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The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês

10.1074/jbc.m208283200

ISSN

1083-351X

Autores

Min‐Chuan Huang, Anna Laskowska, Dietmar Vestweber, Martin K. Wild,

Tópico(s)

Galectins and Cancer Biology

Resumo

Fuc-TIV and Fuc-TVII are the two α(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLex), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLex-like epitopes in transfected Chinese hamster ovary (CHO)-Pro−5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLex. Surprisingly however, Fuc-TIV-generated sLex was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80–90% of Fuc-TVII-generated sLex was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLex on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivospecificity for acceptor substrates with Fuc-TVII generating sLex preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLex is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates. Fuc-TIV and Fuc-TVII are the two α(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLex), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLex-like epitopes in transfected Chinese hamster ovary (CHO)-Pro−5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLex. Surprisingly however, Fuc-TIV-generated sLex was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80–90% of Fuc-TVII-generated sLex was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLex on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivospecificity for acceptor substrates with Fuc-TVII generating sLex preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLex is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates. The three known selectins, l-, E-, and P-selectin are cell adhesion molecules that initiate interactions between leukocytes and endothelial cells during leukocyte extravasation (1Vestweber D. Blanks J.E. Physiol. Rev. 1999; 79: 181-213Google Scholar). The minimal ligand structure for all three selectins is the tetrasaccharide sialyl Lewis X (sLex). 1The abbreviations used are: sLex, sialyl Lewis X (NeuAcα2,3Galβ1,4(Fucα1,3)-GlcNAc); Fuc-T, fucosyltransferase; mFuc-T, mouse Fuc-T; hFuc-T, human Fuc-T; CHO, Chinese hamster ovary; mAb, monoclonal antibody; DHFR, dihydrofolate reductase; ESL-1, E-selectin ligand-1; PSGL-1, P-selectin glycoprotein ligand-1; C2GnT, core-2 β-1,6-N-acetylglucosaminyl-transferase; αMEM, α minimal essential medium; PBS, phosphate-buffered saline; PC4, CHO cells transfected with PSGL-1, core-2 branching enzyme, and Fuc-TIV (for PC4) or Fuc-TVII (for PC7); FITC, fluorescein isothiocyanate; DTAF, dichlorotriazinylaminofluorescein; TRITC, tetramethylrhodamine isothiocyanate; FCS, fetal calf serum; RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium; CTS, cytoplasmic, transmembrane, and stem region 1The abbreviations used are: sLex, sialyl Lewis X (NeuAcα2,3Galβ1,4(Fucα1,3)-GlcNAc); Fuc-T, fucosyltransferase; mFuc-T, mouse Fuc-T; hFuc-T, human Fuc-T; CHO, Chinese hamster ovary; mAb, monoclonal antibody; DHFR, dihydrofolate reductase; ESL-1, E-selectin ligand-1; PSGL-1, P-selectin glycoprotein ligand-1; C2GnT, core-2 β-1,6-N-acetylglucosaminyl-transferase; αMEM, α minimal essential medium; PBS, phosphate-buffered saline; PC4, CHO cells transfected with PSGL-1, core-2 branching enzyme, and Fuc-TIV (for PC4) or Fuc-TVII (for PC7); FITC, fluorescein isothiocyanate; DTAF, dichlorotriazinylaminofluorescein; TRITC, tetramethylrhodamine isothiocyanate; FCS, fetal calf serum; RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium; CTS, cytoplasmic, transmembrane, and stem region The biosynthesis of sLex requires the sequential action of a number of glycosyltransferases of which the final reaction is mediated by α(1, 3)-fucosyltransferases (2Holmes E.H. Ostrander G.K. Hakomori S. J. Biol. Chem. 1986; 261: 3737-3743Google Scholar, 3Hanisch F.G. Mitsakos A. Schroten H. Uhlenbruck G. Carbohydr. Res. 1988; 178: 23-28Google Scholar). Of the six known human α(1, 3)-fucosyltransferases (Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, Fuc-TVII, and Fuc-TIX), Fuc-TIV and Fuc-TVII have been implicated in the generation of selectin ligands (4Lowe J.B. Kidney Int. 1997; 51: 1418-1426Google Scholar). Fuc-TVII-deficient mice exhibit severe defects in the transmigration of neutrophils into inflamed peritoneum, demonstrating the central importance of this enzyme for the generation of E- and P-selectin ligands on neutrophils (5Maly P. Thall A.D. Petryniak B. Rogers C.E. Smith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Google Scholar). In addition, the lack of Fuc-TVII severely reduced the expression ofl-selectin ligands on high endothelial venules in lymph nodes, leading to strong inhibition of lymphocyte homing (5Maly P. Thall A.D. Petryniak B. Rogers C.E. Smith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Google Scholar). The ability of Th1 cells to bind to E- and P-selectin is induced during the course of differentiation of these cells (6Borges E. Tietz W. Steegmaier M. Moll T. Hallmann R. Hamann A. Vestweber D. J. Exp. Med. 1997; 185: 573-578Google Scholar), and this correlates with the induction of Fuc-TVII (7Wagers A.J. Waters C.M. Stoolman L.M. Kansas G.S. J. Exp. Med. 1998; 188: 2225-2231Google Scholar). Human Fuc-TIV, also termed ELFT for ELAM-1ligand fucosyltransferase, was reported to generate E-selectin binding carbohydrate modifications (8Goelz S.E. Hession C. Goff D. Griffiths B. Tizard R. Newman B. Chi Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Google Scholar). However, this activity has been observed only when the enzyme is expressed at a certain level and in cells with a certain glycosylation phenotype (9Lowe J.B. Kukowska Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Google Scholar, 10Kumar R. Potvin B. Muller W.A. Stanley P. J. Biol. Chem. 1991; 266: 21777-21783Google Scholar, 11Goelz S. Kumar R. Potvin B. Sundaram S. Brickelmaier M. Stanley P. J. Biol. Chem. 1994; 269: 1033-1040Google Scholar, 12Knibbs R.N. Craig R.A. Natsuka S. Chang A. Cameron M. Lowe J.B. Stoolman L.M. J. Cell Biol. 1996; 133: 911-920Google Scholar, 13Wagers A.J. Stoolman L.M. Kannagi R. Craig R. Kansas G.S. J. Immunol. 1997; 159: 1917-1929Google Scholar). Transfection of Chinese hamster ovary (CHO) cells of the DHFR− strain with human Fuc-TIV led to the expression of CSLEX-1-reactive sLex ,although no sLexcould be generated by this enzyme in CHO-Pro−5 cells (10Kumar R. Potvin B. Muller W.A. Stanley P. J. Biol. Chem. 1991; 266: 21777-21783Google Scholar). In BHK-21 cells, human Fuc-TIV generates Lex seven times more efficiently than sLex (14Grabenhorst E. Nimtz M. Costa J. Conradt H.S. J. Biol. Chem. 1998; 273: 30985-30994Google Scholar). In COS cells, human as well as mouse Fuc-TIV could not generate sLex-epitopes on the cell surface as defined by the mAb CSLEX-1 (15Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hiraiwa N. Jenkins N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Google Scholar). In contrast, inin vitro enzyme assays mouse Fuc-TIV transfected in COS cells, but not human Fuc-TIV, could efficiently accept 3′-sialyl-N-acetyllactosamine to form sLex (15Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hiraiwa N. Jenkins N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Google Scholar). The biochemical basis for the selective generation of sLex-like structures by Fuc-TIV is, at present, unexplained. Yet, Fuc-TIV does participate in the generation of physiologically relevant selectin ligands, although it clearly plays a minor role compared with Fuc-TVII. Although the fraction of rolling leukocytes in non-inflamed venules of the skin was normal in Fuc-TIV−/− animals, an increase of the rolling velocity was observed (16Weninger W. Ulfman L.H. Cheng G. Souchkova N. Quackenbush E.J. Lowe J.B. von Andrian U.H. Immunity. 2000; 12: 665-676Google Scholar). Furthermore, leukocytes rolling at velocities below 10 μm/sec were absent in Fuc-TIV−/− mice, and Fuc-TIV deficiency in the context of Fuc-TVII deletion extinguished residual selectin ligand activities observed in Fuc-TVII−/− mice (17Homeister J.W. Thall A.D. Petryniak B. Maly P. Rogers C.E. Smith P.L. Kelly R.J. Gersten K.M. Askari S.W. Cheng G. Smithson G. Marks R.M. Misra A.K. Hindsgaul O. von Andrian U.H. Lowe J.B. Immunity. 2001; 15: 115-126Google Scholar). Thus, it is important to further analyze the relative contribution of Fuc-TVII and Fuc-TIV to the generation of sLex-modified glycoconjugates that might serve as selectin ligands. We have recently analyzed the selective contribution of Fuc-TVII and Fuc-TIV to the generation of the glycoprotein ligands E-selectin ligand-1 (ESL-1) (18Levinovitz A. Mühlhoff J. Isenmann S. Vestweber D. J. Cell Biol. 1993; 121: 449-459Google Scholar, 19Steegmaier M. Levinovitz A. Isenmann S. Borges E. Lenter M. Kocher H.P. Kleuser B. Vestweber D. Nature. 1995; 373: 615-620Google Scholar) and P-selectin glycoprotein ligand-1 (PSGL-1) (20Moore K.L. Stults N.L. Diaz S. Smith D.F. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Google Scholar, 21Sako 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: 1179-1186Google Scholar, 22Yang J. Galipeau J. Kozak C.A. Furie B.C. Furie B. Blood. 1996; 87: 4176-4186Google Scholar, 23McEver R.P. J. Clin. Invest. 1997; 100: 485-491Google Scholar). Each of these two ligands requires sLex for binding, with the difference that ESL-1 requires sLex on N-glycans (18Levinovitz A. Mühlhoff J. Isenmann S. Vestweber D. J. Cell Biol. 1993; 121: 449-459Google Scholar), whereas PSGL-1 requires it on O-glycans that carry a core-2 branch (21Sako 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: 1179-1186Google Scholar, 24Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar). In addition, PSGL-1 requires sulfation of the tyrosine residues within its N terminus for binding to P-selectin (25Pouyani T. Seed B. Cell. 1995; 83: 333-343Google Scholar, 26Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Google Scholar, 27Wilkins P.P. Moore K.L. McEver R.P. Cummings R.D. J. Biol. Chem. 1995; 270: 22677-22680Google Scholar, 28Leppänen 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: 24838-24848Google Scholar, 29Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Google Scholar). The physiological relevance of PSGL-1 in leukocyte extravasation is well established (23McEver R.P. J. Clin. Invest. 1997; 100: 485-491Google Scholar). Antibodies against mouse PSGL-1 inhibit neutrophil recruitment into inflamed peritoneum (30Borges E. Eytner R. Moll T. Steegmaier M. Matthew A. Campbell P. Ley K. Mossmann H. Vestweber D. Blood. 1997; 90: 1934-1942Google Scholar), and the migration of Th1 cells into inflamed skin (6Borges E. Tietz W. Steegmaier M. Moll T. Hallmann R. Hamann A. Vestweber D. J. Exp. Med. 1997; 185: 573-578Google Scholar). Similar results were obtained with mice deficient for the PSGL-1 gene (31Yang J. Hirata T. Croce K. Merrill-Skoloff G. Tchernychev B. Williams E. Flaumenhaft R. Furie B.C. Furie B. J. Exp. Med. 1999; 190: 1769-1782Google Scholar, 32Hirata T. Merrill-Skoloff G. Aab M. Yang J. Furie B.C. Furie B. J. Exp. Med. 2000; 192: 1669-1676Google Scholar). After analyzing mouse neutrophils from Fuc-TIV and Fuc-TVII deficient mice, we have shown recently that Fuc-TVII exclusively directs the expression of PSGL-1 glycoforms that bind with high affinity to P-selectin (33Huang M.C. Zollner O. Moll T. Maly P. Thall A.D. Lowe J.B. Vestweber D. J. Biol. Chem. 2000; 275: 31353-31360Google Scholar). In contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. We could mimic this selectivity in transfected CHO-Pro−5 cells that expressed PSGL-1, ESL-1, and core-2 β-1,6-N-acetylglucosaminyl-transferase (C2GnT) (33Huang M.C. Zollner O. Moll T. Maly P. Thall A.D. Lowe J.B. Vestweber D. J. Biol. Chem. 2000; 275: 31353-31360Google Scholar). The molecular mechanisms in the Golgi that are the basis for this in vivo selectivity are unknown. In addition to glycoprotein counter-receptors, glycolipids have also been described as carriers of sLex and as selectin ligands (34Alon R. Feizi T. Yuen C.T. Fuhlbrigge R.C. Springer T.A. J. Immunol. 1995; 154: 5356-5366Google Scholar, 35Stroud M.R. Handa K. Salyan M.E.K. Ito K. Levery S.B. Hakomori S. Reinhold B.B. Reinhold V.N. Biochemistry. 1996; 35: 758-769Google Scholar, 36Handa K. Stroud M.R. Hakomori S. Biochemistry. 1997; 36: 12412-12420Google Scholar). However, selectin-mediated cell binding to glycolipid counter-receptors has always been tested with immobilized glycolipids. Hence, the relevance of glycolipids as selectin ligands in the physiological context of a cell surface is still unclear. Here, we have analyzed whether Fuc-TVII and Fuc-TIV would differ in their ability to generate sLex-carrying glycolipids or glycoproteins in transfected CHO-Pro−5 cells. Surprisingly, we found that Fuc-TIV preferentially generates sLex on glycolipids, whereas Fuc-TVII preferentially decorates sLex on glycoproteins. A comparison of the E-selectin binding efficiency of these transfectants suggests the possibility that this unexpected acceptor specificity could be a reason for the lower efficiency with which Fuc-TIV generates E-selectin ligands as compared with Fuc-TVII. Furthermore, our results demonstrate that the Golgi environment of CHO-Pro−5 cells provides conditions under which each enzyme is able to generate sLex-epitopes. However, the enzymes preferentially synthesize these epitopes on different classes of carrier moieties in vivo. CHO-Pro−5 cells (11Goelz S. Kumar R. Potvin B. Sundaram S. Brickelmaier M. Stanley P. J. Biol. Chem. 1994; 269: 1033-1040Google Scholar) were obtained from Dr. A. Hasilik (University of Marburg) and grown in α-minimal essential medium (αMEM) (Invitrogen) containing 10% fetal bovine serum, 100 μg/mll-glutamine, and 100 units/ml penicillin/streptomycin at 37 °C in a humidified atmosphere of 10% CO2. Stable transfection was performed as described previously (37Zöllner O. Vestweber D. J. Biol. Chem. 1996; 271: 33002-33008Google Scholar) with slight modifications. Briefly, 1 × 107 CHO-Pro−5 cells harvested in PBS (containing 5 mm EDTA) at 90% confluency were electroporated in 0.6 ml of PBS either with 10 μg of pcDNA3 vector containing mouse Fuc-TIV cDNA (38Borges E. Pendl G. Eytner R. Steegmaier M. Zöllner O. Vestweber D. J. Biol. Chem. 1997; 272: 28786-28792Google Scholar) or 20 μg of pcDNA1 vector containing human Fuc-TIV cDNA (37Zöllner O. Vestweber D. J. Biol. Chem. 1996; 271: 33002-33008Google Scholar) (both kindly provided by Dr. John Lowe, University of Michigan, Ann Arbor, MI) in a 0.4 cm cuvette at 950 μF and 0.25 kV. The human Fuc-TIV plasmid was co-transfected with 5 μg of pAH58 vector (39Riele H. Maandag E.R. Clarke A. Hooper M. Berns A. Nature. 1990; 348: 649-651Google Scholar) for hygromycin B resistance. Cells transfected with mouse Fuc-TIV were selected with 800 μg/ml G418; cells transfected with human Fuc-TIV were selected with 300 μg/ml hygromycin. Mock transfected cells were generated with the same vectors lacking the Fuc-T cDNA inserts. CHO-Pro−5 cells co-transfected with mouse PSGL-1 and human C2GnT were called PC and have been described (33Huang M.C. Zollner O. Moll T. Maly P. Thall A.D. Lowe J.B. Vestweber D. J. Biol. Chem. 2000; 275: 31353-31360Google Scholar). These cells, further transfected in a second round either with mouse Fuc-TIV in pcDNA3 or mouse Fuc-TVII in pcDNA3 and co-transfected with the pAH58 vector for hygromycin B resistance, were also described previously and are named PC4 and PC7, respectively (33Huang M.C. Zollner O. Moll T. Maly P. Thall A.D. Lowe J.B. Vestweber D. J. Biol. Chem. 2000; 275: 31353-31360Google Scholar). A hybrid cDNA encoding the cytoplasmic, transmembrane domain and stem region of mouse Fuc-TIV fused to the catalytic domain of mouse Fuc-TVII was constructed according to a strategy taken from Ref. 40Grabenhorst E. Conradt H.S. J. Biol. Chem. 1999; 274: 36107-36116Google Scholar as follows. In a first PCR, a 303-bp fragment was generated using sense primer 5′-TGG AAT TCT GCA GAT CA-3′, antisense primer 5′-CCA GAT AAG GAT GGT GAG CAG GCG TTG CGG AGC TGG-3′, and the pcDNA3 vector containing mouse Fuc-TIV cDNA as a template (PCR conditions were 3 min at 94 °C, 25 cycles with 15s at 94 °C, 20s at 55 °C, 30s at 72 °C, and 10 min at 72 °C). The purified PCR product was then used as a 5′-megaprimer in a second PCR together with antisense primer 5′-GTC AAG CCT GGA ACC AGC TT-3′ and the pcDNA3 vector containing mouse Fuc-TVII cDNA as a template (PCR conditions were 3 min at 94 °C, 30 cycles with 20s at 94 °C, 20s at 50 °C, 30s at 72 °C, and 10 min at 72 °C). The PCR product was cloned into a pcDNA3 vector using the TOPO-TA system (Invitrogen). PC cells were transfected with this construct (plus co-transfection with pAH58 vector for hygromycin B resistance) and were referred to as PC 4/7 chimera cells. Following transfection, CHO cells were seeded into 90-mm culture dishes. After 6–10 days, individual clones were surrounded by glass rings, sealed with sterile grease, and released with trypsin/EDTA solution (Invitrogen). All cell lines were subcloned twice in microtiter plates by seeding the cells at a statistical density of 0.5 cells/well followed by analysis for the expression of transfected cDNAs using PSGL-1- and sLex-specific antibodies in flow cytometry. The following antibodies were used. HECA-452 (anti-sLex, rat IgM) (41Duijvestijn A.M. Horst E. Pals S.T. Rouse B.N. Steere A.C. Picker L.J. Meijer C.J. Butcher E.C. Am. J. Pathol. 1988; 130: 147-155Google Scholar) and CSLEX-1 (anti-sLex, mIgM) (42Fukushima K. Hirota M. Terasaki P.I. Wakisaka A. Togashi H. Chia D. Suyama N. Fukushi Y. Nudelman E. Hakomori S. Cancer Res. 1984; 44: 5279-5285Google Scholar) were purchased from the ATCC. Anti-CD65s (a variant of sLex; NeuAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4(Fucα1,3)-GlcNAc (clone VIM-2, mIgM)) (43Macher B.A. Buehler J. Scudder P. Knapp W. Feizi T. J. Biol. Chem. 1988; 263: 10186-10191Google Scholar) was purchased from Bio Research (Kaumberg, Austria); 2F3 (anti-sLex, mIgM) (44Ohmori K. Takada A. Ohwaki I. Takahashi N. Furukawa Y. Maeda M. Kiso M. Hasegawa A. Kannagi M. Kannagi R. Blood. 1993; 82: 2797-2805Google Scholar) and anti-CD15 (Lex) mAb clone HI98 (mIgM) were purchased from BD Pharmingen. FITC-conjugated rabbit anti-mouse IgG, FITC-conjugated rabbit anti-rat IgG and IgM, FITC-conjugated rabbit anti-mouse IgM, DTAF-conjugated goat anti-human IgG, TRITC-conjugated goat anti-mouse IgG and IgM, peroxidase-conjugated anti-rabbit IgG and peroxidase-conjugated anti-rat IgG and IgM were all purchased from Dianova (Hamburg, Germany). Polyclonal antibodies against ESL-1 (Affi-60) and polyclonal antibodies against mPSGL-1 (Affi-124) were generated as described previously (6Borges E. Tietz W. Steegmaier M. Moll T. Hallmann R. Hamann A. Vestweber D. J. Exp. Med. 1997; 185: 573-578Google Scholar, 19Steegmaier M. Levinovitz A. Isenmann S. Borges E. Lenter M. Kocher H.P. Kleuser B. Vestweber D. Nature. 1995; 373: 615-620Google Scholar). Anti-mPSGL-1 mAb 4RA10 (rat IgG) was described previously (45Pendl G.G. Robert C. Steinert M. Thanos R. Eytner R. Borges E. Wild M.K. Lowe J.B. Fuhlbrigge R.C. Kupper T.S. Vestweber D. Grabbe S. Blood. 2002; 99: 946-956Google Scholar). The GM3-specific antibody GMR6 (mIgM) was purchased from Seikagaku (Tokyo, Japan). Isotype-matched negative control antibodies were R4–22 (rat IgM, BD Pharmingen), C48.6 (mIgM, BD Pharmingen), and anti-mP-selectin antibody RB40.34 (rat IgG (46Bosse R. Vestweber D. Eur. J. Immunol. 1994; 24: 3019-3024Google Scholar)). E-selectin-IgG and vascular endothelial cadherin-IgG chimeras were produced as described previously (47Hahne M. Jäger U. Isenmann S. Hallmann R. Vestweber D. J. Cell Biol. 1993; 121: 655-664Google Scholar, 48Gotsch U. Borges E. Bosse R. Böggemeyer E. Simon M. Mossmann H. Vestweber D. J. Cell Sci. 1997; 110: 583-588Google Scholar). HECA-452 and CSLEX-1 were prepared from supernatants of hybridomas (obtained from the ATCC) cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum. Flow cytometry was essentially performed as described (37Zöllner O. Vestweber D. J. Biol. Chem. 1996; 271: 33002-33008Google Scholar, 38Borges E. Pendl G. Eytner R. Steegmaier M. Zöllner O. Vestweber D. J. Biol. Chem. 1997; 272: 28786-28792Google Scholar). Briefly, 5 × 105cells were incubated with 10–20 μg/ml mAb in flow cytometry buffer (Hanks' buffer, 3% FCS, 0.04% azide) at 4 °C or 37 °C for 20 min. For detection of selectin-IgG binding, 20 μg/ml E-selectin-IgG construct in flow cytometry buffer was used. Cells were washed twice with the same buffer and stained with FITC- or DTAF-conjugated secondary monoclonal antibodies at 1:100 dilutions. After 20 min of incubation at 4 °C, cells were washed twice, counter stained with propidium iodide, and analyzed by flow cytometry (FACScalibur, BD Life Sciences). Data were collected by gating for propidium iodide-negative cells and analyzed using the CellQuest program. 2 × 108 cells were lysed in 1 ml of lysis buffer (1% Triton X-100, 20 mmTris-HCl, pH 8.4, 160 mm NaCl, 1 mmCaCl2, 1 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride, and 0.1 units/ml α2-macroglobulin), and cell debris was pelleted at 10,000 × g for 10 min. The protein concentration was determined by the BCA assay (Pierce). Fucosyltransferase activity assays were carried out in a total volume of 40 μl containing 10 μg of protein extract, 25 mm cacodylate (pH 6.2), 0.25% Triton X-100, 10 mm MnCl2, 5 mmGDP-fucose, 0.07 μCi of GDP-[3H]fucose, and 10 mm N-acetyllactosamine (NAL) as acceptor oligosaccharide (purchased from Dextra Laboratories). Blanks were prepared by omitting the acceptor in the reaction mixture. After incubation at 37 °C for 2 h, 1 ml of a Dowex 1-X8 slurry (1:4 (w/v) in water) was added to the reaction and vortexed. 500 μl of the supernatant was counted in 5 ml of scintillant (Ultima Gold XR, Packard). To obtain values solely due to the fucosylation of the acceptor substrate, total counts of the control (without acceptor substrate) were subtracted from total counts of samples with the acceptor. The specific activity of the fucosyltransferase was calculated as pmol/min/mg. Total cellular RNA was isolated from 107 cells using the Trizol reagent (Invitrogen) according to the manufacturer's protocol. 1 μl of total RNA was used as template in a 20-μl reverse transcription reaction. To reflect the initial mRNA expression levels, PCR amplification of cDNA was carried out with cycle numbers that had been tested to be in the linear range and well below the plateau phase of amplification. The following number of cycles and primers were used. For β-actin: 20 cycles, sense-primer 5′-TGG GTC AGA AGG ACT CCT ATG-3′, antisense-primer 5′-CAG GCA GCT CAT AGC TCT TCT-3′, product of a 591-bp fragment. For mouse Fuc-TIV: 25 cycles, sense-primer 5′-GAC GCT AAC TGG CAA AGC CCT-3′, antisense-primer 5′-GGT GAT GTA ATC CAC GTG CCG-3′, product of a 451-bp fragment. For human Fuc-TIV: 25 cycles, sense primer 5′-TGG ATC TGC GCG TGT TGG ACT-3′, antisense primer 5′-CGG TCA CAT GTT GGC TCA GTT-3′, product of a 360-bp fragment. For mouse Fuc-TVII: 25 cycles, sense primer 5′-CCG TCT GAG TGC TAA CCG GAG-3′, antisense primer 5′-CGC CAG AAC TTC TCA GTG ATG-3′, product of a 501-bp fragment. PCR reactions were performed in a final volume of 50 μl with 2.5 units ofTaq polymerase (Amersham Biosciences) and 4 μl of single-stranded cDNA from the RT reaction as template. The PCR amplification was carried out using a Biometra thermal cycler with the following program: 95 °C for 3 min followed by 25 or 20 cycles of 94 °C for 50 s, 56 °C for 50 s, and 72 °C for 50 s followed by 72 °C for 7 min. To compare the mRNA expression levels in more detail, PCR was carried out as above using 2-fold serial dilutions of the input cDNA. PCR products were separated by 1.5% agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized with 32P-labeled hFuc-TIV, mFuc-TIV, mFuc-TVII, or β-actin cDNA probe (106 cpm/ml). The blots were exposed to Hyperfilm (AmershamBiosciences). For each experiment, cells were harvested in PBS containing 5 mm EDTA and washed once with αMEM. For proteinase K treatment, cells were resuspended in PBS at a density of 1 × 107 cells/ml. 1 × 106 cells (in 100 μl) were aliquoted to each well of 96-well v-bottom plates, and 1 μl of proteinase K stock solution (Roche Molecular Biochemicals; 10 mg/ml in 50 mm Tris-HCl, pH 8.0, and 1 mm CaCl2) was added to each well. After 20 min of incubation at 37 °C, 100 μl of PBS containing 6% FCS and a protease inhibitor mixture containing EDTA (Roche Molecular Biochemicals) was added to inhibit proteinase K activity. After 5 min of incubation at room temperature, cells were washed twice with Hanks' buffer containing 3% FCS and 0.04% azide. In all assays, proteinase K treatment resulted in less than 15% cell death. For trypsin treatment, cells were resuspended in αMEM and plated in V-bottom plates (Greiner) with 7.5 × 105 cells/well. Trypsin (Roche Molecular Biochemicals) was added to give a final concentration of 1.7 mg/ml. Cells were incubated for 1 h at 37 °C and washed three times in αMEM. Trypsin treatment resulted in less than 10% cell death. Cells were plated on chamber slides (Nunc Lab-Tec) 1 day before the experiment, washed once with Hanks' buffer/0.04% azide, and fixed with 4% paraformeldehyde at room temperature for 10 min. To remove glycolipids, fixed cells were incubated with 100 μl of chloroform/methanol (1:1) at room temperature for 10 min. For staining controls, the extraction step was omitted. Cells were then washed twice with Hanks' buffer/0.04% azide and incubated for 1 h at room temperature with 100 μl of primary antibody in DMEM containing 10% FCS and 0.04% azide. After washing three times with Hanks' buffer/0.04% azide, FITC-, DTAF- or TRITC-conjugated secondary antibodies were added and incubated at room temperature for 1 h. Cells were washed five times with Hanks buffer/0.04% azide and mounted with one drop of Dako fluorescent mounting medium. Immunofluorescence images were captured and analyzed by confocal microscopy (Leica, Heidelberg, Germany). Five different areas on the microscope slide were scanned individually by using ×20 objective and 5-milliwatt laser output. Using Leica software, the intensity of immunofluorescence on the image was quantified and divided by the number of cells counted to make quantifications comparable (mean fluorescence intensity). Cells were lysed in lysis buffer (1 × 107 cells/ml lysis buffer) containing 1% Triton X-100, 20 mm Tris-HCl, pH 8.0, 160 mm NaCl, 1 mm CaCl2, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, and 0.1 units/ml α2-macroglobulin, at 4 °C for 30 min. Insoluble material was removed by centrifugation at 14,000 rpm for 15 min. The lysate was incubated with protein A-Sepharose beads loaded with polyclonal mAbs against ESL-1 (Affi-60) or PSGL-1 (Affi-124) at 4 °C overnight. Immunoprecipitated proteins were separated on 6% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schüll). Membranes were incubated with blocking buffer (Tris-buffered saline, 1% Tween 20, 4% nonfat milk) at room temperature for 2 h and probed with the hybridoma supernatant of mAb HECA-452. After washing with blocking buffer, the blot was probed with horseradish peroxidase-conjugated anti-rat IgM in blocking buffer at room temperature for 1 h. The blot was washed with Tris-buffered saline/0.1% Tween 20 and then visualized by chemiluminescence using the ECL reagent (Amersham Biosciences). Adhesion assays were performed in 96-well flat bottom plates (Maxisorp, Nunc) coated with E-selectin-IgG, VE-cadherin-IgG, or human IgG1 diluted in Hanks' balanced salt solution (Hanks' buffer, Biochem, Berlin, Germany) or coated with 10% FCS (49Lenter M.

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