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• W2040307553 abstract "Substitution of the asparagine-linked GlcNAc by α1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6.8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core α1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial α1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn, GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAcβ1-Asn, and GlcNAcβ1–2Manα1–3[Manα1–3(Manα1–6)Manα1–6]Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core α1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Substitution of the asparagine-linked GlcNAc by α1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6.8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core α1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial α1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn, GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAcβ1-Asn, and GlcNAcβ1–2Manα1–3[Manα1–3(Manα1–6)Manα1–6]Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core α1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. The most characteristic features of asparagine-linked oligosaccharides from plants are the substitution of the core pentasaccharide by xylose and α1,3-linked fucose (1Lerouge P. Cabanes-Macheteau M. Rayon C. Fitchette-Lainé A.C. Gomord V. Faye L. Plant Mol. Biol. 1998; 38: 31-48Crossref PubMed Google Scholar, 2Rayon C. Lerouge P. Faye L. J. Exp. Bot. 1998; 49: 1463-1472Crossref Scopus (121) Google Scholar). The resulting heptasaccharide “MMXF3” (Fig. 1) very often constitutes the main oligosaccharide species on a plant glycoprotein (3Kurosaka A. Yano A. Itoh N. Kuroda Y. Nakagawa T. Kawasaki T. J. Biol. Chem. 1991; 266: 4168-4172Abstract Full Text PDF PubMed Google Scholar, 4Wilson I.B.H. Altmann F. Glycoconjugate J. 1998; 15: 1055-1070Crossref PubMed Scopus (86) Google Scholar). According to their biosynthesis, these structures are classified as complex-type N-glycans, even though the terms paucimannosidic or truncated N-glycans appear to be more justified. The α-mannosyl residues may, however, be substituted by GlcNAc and these GlcNAc residues may be further decorated by galactose and fucose to form the same structure as the human Lewis a epitope (Fig. 1) (5Melo N.S. Nimtz M. Conradt H.S. Fevereiro P.S. Costa J. FEBS Lett. 1997; 415: 186-191Crossref PubMed Scopus (82) Google Scholar, 6Fitchette-Lainé A.C. Gomord V. Cabanes M. Michalski J.C. Saint Macary M. Foucher B. Cavelier B. Hawes C. Lerouge P. Faye L. Plant J. 1997; 12: 1411-1417Crossref PubMed Scopus (176) Google Scholar). The antigenicity of “paucimannosidic” plant N-glycans is well documented (7McManus M.T. McKeating J. Secher D.S. Osborne D.J. Ashford D. Dwek R.A. Rademacher T.W. Planta (Basel). 1988; 175: 506-512Crossref PubMed Scopus (64) Google Scholar, 8Wilson I.B.H. Harthill J.E. Mullin N. Ashford D. Altmann F. Glycobiology. 1998; 8: 651-661Crossref PubMed Scopus (179) Google Scholar, 9Faye L. Gomord V. Fitchette-Lainé A.C. Chrispeels M.J. Anal. Biochem. 1993; 209: 104-108Crossref PubMed Scopus (152) Google Scholar, 10Garcia-Casado G. Sanchez-Monge R. Chrispeels M.J. Armentia A. Salcedo G. Gomez L. Glycobiology. 1996; 6: 471-477Crossref PubMed Scopus (180) Google Scholar, 11Petersen A. Vieths S. Aulepp H. Schlaak M. Becker W.M. J. Allergy Clin. Immunol. 1996; 98: 805-815Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Since both xylose and core α1,3-fucose are not seen in mammalian glycoproteins they may form the key component of epitopes for carbohydrate-reactive antibodies (9Faye L. Gomord V. Fitchette-Lainé A.C. Chrispeels M.J. Anal. Biochem. 1993; 209: 104-108Crossref PubMed Scopus (152) Google Scholar, 10Garcia-Casado G. Sanchez-Monge R. Chrispeels M.J. Armentia A. Salcedo G. Gomez L. Glycobiology. 1996; 6: 471-477Crossref PubMed Scopus (180) Google Scholar, 12Faye L. Chrispeels M.J. Glycoconjugate J. 1988; 5: 245-256Crossref Scopus (111) Google Scholar). There is, however, evidence that the α1,3-linked fucosyl residue is the predominant antibody binding structural element (3Kurosaka A. Yano A. Itoh N. Kuroda Y. Nakagawa T. Kawasaki T. J. Biol. Chem. 1991; 266: 4168-4172Abstract Full Text PDF PubMed Google Scholar, 8Wilson I.B.H. Harthill J.E. Mullin N. Ashford D. Altmann F. Glycobiology. 1998; 8: 651-661Crossref PubMed Scopus (179) Google Scholar, 11Petersen A. Vieths S. Aulepp H. Schlaak M. Becker W.M. J. Allergy Clin. Immunol. 1996; 98: 805-815Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 13Tretter V. Altmann F. Kubelka V. März L. Becker W.M. Int. Arch. Allergy Immunol. 1993; 102: 259-266Crossref PubMed Scopus (193) Google Scholar). Due to the ubiquitous occurrence of such paucimannosidic N-glycans throughout the plant kingdom, they are responsible for the frequently observed cross-reactivity of antibodies raised against plant glycoproteins and are therefore termed “cross-reactive carbohydrate determinants” (12Faye L. Chrispeels M.J. Glycoconjugate J. 1988; 5: 245-256Crossref Scopus (111) Google Scholar, 14Aalberse R.C. van Ree R. Clin. Rev. Allergy Immunol. 1997; 15: 375-387Crossref PubMed Scopus (93) Google Scholar, 15Aalberse R.C. Allergy. 1998; 53: 54-57Crossref PubMed Scopus (44) Google Scholar). Anti-cross-reactive carbohydrate determinants antibodies of the IgE class have been found in sera of many allergic patients (8Wilson I.B.H. Harthill J.E. Mullin N. Ashford D. Altmann F. Glycobiology. 1998; 8: 651-661Crossref PubMed Scopus (179) Google Scholar, 11Petersen A. Vieths S. Aulepp H. Schlaak M. Becker W.M. J. Allergy Clin. Immunol. 1996; 98: 805-815Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 13Tretter V. Altmann F. Kubelka V. März L. Becker W.M. Int. Arch. Allergy Immunol. 1993; 102: 259-266Crossref PubMed Scopus (193) Google Scholar, 14Aalberse R.C. van Ree R. Clin. Rev. Allergy Immunol. 1997; 15: 375-387Crossref PubMed Scopus (93) Google Scholar, 16Jankiewicz A. Aulepp H. Altmann F. Fötisch K. Vieths S. Allergo J. 1998; 7: 87-95Google Scholar, 17Prenner C. Mach L. Glössl J. März L. Biochem. J. 1992; 284: 377-380Crossref PubMed Scopus (92) Google Scholar). While the clinical role of cross-reactive carbohydrate determinants remains controversial, they are suspected to obscure (at least in vitro) allergy diagnosis. Anti-cross-reactive carbohydrate determinants antibodies will also react with many insect glycoproteins such as honeybee venom phospholipase A2 or neuronal membrane glycoproteins from insect embryos because insects, like plants, are capable of synthesizing the core α1,3-fucose epitope (3Kurosaka A. Yano A. Itoh N. Kuroda Y. Nakagawa T. Kawasaki T. J. Biol. Chem. 1991; 266: 4168-4172Abstract Full Text PDF PubMed Google Scholar, 11Petersen A. Vieths S. Aulepp H. Schlaak M. Becker W.M. J. Allergy Clin. Immunol. 1996; 98: 805-815Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 12Faye L. Chrispeels M.J. Glycoconjugate J. 1988; 5: 245-256Crossref Scopus (111) Google Scholar, 13Tretter V. Altmann F. Kubelka V. März L. Becker W.M. Int. Arch. Allergy Immunol. 1993; 102: 259-266Crossref PubMed Scopus (193) Google Scholar, 18Wang X. Sun B. Yasuyama K. Salvaterra P.M. Insect Biochem. Mol. Biol. 1994; 24: 233-242Crossref PubMed Scopus (24) Google Scholar, 19Altmann F. Staudacher E. Wilson I.B.H. März L. Glycoconjugate J. 1999; 16: 109-123Crossref PubMed Scopus (283) Google Scholar). In contrast to the blood group-related fucosyltransferases which act on the nonreducing terminus of N-glycans, O-glycans, or glycolipids (20Staudacher E. Trends Glycosci. Glycotechn. 1996; 8: 391-408Crossref Scopus (21) Google Scholar), core fucosyltransferases have received little attention. Only recently, the molecular cloning of GDP-l-Fuc 1The abbreviations used are: Fuc, l-Fucose; Fuc-T C3, GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase); Fuc-T C6, GDP-l-Fuc:Asn-linked GlcNAc α1,6-fucosyltransferase (core α1,6-fucosyltransferase); GnGn, GnGnF3, and GnGnF6, N-glycans, for structures, see Fig. 1; HPLC, high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; GnGn, GnGnF6, GnGnF3F6, GnGnF3, MMF3, GalGal, GalGnF3, GnGal, MM, M5Gn, and M5GnF3,N-glycans, for structures see Fig. 1, PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; Mes, 2-(N-morpholino)ethanesulfonic acid; bp, base pair(s)1The abbreviations used are: Fuc, l-Fucose; Fuc-T C3, GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase); Fuc-T C6, GDP-l-Fuc:Asn-linked GlcNAc α1,6-fucosyltransferase (core α1,6-fucosyltransferase); GnGn, GnGnF3, and GnGnF6, N-glycans, for structures, see Fig. 1; HPLC, high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; GnGn, GnGnF6, GnGnF3F6, GnGnF3, MMF3, GalGal, GalGnF3, GnGal, MM, M5Gn, and M5GnF3,N-glycans, for structures see Fig. 1, PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; Mes, 2-(N-morpholino)ethanesulfonic acid; bp, base pair(s): Asn-linked GlcNAc α1,6-fucosyltransferase (core α1,6-fucosyltransferase, Fuc-T C6, Fuc-T VIII) from porcine brain and from human gastric cancer cells has been reported (21Uozumi N. Yanagidani S. Miyoshi E. Ihara Y. Sakuma T. Gao C.X. Teshima T. Fujii S. Shiba T. Taniguchi N. J. Biol. Chem. 1996; 271: 27810-27817Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 22Yanagidani S. Uozumi N. Ihara Y. Miyoshi E. Yamaguchi N. Taniguchi N. J. Biochem. (Tokyo). 1997; 121: 626-632Crossref PubMed Scopus (134) Google Scholar). As regards core α1,3-fucosyltransferase (Fuc-T C3), a first characterization of the enzyme from mung bean seedlings revealed its dependence on the presence of nonreducing terminal GlcNAc (23Staudacher E. Dalik T. Wawra P. Altmann F. März L. Glycoconjugate J. 1995; 12: 780-786Crossref PubMed Scopus (38) Google Scholar). In this paper, we report the purification to homogeneity of Fuc-T C3 from mung bean seedlings, the cloning of its cDNA by a PCR-based approach, and the expression of active recombinant Fuc-T C3 in baculovirus infected insect cells. Mung bean seedlings (germinated for 3 days in the dark) were kindly donated by Dr. Zun-Ho Wu (Vienna, Austria) and by Evergreen Co. (Oeynhausen, Austria). Activated CH-Sepharose 4B, S-Sepharose, and GDP-l-[U-14C]fucose were obtained from Amersham Pharmacia Biotech. “GnGn-Sepharose” (see Fig. 1 for glycan structures) was prepared by coupling of GnGn-peptide (see below) to activated CH-Sepharose 4B according to the manufacturer's instructions. GDP-l-fucose, bovine kidneyN-acetyl-β-glucosaminidase, N-acetyllactosamine (Galβ1–4GlcNAc), lacto-N-biose (Galβ1–3GlcNAc), lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), IEF standard mixture, and IPL-41 medium were purchased from Sigma. GDP-hexanolamine-agarose was purchased from Calbiochem. Sequencing grade trypsin, N-glycosidase A, N-glycosidase F, alkaline phosphatase, PstI, and BamHI were from Roche Molecular Biochemicals. 2,5-Dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, Dowex 1-X8, and Dowex 50W-X2 (H+-form) were purchased from Fluka. β-Galactosidase from Aspergillus oryzae was prepared as described (24Zeleny R. Altmann F. Praznik W. Anal. Biochem. 1997; 246: 96-101Crossref PubMed Scopus (57) Google Scholar). Biantennary asialo- and agalacto-glycopeptide (GnGn-peptide; see Fig. 1) were prepared from bovine fibrin by Pronase digestion, chemical desialylation, and enzymatic degalactosylation as described (23Staudacher E. Dalik T. Wawra P. Altmann F. März L. Glycoconjugate J. 1995; 12: 780-786Crossref PubMed Scopus (38) Google Scholar). The core 6-fucosylated GnGnF6-peptide was similarly derived from human IgG (25Staudacher E. März L. Glycoconjugate J. 1998; 15: 355-360Crossref PubMed Scopus (38) Google Scholar). MM-peptide was prepared by treatment of GnGn-peptide with N-acetyl-β-glucosaminidase (25Staudacher E. März L. Glycoconjugate J. 1998; 15: 355-360Crossref PubMed Scopus (38) Google Scholar, 26Kubelka V. Altmann F. Staudacher E. Tretter V. März L. Hård K. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (220) Google Scholar). Dabsylated GnGn-hexapeptide was derived by β-galactosidase degradation of dabsylated GalGal-hexapeptide which was available from a previous study (27Altmann F. Schweiszer S. Weber C. Glycoconjugate J. 1995; 12: 84-93Crossref PubMed Scopus (89) Google Scholar). Man5GlcNAc2-Asn (M5-Asn) was obtained by pronase digestion of α-amylase (27Altmann F. Schweiszer S. Weber C. Glycoconjugate J. 1995; 12: 84-93Crossref PubMed Scopus (89) Google Scholar). From this substrate, M5Gn-Asn (see Fig. 1 for structure) was prepared using recombinant GlcNAc transferase I from tobacco which was kindly provided by Dr. Herta Steinkellner (28Strasser R. Mucha J. Schwihla H. Altmann F. Glössl J. Steinkellner H. Glycobiology. 1999; (in press)PubMed Google Scholar). The structure and purity of these acceptors was checked by MALDI-TOF MS (see below). SV Total RNA Isolation System, avian myeloblastosis virus reverse transcriptase, and Taq Polymerase were purchased from Promega. Lipofectin, fetal calf serum, 3′-5′ RACE System (version 2.0) for rapid amplification of cDNA ends, and degenerate oligonucleotides were purchased from Life Technologies, Inc., whereas Fuc-T C3 specific primers were synthesized by Vienna Biocenter Genomics. The TA Cloning Kit was obtained from Invitrogen. Enzymatic activity of Fuc-T C3 was determined using GnGn-peptide and GDP-l-[U-14C]fucose at substrate concentrations of 0.5 and 0.25 mm, respectively, in the presence of Mes-HCl buffer, Triton X-100, MnCl2, GlcNAc, and AMP as described (25Staudacher E. März L. Glycoconjugate J. 1998; 15: 355-360Crossref PubMed Scopus (38) Google Scholar, 29Staudacher E. Altmann F. Glössl J. März L. Schachter H. Kamerling J.P. Hård K. Vliegenthart J.F.G. Eur. J. Biochem. 1991; 199: 745-751Crossref PubMed Scopus (45) Google Scholar). Where specified, other acceptors were used. All purification steps were performed at 4 °C. Mung bean seedlings were homogenized with a kitchen blender using 0.75 volumes of extraction buffer per kg of beans. The extraction buffer consisted of 0.5 mm dithiothreitol, 1 mm EDTA, 0.5% polyvinylpyrrolidone, 0.25 m sucrose, and 50 mmTris-HCl buffer, pH 7.3. The resulting homogenate was filtered through two layers of cloth and the filtrate was centrifuged at 30,000 ×g for 40 min. The supernatant was discarded and the pellet was extracted with solubilization buffer consisting of 0.5 mm dithiothreitol, 1 mm EDTA, 1.5% Triton X-100, and 50 mm Tris-HCl, pH 7.3, by stirring overnight. Subsequent centrifugation at 30,000 × g for 40 min yielded the Triton X-100 extract which was further purified as follows. Step 1: the Triton X-100 extract was applied to a column (5 × 28 cm) of DE52 cellulose (Whatman) previously equilibrated with buffer A (25 mm Tris-HCl buffer, pH 7.3, containing 0.1% Triton X-100 and 0.02% NaN3). The non-binding fraction was directly used for step 2. Step 2: the sample was applied to a column (2.5 × 32 cm) of Affi-Gel Blue (Bio-Rad) equilibrated with buffer A. After washing the column with this buffer, adsorbed protein was eluted with buffer A containing 0.5 m NaCl. Step 3: following dialysis of the eluate from step 2 against buffer B (25 mm sodium citrate buffer, pH 5.3, containing 0.1% Triton X-100 and 0.02% NaN3) it was loaded onto a column (1.5 × 18 cm) of S-Sepharose equilibrated with the same buffer. Bound protein was eluted with a linear gradient from 0 to 0.5m NaCl in buffer B. Fractions containing Fuc-T C3 were pooled and dialyzed against buffer C (25 mm Tris-HCl buffer, pH 7.3, containing 5 mm MnCl2 and 0.02% NaN3). Step 4: the dialyzed sample was applied to a column (0.5 × 4.5 cm) of GnGn-Sepharose previously equilibrated with buffer C. Elution of the bound protein was accomplished with buffer C containing 1m NaCl instead of MnCl2. Step 5: the enzyme was then dialyzed against buffer D (25 mm Tris-HCl, pH 7.3, containing 10 mmMgCl2, 0.1 m NaCl, and 0.02% NaN3) and subsequently loaded onto a column (0.5 × 4.5 cm) of GDP-hexanolamine-Sepharose. After washing the column with buffer D, Fuc-T C3 was eluted by substituting MgCl2 and NaCl with 0.5 mm GDP. Active fractions were pooled, dialyzed against 20 mm Tris-HCl buffer of pH 7.3, and lyophilized. SDS-PAGE was performed in a Bio-Rad Mini Protean Cell on gels containing 12.5% acrylamide and 1% bisacrylamide. Gels were either stained with Coomassie Brilliant Blue R-250 or silver. Isoelectric focusing of Fuc-T C3 was carried out on precast gels with a pI range from 6 to 9 (Servalyt precotes 6–9, Serva) and gels were silver stained according to the manufacturers instructions. For two-dimensional electrophoresis, lanes from the focusing gel were excised, treated with S-alkylation reagents and SDS, and subject to SDS-PAGE as described previously (30Görg A. Postel W. Günther S. Electrophoresis. 1988; 9: 681-692Crossref PubMed Scopus (103) Google Scholar). Protein bands were excised from Coomassie-stained SDS-polyacrylamide gels, carboxyamidomethylated, and digested with sequencing grade trypsin according to the in-gel digestion procedure described previously (31Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1298) Google Scholar). The tryptic peptides were separated by reverse phase HPLC on a 1.0 × 250-mm Vydac C18 at 40 °C with a flow rate of 0.05 ml/min using a HP 1100 apparatus (Hewlett-Packard). Isolated peptides were sequenced with a Hewlett-Packard G1005A protein sequencing system according to the manufacturer's protocol. In addition, the peptide mixture obtained by in-gel digestion was analyzed by MALDI-TOF MS (see below). Total RNA was isolated from 3-day-old mung bean hypocotyls using the SV Total RNA Isolation System from Promega according to the supplier's instructions. To achieve first strand cDNA synthesis, total RNA was incubated for 1 h at 48 °C with avian myeloblastosis virus reverse transcriptase and oligo(dT) primer using the Reverse Transcription System (Promega). First strand cDNA was subjected to PCR using as the sense primer 5′-GCIGARTAYTAYGCIGARAAYAAYATHGC-3′ (S1) and as the antisense primer 5′-CRTADATRTGRTAIACIGTYTC-3′ (S2) or 5′-TADATISWYTCCATYTCRAA-3′ (S3), where I stands for inosin; R for G + A; Y for T + C; H for T + C + A; D for T + G + A; S for G + C; and W for A + T. PCR was performed on 10 μl of the reverse transcriptase reaction in a volume of 50 μl containing 0.1 μmol of each primer, 0.1 mmdNTPs, 2 mm MgCl2, 10 mm Tris-HCl buffer of pH 9.0, 50 mm KCl, and 0.1% Triton X-100. After an initial denaturation step at 95 °C for 2 min, 40 cycles of 1 min at 95 °C, 1 min at 49 °C, and 2 min at 72 °C were run. The final extension step at 72 °C was carried out for 8 min. PCR products were subcloned into pCR2.1 vector using the TA Cloning Kit (Invitrogen) and sequenced. On the basis of the sequence of PCR product(s), the missing 5′ and 3′ regions of the cDNA coding for Fuc-T C3 were obtained by 5′- and- 3′-rapid amplification of cDNA ends (RACE) using the RACE kit from Life Technologies, Inc. according to the manufacturer's recommendations. 3′-RACE was performed with hemi-nested PCR using as antisense primer the universal amplification primer supplied with the kit and as sense primers at first 5′-CTGGAACTGTCCCTGTGGTT-3′ and then 5′-AGTGCACTAGAGGGCCAGAA-3′. Likewise, 5′-RACE was performed by means of hemi-nested PCR using as the sense primer the abridged anchor primer supplied with the kit and as antisense primers either 5′-GAATGCAAAGACGGCACGATGAAT-3′ and then 5′-TTCGAGCACCACAATTGGAAAT-3′ or PCR was performed with an annealing temperature of 55 °C under conditions otherwise as described above. Both 5′- and 3′-RACE products were subcloned into pCR2.1 vector and sequenced. Genomic DNA was prepared out of lyophilized mung bean hypocotyls by means of the DNeasy Plant Kit (Qiagen) following the manufacturer's instructions. PCR was performed on 200 ng of DNA in 50 μl of solution containing 20 nmol each of fucosyltransferase-specific primers (see below) essentially as described above except that the annealing temperature was raised to 58 °C. The three resulting PCR products (FSP34–59, FSP37–515, and FSP 32–511) were subcloned into pCR2.1 vector using the TA cloning Kit (Invitrogen) and sequenced. Forward primers 5′-GGAACCATCCACCCATAAC-3′, 5′-AGTCGTGTTCGGTTGGATGT-3′, and 5′-CTGGAACTGTCCCTGTGGTT-3′ and reverse primers 5′-CTCAGCATAGTATTCTGCTG-3′, 5′- GAAGGAGCAAAGTCCTGAATA-3′, and 5′-GTACCATTTAGCGCAT-3′ were used to cover cDNA regions from −174 to 522, 392 to 944, and 890 to 1550 bp, respectively. Sequences of subcloned fragments were determined by the dideoxynucleotide chain termination method using an ABI PRISM Dye Terminator Cycle Sequencing Ready reaction Kit and an ABI PRISM 310 Genetic analyzer (Perkin-Elmer). T7 and M13 forward primers were used for sequencing the PCR products cloned in pCR2.1. Sequencing of both strands of the complete coding region was performed by the Vienna VBC Genomics-Sequencing Service using the cycle sequencing method with infrared labeled primers (IRD700 and IRD800) and a LI-COR Long Read IR 4200 sequencer (Lincoln, NE). The coding region of the putative Fuc-T C3 cDNA including the cytoplasmic and the transmembrane regions was amplified using the forward primer 5′-cggcggatcCGCAATTGAATGATG-3′ and the reversal primer 5′-ccggctgcaGTACCATTTAGCGCAT-3′ by means of the Expand High Fidelity PCR System (Roche Molecular Biochemicals). The PCR product was double digested with PstI and BamHI and subcloned into alkaline phosphatase-treated baculovirus transfer vector pVL1393 previously double digested with PstI and BamHI. To allow homologous recombination, the transfer vector was co-transfected with BaculoGold viral DNA (PharMingen, San Diego, CA) into Sf9 insect cells in IPL-41 medium containing Lipofectin. After 5 days of incubation at 27 °C, various volumes of supernatant containing recombinant virus were used for infection of Sf21 insect cells. After incubation for 4 days at 27 °C in IPL-41 medium containing 5% fetal calf serum, the Sf21 cells were harvested and washed twice with phosphate-buffered saline. The cells were resuspended in 25 mm Tris-HCl buffer of pH 7.4 containing 2% Triton X-100 and disrupted by sonication on ice. This homogenate as well as the culture supernatant were assayed for Fuc-T C3 activity. Mock infections were performed with recombinant baculovirus encoding tobacco GlcNAc transferase I (28Strasser R. Mucha J. Schwihla H. Altmann F. Glössl J. Steinkellner H. Glycobiology. 1999; (in press)PubMed Google Scholar). Dabsylated GnGn-hexapeptide (2 nmol) was incubated with insect cell homogenate containing recombinant Fuc-T C3 (0.08 milliunit) in the presence of non-radioactive GDP-l-fucose (10 nmol) under conditions otherwise identical to those described for determination of transferase activity (see above). A control experiment was performed with homogenate from mock-infected insect cells. After incubation for 16 h at 37 °C, aliquots of 0.5 μl were diluted 20-fold and analyzed by MALDI-TOF MS. In addition, aliquots of both samples were mixed to give similar concentrations of substrate and product. This mixture was diluted with 0.1 m ammonium acetate of pH 4.0 containing 10 microunits of N-glycosidase A or with 50 mm Tris/HCl of pH 8.5 containing 100 microunits (1 unit hydrolyzing 1 μmol of substrate/min) of N-glycosidase F, respectively. After 2 and 20 h, small aliquots of these mixtures were removed and analyzed by MALDI-TOF MS. The remaining half of the sample containing the Fuc-T C3 product was digested withN-glycosidase A. The resulting oligosaccharides were pyridylaminated and analyzed by reverse phase HPLC (8Wilson I.B.H. Harthill J.E. Mullin N. Ashford D. Altmann F. Glycobiology. 1998; 8: 651-661Crossref PubMed Scopus (179) Google Scholar, 32Kubelka V. Altmann F. März L. Arch. Biochem. Biophys. 1994; 308: 148-157Crossref PubMed Scopus (128) Google Scholar, 33Hase S. Ibuki T. Ikenaka T. J. Biochem. (Tokyo). 1984; 95: 197-203Crossref PubMed Scopus (390) Google Scholar). The transferase product was degraded usingN-acetyl-β-glucosaminidase and again analyzed by HPLC. Pyridylaminated GnGnF6 derived from human IgG, MMF3 from honeybee venom phospholipase A2, GnGn, GnM, MGn, and MM from bovine fibrin were used as reference substances (23Staudacher E. Dalik T. Wawra P. Altmann F. März L. Glycoconjugate J. 1995; 12: 780-786Crossref PubMed Scopus (38) Google Scholar, 26Kubelka V. Altmann F. Staudacher E. Tretter V. März L. Hård K. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 213: 1193-1204Crossref PubMed Scopus (220) Google Scholar, 34Altmann F. Schwihla H. Staudacher E. Glössl J. März L. J. Biol. Chem. 1995; 270: 17344-17349Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Additionally, the mass of the pyridylaminated product was determined by MALDI-TOF MS (see below). Mass spectrometry was performed on a DYNAMO (Thermo BioAnalysis, Santa Fe, NM), a linear MALDI-TOF MS capable of dynamic extraction (a synonym for delayed extraction). Two types of sample-matrix preparations were employed. Peptides and dabsylated glycopeptides were dissolved in 5% formic acid and aliquots were spotted on the target, air dried, and overlaid with 1% α-cyano-4-hydroxycinnamic acid. Pyridylaminated glycans, reducing oligosaccharides, and underivatized glycopeptides were properly diluted with water, spotted on the target, and air dried. After addition of 2% 2,5-dihydroxybenzoic acid, the samples were immediately dried by application of vacuum. Protein concentrations were determined by the bicinchoninic acid method (Pierce) or, at the final steps of enzyme purification, by amino acid analysis (35Altmann F. Anal. Biochem. 1992; 204: 215-219Crossref PubMed Scopus (93) Google Scholar). Fuc-T C3 was purified from mung bean seedlings by Triton X-100 extraction of a crude microsomal preparation and several chromatographic steps including cation exchange and two types of affinity chromatography. The typical elution profile of activity from the S-Sepharose is shown in Fig. 2. Conveniently, this step provided separation from N-acetyl-β-glucosaminidase which otherwise would have degraded the ligand of the subsequent affinity chromatography on GnGn-Sepharose (Fig. 2). After the last purification step on GDP-hexanolamine-Sepharose, the final yield was 18 μg of protein from 5 kg of mung beans (TableI). SDS-PAGE revealed two bands, a major band at 54 kDa and one at 56 kDa (Fig. 3). In order to check whether the two polypeptides are distinct or just different forms of the same enzyme, the bands were compared by MALDI-TOF MS of tryptic peptides obtained by in-gel digestion. The mass spectra of the 54- and 56-kDa band were indistinguishable indicating that both b" @default.
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• W2040307553 title "Purification, cDNA Cloning, and Expression of GDP-l-Fuc:Asn-linked GlcNAc α1,3-Fucosyltransferase from Mung Beans" @default.
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