Invitrogen).The human Fuc-TIII and Fuc-TIV cDNAs were amplified by the polymerase chain reaction from genomic DNA. ( 3Q. Zhou, T. Fujimoto, R. P. McEver, and R. D. Cummings, manuscript in preparation.) The amplified products were sequenced to confirm that they matched the published sequences (22.Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes & Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 23.Goelz S.E. Hession C. Goff D. Griffiths B. Tizard R. Newman B. Chi-Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Abstract Full Text PDF PubMed Scopus (286) Google Scholar) and were then ligated into the expression vector pRC/RSV (Invitrogen). The cDNA encoding human Fuc-TVII in the plasmid pCDM8 (24.Natsuka S. Gersten K.M. Zenitas K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar) was a generous gift from Dr. John Lowe.TransfectionsCHO DHFR(-) cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37°C in an atmosphere containing 5% CO2. CHO DHFR(-) cells express sLex when transfected with cDNAs for Fuc-TIII or Fuc-TIV(25.Goelz S. Kumar R. Potvin B. Sundaram S. Brickelmaier M. Stanley P. J. Biol. Chem. 1994; 269: 1033-1040Abstract Full Text PDF PubMed Google Scholar). CHO cells were transfected with Fuc-TIII or Fuc-TIV cDNA in pRC/RSV using the Stratagene Transfection MBS kit according to the suggested protocol. CHO cells were transfected with C2GnT cDNA in pcDNA3, or C2GnT cDNA plus Fuc-TIII cDNA, using calcium phosphate precipitation. The transfected cells were selected in medium containing 400 μg/ml of G418 (Life Technologies, Inc.). Individual colonies were isolated and recloned by limiting dilution. In this manner, permanently transfected CHO cells were isolated that expressed Fuc-TIII, Fuc-TIV, C2GnT, or Fuc-TIII plus C2GnT. Portions of these cells were then transfected with PSGL-1 cDNA in pZeoSV using Lipofectamine(™). Some of the Fuc-TIV-expressing cells were transfected with both PSGL-1 cDNA and C2GnT cDNA. All cells transfected a second time were selected in medium containing both G418 (400 μg/ml) and Zeocin(™) (250 μg/ml). Individual colonies were expanded and, in some cases, recloned by limiting dilution. In this manner, permanently transfected CHO cells were isolated that expressed various combinations of PSGL-1, C2GnT, Fuc-TIII, and/or Fuc-TIV.Fuc-TVII cDNA was transiently transfected into CHO cells permanently transfected with PSGL-1 cDNA, or with PSGL-1 cDNA plus C2GnT cDNA. In other experiments, wild-type or mutated PSGL-1 cDNA was transiently transfected into CHO cells permanently transfected with Fuc-TIII cDNA or C2GnT cDNA plus Fuc-TIII cDNA. Transiently transfected CHO cells were analyzed 2 days after transfection.COS-7 cells were transfected with Fuc-TIII or Fuc-TIV cDNA using Lipofectamine(™) and selected in medium containing 400 μg/ml G418 to obtain cells permanently expressing Fuc-TIII or Fuc-TIV.3 COS cells expressing Fuc-TIII were transiently transfected with PSGL-1 cDNA, or PSGL-1 plus C2GnT cDNA using Lipofectamine(™). The cells were analyzed 2 days after transfection.C2GnT AssayCells were detached with 0.02% EDTA in phosphate-buffered saline, washed twice with phosphate-buffered saline, centrifuged, and stored as cell pellets at −20°C until they were analyzed. Frozen cell pellets were typically resuspended in 100 μl of 10 mM sodium cacodylate, pH 7.2, containing 0.1 mg/ml leupeptin (Sigma), 0.2 mg/ml aprotinin (Sigma), and 1 mM Pefabloc SC (AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride HCL) (Boehringer Manneheim), and homogenized with 30 strokes of a hand-held Teflon Potter-Elvehjem homogenizer. Homogenates were brought to 1% Triton X-100 by addition of 0.1 volume of 10% Triton X-100 in buffer. Protein concentrations were determined using the BCA protein assay (reagents from Sigma) with bovine serum albumin as standard. C2GnT activities were assayed with a modification of a previously described procedure(26.Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconj. J. 1988; 5: 49-63Crossref Scopus (277) Google Scholar). Briefly, C2GnT activities were measured by mixing 2 μl of cell lysate with 5 μl of assay mixture containing 25 mM sodium cacodylate, pH 6.5, 5 mM EDTA, 333 mM GlcNAc, 10% glycerol, 0.8 mM UDP-GlcNAc (100,000 cpm/nmol) (unlabeled UDP-GlcNAc from Sigma, and UDP-6-[3H]-N-acetylglucosamine from American Radiolabeled Chemicals, Inc.), and 8 mM benzyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranoside from Sigma. Mixtures were incubated at 37°C for 4 h. Each reaction mixture was diluted with 0.3 ml of water and loaded onto a C18 SepPak cartridge (Millipore) mounted on a vacuum manifold. The cartridge was washed with 25 ml of water to remove unreacted radiolabeled sugar-nucleotide. The hydrophobic glucosylated product was then eluted with 3 ml of methanol and counted in a scintillation counter. Blank values were obtained by carrying out assays without acceptor and were subtracted from the values obtained with acceptor.Flow CytometryHL-60 cells were maintained in RPMI 1640 containing 20% fetal calf serum, 4 mML-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Binding of mAbs to intact HL-60, CHO, or COS cells was measured as described previously(3.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar). Binding of fluid-phase P-selectin to intact cells was assessed as described previously(27.Moore K.L. Thompson L.F. Biochem. Biophys. Res. Commun. 1992; 186: 173-181Crossref PubMed Scopus (88) Google Scholar).Cell Adhesion AssayConfluent or near-confluent monolayers of CHO cells were labeled for 16 h in medium containing 10 μCi/ml [3H]thymidine. The cells were washed with Hanks' balanced salt solution (HBSS, Life Technologies, Inc.) and then detached from the substratum by incubation in 0.02% EDTA for 10 min. The cells were washed twice in serum-free Dulbecco's modified Eagle's medium and then suspended at 2 × 106 cells/ml in Dulbecco's modified Eagle's medium containing 1% bovine serum albumin and 1% fetal bovine serum. The cells were labeled to a specific activity of 2-7 × 10−6μCi/cell; when the cells were centrifuged, greater than 95% of the radioactivity remained associated with the cell pellet.Microtiter wells (Immulon I Removawell(™) strips, Dynatech Laboratories) were coated with soluble P-selectin or soluble E-selectin (each at 2.5 μg/ml) in 100 μl of HBSS overnight at 4°C. The wells were then blocked with 1% human serum albumin in HBSS for 2 h at room temperature. Labeled CHO cells (100 μl, 2 × 105 cells) were added to each well and incubated on an orbital shaker for 20 min at room temperature. In some experiments, the cells were preincubated with 10 μg/ml of the mAbs PL1, PL2, G1, or S12. After aspirating the unbound cells, the wells were gently washed three times with HBSS. Each well was then cut out, and the bound radioactivity was determined by liquid scintillation counting. The number of bound cells was derived from a standard curve generated with known concentrations of cells.Metabolic Labeling and Immunoprecipitation of PSGL-1CHO cells were metabolically labeled with [3H]glucosamine or [35S]sulfate as described previously for HL-60 cells(6.Moore K.L. Stults N.L. Diaz S. Smith D.L. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (421) Google Scholar, 13.Wilkins 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). Cell extracts were immunoprecipitated as described previously(8.Moore 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, 13.Wilkins 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), using the rabbit antiserum to a peptide corresponding to residues 42-56 of PSGL-1 or, as a control, normal rabbit serum. The immunoprecipitates were analyzed by SDS-PAGE under reducing or nonreducing conditions, followed by fluorography. In some gels, 125I-labeled PSGL-1 from human neutrophils was analyzed in parallel.Glycosidase Treatment of PSGL-1Immunoprecipitates of [3H]glucosamine-labeled PSGL-1 were either mock-treated or treated with 10 milliunits/ml sialidase, alone or with 0.1 milliunit/ml endo-α-galactosaminidase, in 20 μl of 50 mM sodium acetate, pH 5.5, for 18 h at 37°C. The samples were then analyzed by SDS-PAGE under nonreducing conditions, followed by fluorography.Structural Analysis of PSGL-1 OligosaccharidesFollowing identification of [3H]glucosamine-labeled PSGL-1 by fluorography, the dried gel was aligned with the exposed x-ray film, and the band from the nonreducing lane was subjected to β-elimination (28.Cummings R.D. Merkle R.K. Stults N.L. Methods Cell Biol. 1989; 32: 141-183Crossref PubMed Scopus (43) Google Scholar). The released oligosaccharides were treated with sialidase (2.5 milliunits/ml) in 20 μl of 50 mM sodium acetate, pH 5.5, for 18 h at 37°C(28.Cummings R.D. Merkle R.K. Stults N.L. Methods Cell Biol. 1989; 32: 141-183Crossref PubMed Scopus (43) Google Scholar). In some experiments, neutral β-eliminated oligosaccharides from PSGL-1 co-expressed with C2GnT and Fuc-TIV were treated with 0.25 milliunit/ml of α1-3/4-L-fucosidase in 20 μl of 50 mM KH2PO4, pH 6.0, 0.1 M NaCl, 0.02% sodium azide at 37°C for 18 h. A portion of the oligosaccharides was analyzed by amine-adsorption high performance liquid chromatography on a Varian AX-5 column (4 mm × 30 cm). The column was equilibrated in 65:35 (v/v) acetonitrile:deionized water and was eluted with a linear gradient of increasing water (0.33%/min)(29.Blanken W.M. Bergh M.L.E. Koppen P.L. Van den Eijnden D.H. Anal. Biochem. 1985; 145: 322-330Crossref PubMed Scopus (87) Google Scholar). Fractions (0.5 ml) were collected, and radioactivity in each fraction was determined by scintillation counting.The O-linked core 1 disaccharide standard, Galβ1-3GalNAcOH, was prepared by reduction of Galβ1-3GalNAc with NaB[3H]4. Other [3H]glucosamine-labeled standards were prepared from β-eliminated oligosaccharides from radiolabeled glycoproteins of HL-60 cells. Each O-linked standard was authenticated by sequential exoglycosidase digestion to yield the Galβ1-3GalNAcOH structure(28.Cummings R.D. Merkle R.K. Stults N.L. Methods Cell Biol. 1989; 32: 141-183Crossref PubMed Scopus (43) Google Scholar). Columns were also calibrated using chitin-derived oligosaccharides (GlcNAc1−7) prepared as described previously(28.Cummings R.D. Merkle R.K. Stults N.L. Methods Cell Biol. 1989; 32: 141-183Crossref PubMed Scopus (43) Google Scholar).Detection of Tyrosine SulfateTyrosine sulfation of PSGL-1 was determined as described previously(13.Wilkins 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). Briefly, immunoprecipitated 35S-PSGL-1 was subjected to base hydrolysis, then analyzed by chromatography on a Varian AX-5 column (4 mm × 30 cm) that was eluted with a gradient of NaH2PO4.RESULTSPSGL-1 Expressed on CHO Cells Binds Fluid-phase P-selectin Only When It Is Co-expressed with C2GnT and an α1-3 FucosyltransferaseTo study the modifications required for recombinant PSGL-1 to bind P- and E-selectin, we used CHO cells because they have well characterized oligosaccharide structures. CHO cells synthesize complex N-linked glycans with type 2 polylactosamine sequences(30.
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