Activity, Splice Variants, Conserved Peptide Motifs, and Phylogeny of Two New α1,3-Fucosyltransferase Families (FUT10 and FUT11)
2008; Elsevier BV; Volume: 284; Issue: 7 Linguagem: Inglês
10.1074/jbc.m809312200
ISSN1083-351X
AutoresRosella Mollicone, Stuart Moore, Nicolai V. Bovin, Marcela Garcia-Rosasco, Jean‐Jacques Candelier, Iván Martínez-Duncker, Rafaël Oriol,
Tópico(s)Ubiquitin and proteasome pathways
ResumoWe report the cloning of three splice variants of the FUT10 gene, encoding for active α-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related α-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other α1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH2 termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link α-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic α1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an α1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude α1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta α-N-acetylglucosaminidase. We report the cloning of three splice variants of the FUT10 gene, encoding for active α-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related α-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other α1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH2 termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link α-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic α1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an α1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude α1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta α-N-acetylglucosaminidase. Fucosyltransferases are globular type II trans-membrane Golgi-resident proteins that catalyze the transfer of α-l-fucose from GDP-Fuc onto N- and O-linked glycans, free oligosaccharides, or lipids (1Oriol R. Mollicone R. Cailleau A. Balanzino L. Breton C. Glycobiology. 1999; 9: 323-334Crossref PubMed Scopus (199) Google Scholar) or directly onto proteins (2Wang Y. Shao L. Shi S. Harris R.J. Spellman M.W. Stanley P. Haltiwanger S. J. Biol. Chem. 2001; 276: 40338-40345Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). These fucosylations are involved in a variety of biological processes, including selectin-mediated leukocyte-endothelial adhesion, lymphocyte homing, ABO blood group histocompatibility, notch receptor signaling (3Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (204) Google Scholar), embryo-fetal development, and host-microbe interactions (4Becker D.J. Lowe J.B. Glycobiology. 2003; 13: 41R-53RCrossref PubMed Scopus (671) Google Scholar). Changes in the glycosylation pattern of proteins may interfere with cellular functions and may thus lead to health disorders, such as cancers or rare autosomal recessive diseases, such as congenital disorders of glycosylation, characterized by glycosylation deficiencies (5Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Fucosyl residues in mammals are found linked to an oligosaccharide acceptor in α1,2-, α1,3-, α1,4-, and α1,6-orientations (6Costache M. Cailleau A. Fernandez-Mateos P. Oriol R. Mollicone R. Transfus. Clin. Biol. 1997; 4: 365-380Crossref Scopus (45) Google Scholar) or directly to serine or threonine as protein-O-fucosylations (7Loriol C. Dupuy F. Rampal R. Dlugosz M.A. Haltiwanger R.S. Maftah A. Germot A. Glycobiology. 2006; 16: 736-747Crossref PubMed Scopus (20) Google Scholar, 8Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. 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Plenum Press, London, UK1995: 37-73Google Scholar), and they are encoded by monoexonic genes (1Oriol R. Mollicone R. Cailleau A. Balanzino L. Breton C. Glycobiology. 1999; 9: 323-334Crossref PubMed Scopus (199) Google Scholar). The α1,6-fucosyltransferase encoded by the FUT8 gene transfers α-l-fucose onto the innermost asparagine-linked GlcNAc of the chitobiose disaccharide unit of glycoproteins (14Yanagidani S. Uozumi N. Ihara Y. Miyoshi E. Yamaguchi N. Taniguchi N. J. Biochem. (Tokyo). 1997; 121: 626-632Crossref PubMed Scopus (133) Google Scholar, 15Javaud C. Dupuy F. Maftah A. Julien R. Petit J. Genetica. 2003; 118: 157-170Crossref PubMed Scopus (54) Google Scholar). Previously, we found that α1,2-fucosyltransferases, α1,6-fucosyltransferases, and protein-O-fucosyltransferases share three main conserved peptide motifs (16Martinez-Duncker I. Mollicone R. Candelier J.J. Breton C. Oriol R. Glycobiology. 2003; 13: 1C-5CCrossref PubMed Scopus (62) Google Scholar) and constitute a new superfamily. Human α1,3/4-fucosyltransferases and their genetic expression are developmentally regulated (17Candelier J.J. Mollicone R. Mennesson B. Coullin P. Oriol R. Histochem. Cell Biol. 2000; 114: 113-124Crossref PubMed Scopus (19) Google Scholar, 18Mollicone R. Candelier J.J. Mennesson B. Couillin P. Venot A.P. Oriol R. Carbohydrate Res. 1992; 228: 265-276Crossref PubMed Scopus (69) Google Scholar). We have previously shown that FUT4 and FUT9 genes are derepressed early in human embryogenesis, whereas FUT6 and FUT3 appear sequentially after the 8th week of development (19Cailleau-Thomas A. Couillin P. Candelier J.J. Balanzino L. Mennesson B. Oriol R. Mollicone R. Glycobiology. 2000; 10: 789-802Crossref PubMed Scopus (60) Google Scholar). This suggests that during development, the Lex or CD15 antigen (generated by FUT4 or FUT9) appears earlier than the sialyl-Lex (made by FUT5, FUT6, or FUT7) or the type 1 Lewis structures (Lea and Leb antigens, made by FUT3 or FUT5). Lex has been found mainly in undifferentiated rapidly dividing cells (20Clarke J.L. Watkins W.M. J. Biol. Chem. 2000; 271: 10317-10328Abstract Full Text Full Text PDF Scopus (59) Google Scholar), whereas sialyl-Lex is more abundant in differentiated cells (17Candelier J.J. Mollicone R. Mennesson B. Coullin P. Oriol R. Histochem. Cell Biol. 2000; 114: 113-124Crossref PubMed Scopus (19) Google Scholar). This is particularly interesting because these glycotopes are implicated in embryo-fetal development, selectin-dependent leukocyte recruitment, and lymphocyte homing (21Lowe J.B. Cell. 2001; 104: 809-812Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). A few years ago, Renkonen and co-workers (22Roos C. Kolmer M. Mattila P. Renkonen R. J. Biol. Chem. 2002; 277: 3168-3175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), using the Drosophila genome-wide bioinformatics approach to identify the proteome involved in α-l-fucosylated glycan metabolism, identified a Drosophila fucosyltransferase and two human orthologous genes, encoding for the fucosyltransferases FUT10 and FUT11. Due to the presence of the two main conserved motifs (23Breton C. Oriol R. Imberty A. Glycobiology. 1998; 8: 87-94Crossref PubMed Scopus (112) Google Scholar), they were assumed to be α1,3-fucosyltransferases, but their activity has not been experimentally demonstrated yet in any species, including humans (22Roos C. Kolmer M. Mattila P. Renkonen R. J. Biol. Chem. 2002; 277: 3168-3175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), mice (24Baboval T. Smith F.I. Mamm. Genome. 2002; 13: 538-541Crossref PubMed Scopus (12) Google Scholar), 5S. K. Patnaik (2007) Nature Precedings, available on the World Wide Web. 5S. K. Patnaik (2007) Nature Precedings, available on the World Wide Web. flies (26Paschinger K. Staudacher E. Stemmer U. Fabini G. Wilson I.B.H. Glycobiology. 2005; 15: 463-474Crossref PubMed Scopus (100) Google Scholar, 27Rendic D. Linder A. Paschinger K. Borth N. Wilson I.B.H. Fabini G. J. Biol. Chem. 2006; 281: 3343-3353Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), or honeybees (29Rendic D. Klaudiny J. Stemmer U. Schmidt J. Paschinger K. Wilson I.B.H. Biochem. J. 2007; 402: 105-115Crossref PubMed Scopus (24) Google Scholar). Four insect α1,3-fucosyltransferases (Fuc-TA, Fuc-TB, Fuc-TC, and Fuc-TD) were first identified in D. melanogaster (26Paschinger K. Staudacher E. Stemmer U. Fabini G. Wilson I.B.H. Glycobiology. 2005; 15: 463-474Crossref PubMed Scopus (100) Google Scholar, 27Rendic D. Linder A. Paschinger K. Borth N. Wilson I.B.H. Fabini G. J. Biol. Chem. 2006; 281: 3343-3353Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The Fuc-TA is a core α1,3-fucosyltransferase (28Fabini G. Freilinger A. Altmann F. Wilson I.B.H. J. Biol. Chem. 2001; 276: 28058-28067Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), the Fuc-TB is orthologous to human FUT10 and FUT11, Fuc-TC is probably involved in the synthesis of Lex (29Rendic D. Klaudiny J. Stemmer U. Schmidt J. Paschinger K. Wilson I.B.H. Biochem. J. 2007; 402: 105-115Crossref PubMed Scopus (24) Google Scholar), and no activity has yet been found for the Fuc-TD. In this work, we cloned three new active splice variants of the human FUT10 gene, two in the embryo and one in the adult. We investigated their subcellular distribution and their fucosyltransferase activity. In addition, we report an α1,3-fucosyltransferase activity for FUT11, with an acceptor pattern similar to FUT10. Fucosyltransferase Assays—Transfected COS7 cells were homogenized on ice in 2% Triton X-100, and protein concentration was measured with the Bio-Rad Bradford protein microassay. Each fucosyltransferase assay was performed, unless otherwise stated, in a total volume of 65 μl, containing 15 μg of cell protein extract, 65 mm cacodylate buffer (pH 7.25), 10 mm l-fucose, 7 μm GDP-[14C]l-fucose (29 × 104 dpm/test at 300 mCi/mmol; Amersham Biosciences), and 5 μl/test of a 1 mg/ml solution of acceptor substrate (Table 1). For α1,3-fucosyltransferase assays, we used the conditions already described for the α1,6-fucosyltransferase FUT8 (30Martinez-Duncker I. Michalski J.C. Bauby C. Candelier J.J. Mennesson B. Codogno P. Oriol R. Mollicone R. Glycobiology. 2004; 14: 13-25Crossref PubMed Scopus (9) Google Scholar), and the activities were compared in the absence or presence of 20 mm MnCl2 as cofactor. The S.D. values of the mean value activities correspond to 10 independent tests for each enzyme. The mean of three independent enzymatic experiments is shown when there is no S.D. For kinetic studies, we used 25 μg of homogenate proteins, 3 μm GDP-[14C]l-fucose (125 × 103 dpm/test at 300 mCi/mmol) plus 125 μm cold GDP-Fuc (kindly provided by Claudine Augé, Paris-XI University of Sciences, Orsay, France).TABLE 1Acceptors used for the α1,3- and α1,6-fucosyltransferase activities Open table in a new tab The reactions were generally continued for 4 h at 37 °C, stopped by the addition of 3 ml of cold water, and centrifuged, and the supernatant was applied to a conditioned Sep-Pak C18 reverse-phase chromatography cartridge (Waters, Milford, MA) attached to a 10-ml syringe (19Cailleau-Thomas A. Couillin P. Candelier J.J. Balanzino L. Mennesson B. Oriol R. Mollicone R. Glycobiology. 2000; 10: 789-802Crossref PubMed Scopus (60) Google Scholar, 31Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconj. J. 1988; 5: 49-63Crossref Scopus (279) Google Scholar). The retained hydrophobic acceptors were separated from the unreacted GDP-[14C]fucose and its hydrolysis products by washing with 25 ml of H2O and then eluted with two 5-ml fractions of methanol and counted with 10 ml of Instagel-Plus (Hewlett-Packard, Evry, France) in a liquid scintillation β-counter (LS-6500; Beckman). The transfer of [14C]fucose was expressed in pmol/h/mg of protein, unless otherwise stated. Preparation of α1,3- and α1,6-Fucosyltransferase Acceptors—The biotin-labeled natural biantennary-glycoasparagine oligosaccharide acceptor was obtained from a pool of human plasma proteins by Pronase digestion followed by gel purification, desialylation, degalactosylation, and binding to biotin (BGA-biotin), as already described (30Martinez-Duncker I. Michalski J.C. Bauby C. Candelier J.J. Mennesson B. Codogno P. Oriol R. Mollicone R. Glycobiology. 2004; 14: 13-25Crossref PubMed Scopus (9) Google Scholar) (Table 1). Synthetic short amphiphilic oligosaccharide acceptors were synthesized as 8-methoxycarbonyl octyl glycosides also called oligosaccharide-O-grease (Ogr). 6The abbreviations used are: Ogr, oligosaccharide-O-grease; BSA, bovine serum albumin; cds, coding sequence; ER, endoplasmic reticulum; GFP, green fluorescent protein; MYA, millions of years ago; ORF, open reading frame; PBS, phosphate-buffered saline; TMD, trans-membrane domain; NAG, β-N-acetylglucosaminidase; contig, group of overlapping clones. These acceptors were kindly provided by Ole Hindsgaul and Monica Palcic (Carlsberg Laboratory, Copenhagen, Denmark) (Table 1). Biantennary or other N-glycan-related acceptors, synthesized as biotinylated monomeric probes (0987-BM, 0988-BM, 0989-BM, 0990-BM, and chitobiose-BM), were bought from Lectinity Corp. (Moscow, Russia). They are synthesized with an elongated spacer linked to biotin (32Korchagina E.Y. Bovin N.V. Bioorg. Khim. 1992; 18: 283-298PubMed Google Scholar), giving them the possibility of being adsorbed on Sep-Pak C18 cartridges as described for the above mentioned hydrophobic acceptors. The biantennary derivative, where the innermost GlcNAc residue is substituted by an N-acetyl-mannosamine residue (referred as 7OS-ManNAc-BM), was obtained from free 7OS by alkali 2-epimerization followed by 1-amination, chloroacetylation, ammonolysis, and final biotinylation (32Korchagina E.Y. Bovin N.V. Bioorg. Khim. 1992; 18: 283-298PubMed Google Scholar). The "opened" amino-alditol-GlcNAc derivative of biantennary OS (referred as 7OS-amino-alditol-GlcNAc-BM) was obtained by reductive amination of free 7OS with NH3 in the presence of sodium cyanoborohydride, followed by biotinylation (32Korchagina E.Y. Bovin N.V. Bioorg. Khim. 1992; 18: 283-298PubMed Google Scholar) (Table 1). Preparation of the α1,6-Fucosylated 0989-BM Acceptor with the FUT8 Fucosyltransferase—The innermost GlcNAc residue of the 0989-BM acceptor (50 μg) was fucosylated in the α1,6-position with the recombinant FUT8 enzyme. The reaction conditions were as follows: 65 μl containing 80 μg of protein from the homogenate of COS7 cells transfected with FUT8 cDNA, 65 mm cacodylate buffer (pH 7.25), 10 mm l-fucose, 300 μm cold GDP-l-fucose, and 5 μg/test of the 0989-BM acceptor substrate (Table 1). After 4 h at 37 °C, the reaction is more than 97% complete, and the retained Fucα1,6-0989-BM product was purified as described above. The transfer of the [14C]fucose in position 3 of Fucα1,6-0989-BM by the different FUT10 isoforms was performed as described above with a 16-h incubation at 37 °C. The activity was expressed as dpm/reaction. Conalbumin Glycopeptide Acceptor Preparation for Fucosyltransferase Assay—Glycopeptides were prepared from 100 μg of chicken egg white conalbumin (P-7786; Sigma) after digestion with 7 units of Pronase (5 mg/ml; P-5147; Sigma) for 24 h at 37 °C, in 0.1 m Tris/HCl, 4 mm CaCl2, pH 8.0, buffer. The reaction was stopped by heating for 5 min at 100 °C. Conalbumin glycopeptides were recovered from the supernatant, after a 10-min centrifugation at 4000 rpm, column-desalted, dried, and then used as a source for potential glycopeptide acceptors for α(1,3)- or α(1,6)-fucosyltransferase activities, as described above. Another fucosylation reaction was performed as described above, using 100 μg of native chicken egg white conalbumin. After terminating the fucosyltransferase incubation, Pronase was added, and the reactions were incubated overnight at 37 °C. The resulting [14C]fucosylglycopeptides (fractions 9-15) were separated from unreacted GDP-[14C]fucose and liberated [14C]fucose (fractions 18-26) on a Biogel P2 column (27 × 1 cm; Bio-Rad) equilibrated with 100 mm acetic acid. The resulting [14C]fucosyl-conalbumin glycopeptides were quantitated with a liquid scintillation β-counter. TLC—A fucosyltransferase assay using the homogenate derived from cells transfected with the FUT10-419 construct was performed for 6 or 16 h at 37 °C with the BGA-biotin acceptor. The radioactive oligosaccharide product was purified on a Sep-Pak C18 cartridge and eluted with methanol. After lyophilization, two-thirds of the radioactive product was digested overnight at 37 °C with 0.6 units of β-N-acetylglucosaminidase (NAG) from human placenta (Sigma) in 0.1 m citrate/phosphate buffer, pH 5.5. Subsequent to clean-up using Sep-Pak cartridges as described above, standards and NAG-treated 14C-fucosylated acceptor molecules were resolved on silica-coated plastic TLC sheets (Merck) developed in n-propyl alcohol/acetic acid/water (3:3:2 for 24 h) (33Moore S.E.H. Spiro R.G. J. Biol. Chem. 1994; 269: 12715-12721Abstract Full Text PDF PubMed Google Scholar). Radioactive components were visualized by fluorography after spraying the TLC plates with En3hance (PerkinElmer Life Sciences). Standard oligosaccharides separated under the same conditions were Man5GlcNAc2 (34Saint-Pol A. Codogno P. Moore S.E.H. J. Biol. Chem. 1999; 274: 13547-13555Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and 14C-labeled Gal2GlcNAc2Man3GlcNAc2, which was generated by incubating GlcNAc2Man3GlcNAc2 (Dextra Laboratories) with UDP-[14C]galactose and bovine milk galactosyltransferase (Sigma). RNA Isolation and Northern Blot Analysis—Embryos aged from 50 to 70 days were obtained from legal abortions and stored at -80 °C as already described (19Cailleau-Thomas A. Couillin P. Candelier J.J. Balanzino L. Mennesson B. Oriol R. Mollicone R. Glycobiology. 2000; 10: 789-802Crossref PubMed Scopus (60) Google Scholar). Total RNA was extracted with guanidine isothiocyanate and purified by cesium chloride gradient centrifugation. Contaminating DNA was removed by digestion with RNase-free DNase I (10 units/μg of RNA from Roche Applied Science) for 15 min at room temperature, followed by 15 min of inactivation at 70 °C and purification of the RNA by phenol/chloroform extraction. Embryonic poly(A)+ mRNAs were double purified using oligo(dT)-cellulose (type 3; Sigma) chromatography. Poly(A)+ RNAs (4 μg/lane) were denatured and fractionated with 1.2% phosphate-agarose gel electrophoresis, transferred to Hybond-N membranes (Amersham Biosciences), and immobilized by baking at 80 °C for 2 h. Prehybridization and hybridization were performed for 16 h at 42 °C in a buffer containing 50% formamide, 5× SSC, 1× PE, 250 μg/ml denatured salmon sperm DNA, and 10% dextran sulfate with the cds-FUT10 probe of 350 bp, obtained by PCR using primers sense F10-8s and antisense F10-4as (Table 2). The blots were first washed at low stringency: 2 × 15 min (in 2× SSC, 0.1% SDS) at 42 °C, followed by a single wash of 15 min (2× SSC, 0.1% SDS) at 50 °C and autoradiographed. A last 15 min wash in (0.5× SSC, 0.1% SDS) at 60 °C was performed, and another autoradiography was made. The films were developed after 3 days at -80 °C.TABLE 2Oligonucleotide primer sequences used in this study for amplifications and sequencingPrimersas, sense; as, antisense.Sequence 5′-3′PositionsbEx, exon.F10-1s5′-CATGCTGCTTCCTCTCGATGCCAAGC-3′Ex 2, 19-44F10-8s5′-GTTCTTCCATGTGAAGTTGTCTCGGG-3′Ex 2, 180-205F10-479s5′-GTTAGCCCTCTGTCAGTCACCCAGG-3′Ex 2, 239-263F10-391s5′-TAGAAGGTCATGGTTGAGCTGGG-3′Ex 4, 55-60/Ex 5, 1-17F10-4as5′-GAGCATAATGGGGTAGCTGTCCAATTCC-3′Ex 5, 147-174F10-7s5′-ATCCAGCCTCTATGGATGCCGATGGC-3′Ex 6, 397-422F10-as25′-GAATCTATAGCTAGATCGTCTCCCTCC-3′Ex 6, 505-528F10-GFPas5′-GAGATGGCCAGCCTGCTGGCTCT-3′Ex 6, 860-882F10-12as5′-GCAGGAGAGCAGAGAACATCAAGATGG-3′Ex 6, 860-894F10-13as5′-CACTGATGAGGTGGCAACAACAGAAGG-3′Ex 6, 907-933F10-KIGFPas5′-TTCGGTCCTTGAATACTAGGCCCC-3′Ex 7, 206-230F11-ints5′-CGCCACACTCCGGTAGACTCC-3′Ex 1, 740-753/Ex 2, 1-7F11-intas5′-GAGTGATTGTTCGGCATCCAGTCC-3′Ex 2, 226-249a s, sense; as, antisense.b Ex, exon. Open table in a new tab Construction of cDNA Libraries—Poly(A)+ mRNAs (1 μg) from a single 50-day embryo and from an adult brain were used to initiate the first strand cDNA synthesis. They were reverse transcribed at 42 °C for 90 min, using the oligo(dT)-cDNA synthesis primer (52-mer) and 200 units of the Superscript-II RNase H-reverse transcriptase from the Superscript first strand cDNA synthesis system kit (Invitrogen). The embryonic and adult cDNA libraries were stored at -20 °C until used. The PCRs were carried out with primers specific for FUT10 (Table 2), the Klentaq mixture (Clontech), and 1 μl of cDNA templates from the 50-day embryo or from adult brain cDNA libraries for the first PCR and 1 μl of the first PCR product diluted 1:10 for the second PCR. The same amplification program with the Advantage cDNA amplification kit mix (BD Clontech, Palo Alto, CA) was used. All PCRs were performed in 50 μl, with 1× Klentaq buffer, a 0.2 μm concentration of each primer, 1 unit of Klentaq DNA polymerase, and 0.2 mm dNTP with the touch-down-RACE program: initial denaturation 94 °C for 90 s, followed by five cycles of 94 °C for 30 s and 72 °C for 4 min, 5 cycles of 94 °C for 30 s and 70 °C for 4 min, and 25 cycles of 94 °C for 30 s and 68 °C for 4 min. Cloning of the FUT10 Transcripts—Four FUT10 cDNA isoforms (FUT10-357, FUT10-391, FUT10-419, and FUT10-479) were amplified by a double PCR using the embryo and the adult cDNA libraries as templates. The FUT10-357 was a truncated variant of FUT10-391, lacking the conserved motifs I and II. It was devoid of enzyme activity and was not further analyzed. The primer associations F10-1s and F10-13as or F10-1s and F10-as2 were used for the first PCR, and distinct combinations of nested primers as F10-8s and F10-12as or F10-8s and F10-as2 were used for the second PCR (Table 2). With these two nested primer combinations, we obtained a broad PCR product of 1400 bp in the embryo cDNA with F10-8s and F10-12as and a product of 1800 bp from the adult cDNA library, with F10-8s and F10-as2 primers. These final PCR products were gel-purified and cloned into the TA cloning vector PCR3.1 (Eukaryotic TA cloning kit; Invitrogen), and 20 plasmid clones were PCR-selected from each positive ligation and sequenced. To generate the FUT10-GFP-tagged transcripts, we amplified the three selected FUT10 constructs: FUT10-391 with primers F10-391s and F10-GFPas, FUT10-419 with primers F10-8s and F10-GFPas, and FUT10-479 with primers F10-479s and F10-K1GFPas, in the presence of the high fidelity Hotstart DNA polymerase (Accuprime-pFx DNA polymerase; Invitrogen). The PCR products were gel-purified and inserted into the mammalian pcDNA3.1-GFP vector (TOPO-CT-GFP-cloning kit; Invitrogen). The resulting GFP-COOH-tagged plasmids were selected by PCR, sequenced, and called FUT10-391-GFP, FUT10-419-GFP, and FUT10-479-GFP. Isolation of FUT11 cDNA Clones—The IMAGE clone 40005868 (BC100994) in a pCR-Blunt-TOPO vector encoding for a 476-amino acid protein and the IMAGE clone 5271548 (BC036037) in a pBluescriptR vector, encoding for a 492-amino acid protein, were obtained from Gene-service Ltd. (Cambridge, UK). The IMAGE clones were digested with EcoRI (NEB-Biolabs, Ozyme, Saint Quentin Yvelines, France). Two DNA fragments of 1730 and 1660 bp, corresponding to FUT11-476 and FUT11-492 cDNA, respectively, were purified and subcloned into the PCR3.1-TA cloning vector (Invitrogen). The presence of the inserts was verified by PCR with Taq polymerase (MBI Fermentas, Euromedex, Strasbourg, France) and the internal primers F11-ints and F11-intas (Table 2). The correct orientation of FUT11-476 and FUT11-492 plasmids was checked with BamHI and EcoRI. DNA Sequencing—The different FUT10, FUT11, and pcDNA3.1-FUT10-GFP expression vectors were sequenced in both directions (Biofidal SARL, Vaulx-en-Velin, France). All of the full-length isoform constructs devoid of PCR errors were selected and used for transfection analysis. Transient Expression of FUT10, FUT11, and GFP-tagged-FUT10 cDNA Constructs in COS7 Cells—cDNA transcripts were inserted into PCR3.1 (TA-cloning System; Invitrogen) and transiently transfected into COS7 cells (20 μg of plasmid) with DEAE-dextran (19Cailleau-Thomas A. Couillin P. Candelier J.J. Balanzino L. Mennesson B. Oriol R. Mollicone R. Glycobiology. 2000; 10: 789-802Crossref PubMed Scopus (60) Google Scholar). After 12, 18, 24, or 48 h, the cells were washed with phosphate-buffered saline (PBS) and harvested for fucosyltransferase assays or indirect immunofluorescence microscopy. Immunofluorescent Localization of the GFP-tagged FUT10 Fusion Proteins—2 × 105 cells were seeded on glass coverslips in 35-mm cell culture Petri dishes 24 h before transfection. Five μg of the GFP-tagged cDNA constructs were transfected into COS7 cells. After 12, 18, 24, or 48 h of growth, they were washed with PBS and then fixed for 15 min with 2% paraformaldehyde in PBS. To quench residual paraformaldehyde, cells were incubated for 20 min in 50 mm NH4Cl in PBS. Thereafter, they were permeabilized with 0.075% saponin, 0.1% bovine serum albumin (BSA) in PBS for 15 min, followed by 1 h of incubation with primary antibodies at room temperature. The GFP-tagged FUT10 recombinant proteins were visualized with a Leica DMR epifluorescence microscope, with a PLAN-APO ×63/1.32-0.6 oil objective lens and an HC-PLAN ×10/25 ocular lens (Leica Microsystems, Wetzlar, Germany). Images were captured with a LEI-750 CE digital camera and LIDA volume 54 Leica software and further processed with Adobe Photoshop 5.0 (Adobe, San Jose, CA). Double immunofluorescence experiments were conducted using a Golgi-specific mouse monoclonal anti-giantin antibody kindly given by Hans-Peter Hauri (35Linstedt A.D. Hauri H.P. Mol. Biol. Cell. 1993; 4: 679-693Crossref PubMed Scopus (355) Google Scholar) (1:1000 in PBS plus 1% BSA) or an endoplasmic reticulum (ER)-specific rabbit polyclonal anti-calnexin antibody (1:200 in PBS plus 1% BSA; StressGen, Assay Designs, Inc., Ann Arbor, MI) or the lysosomal specific mouse monoclonal anti-Lamp-1 (BB6) antibody, kindly given by Sven Carlsson (1:1000 in PBS plus 1% BSA), and the GFP-tagged-FUT10 recombinant protein. After washing the cells three times in PBS plus 0.1% BSA, the anti-giantin and the anti-Lamp-1 antibodies were revealed with conjugated anti-mouse Ig-Cya3 red fluorochrome, diluted 1:200 (Jackson Laboratories, L'Arbresle, France), and the anti-calnexin antibody with anti-rabbit Ig-Cya3, diluted 1:200 (Jackson Laboratories). The secondary labeled antibodies were incubated for 1 h at room temperature, and the reaction was stopped by washing three times in PBS. The coverslips with the labeled cells were mounted on slides with Mowiol and observed using a Leica DMR epifluorescence microscope. Bioinformatics—Ten α1,3-fucosyltransferase-like sequences from different species were retrieved from data banks by Psi-Blast (36Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang
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