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• W2045133714 abstract "In the present experiments the cDNA coding for a truncated form of the β1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67–74-kDa fusion protein. Typically a yield of 750 μg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, converting the acceptor to the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Accordingly, we call the recombinant β1,6-GlcNAc transferase cIGnT6 to emphasize its action atcentral rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, β1,4galactosyltransferase and β1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes. In the present experiments the cDNA coding for a truncated form of the β1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67–74-kDa fusion protein. Typically a yield of 750 μg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, converting the acceptor to the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Accordingly, we call the recombinant β1,6-GlcNAc transferase cIGnT6 to emphasize its action atcentral rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, β1,4galactosyltransferase and β1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes. d-galactose N-acetyl-d-glucosamine matrix-assisted laser desorption/ionization time-of-flight glutathione S-transferase polymerase chain reaction 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis. Human embryonal carcinoma cells of line PA1 express large amounts of polylactosamines covalently bound to proteins (1Rasilo M. Wartiovaara J. Renkonen O. Pure Appl. Chem. 1979; 52: 55-63Crossref Scopus (7) Google Scholar, 2Muramatsu T. Avner P. Fellous M. Gachelin G. Jacob F. Somatic Cell Genet. 1979; 5: 753-761Crossref PubMed Scopus (15) Google Scholar, 3Rasilo M.L. Wartiovaara J. Renkonen O. Can. J. Biochem. 1980; 58: 384-393Crossref PubMed Scopus (12) Google Scholar, 4Rasilo M.L. Renkonen O. Eur. J. Biochem. 1982; 123: 397-405Crossref PubMed Scopus (11) Google Scholar). Human and murine embryonal carcinoma cells synthesize polylactosamines, which resembled each others in size, monosaccharide composition (5Muramatsu T. Gachelin G. Nicolas J.F. Condamine H. Jakob H. Jacob F. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2315-2319Crossref PubMed Scopus (180) Google Scholar), and branching (6Renkonen O. Biochem. Soc. Trans. 1983; 11: 265-267Crossref PubMed Scopus (8) Google Scholar, 7Fukuda M.N. Dell A. Oates J.E. Fukuda M. J. Biol. Chem. 1985; 260: 6623-6631Abstract Full Text PDF PubMed Google Scholar). In human erythrocytes, linear poly-N-acetyllactosamines (Galβ1–4GlcNAcβ1–3)n1are converted to branched arrays, Galβ1–4GlcNAcβ1–3(Galβ1–4GlcNAcβ1–6)Galβ1-R after birth (8Feizi T. Childs R.A. Watanabe K. Hakomori S.I. J. Exp. Med. 1979; 149: 975-980Crossref PubMed Scopus (134) Google Scholar, 9Fukuda M. Fukuda M.N. Hakomori S. J. Biol. Chem. 1979; 254: 3700-3703Abstract Full Text PDF PubMed Google Scholar). These two types of poly-N-acetyllactosamine backbones represent i and I blood group antigens, respectively (8Feizi T. Childs R.A. Watanabe K. Hakomori S.I. J. Exp. Med. 1979; 149: 975-980Crossref PubMed Scopus (134) Google Scholar,9Fukuda M. Fukuda M.N. Hakomori S. J. Biol. Chem. 1979; 254: 3700-3703Abstract Full Text PDF PubMed Google Scholar). Two types of branching enzymes, IGnT6, appear to be involved in the biosynthesis of branched polylactosamine backbones. One of them, dIGnT6, adds β1,6N-acetyllactosamine at the peridistal galactose residue of GlcNAcβ1–3Galβ1–4GlcNAcβ1-OR, forming GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAcβ1-OR (10Piller F. Cartron J.-P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 11Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H. van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 12Koenderman A.H.L. Koppen P.L. van den Eijnden D.H. Eur. J. Biochem. 1987; 166: 199-208Crossref PubMed Scopus (51) Google Scholar, 13Ropp P. Little M.R. Cheng P.-W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 14Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar). This enzyme does not act at the central galactose of GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc (15Helin J. Penttilä L. Leppänen A. Maaheimo H. Lauri S. Costello C.E. Renkonen O. FEBS Lett. 1997; 412: 637-642Crossref PubMed Scopus (14) Google Scholar). The other IGnT6 (cIGnT6) catalyzes the generation of β1,6N-acetylglucosaminyl branches at the central galactose residue of Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1-OR (14Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar, 16Leppänen A. Penttilä L. Niemelä R. Helin J. Seppo A. Lusa S. Renkonen O. Biochemistry. 1991; 30: 9287-9296Crossref PubMed Scopus (31) Google Scholar, 17Niemelä R. Rabinä J. Leppänen A. Maaheimo H. Costello C.E. Renkonen O. Carbohydr. Res. 1995; 279: 331-338Crossref PubMed Scopus (18) Google Scholar) and at both central galactose residues of Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc (18Leppänen A. Niemelä R. Renkonen O. Biochemistry. 1997; 36: 13729-13735Crossref PubMed Scopus (13) Google Scholar). This enzyme does not react at the GlcNAcβ1–3Galβ1–4GlcNAcβ1-OR (15Helin J. Penttilä L. Leppänen A. Maaheimo H. Lauri S. Costello C.E. Renkonen O. FEBS Lett. 1997; 412: 637-642Crossref PubMed Scopus (14) Google Scholar). The cDNA coding for the enzyme responsible for the key reaction in the biosynthesis of the branched polylactosamine backbones has been isolated (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar), but it is not known whether it codes for a cIGnT6 or a dIGnT6 enzyme or perhaps for an unknown branch-generating enzyme. In the present experiments, a fusion protein representing the catalytic ectodomain of the branch-forming enzyme and glutathioneS-transferase was functionally expressed in Baculovirus-infected insect cells and purified. Analysis of the substrate specificity of the purified recombinant enzyme showed that it possesses the activity of the cIGnT6 type, but not of the dIGnT6 type. The data suggest that the recombinant cIGnT6 is able to transfer multiple GlcNAc branches to long linear polylactosamines, a prerequisite for improving enzyme-assisted in vitrosynthesis of a type of multivalent sialyl Lewis x glycans (21Renkonen O. Toppila S. Penttilä L. Salminen H. Helin J. Maaheimo H. Costello C.E. Turunen J.P. Renkonen R. Glycobiology. 1997; 7: 453-461Crossref PubMed Scopus (48) Google Scholar, 22Salminen H. Ahokas K. Niemelä R. Penttilä L. Maaheimo H. Helin J. Costello C.E. Renkonen O. FEBS Lett. 1997; 419: 220-226Crossref PubMed Scopus (14) Google Scholar) that are high affinity inhibitors of lymphocyte L-selectin. The human PA1 cell β1,6N-acetylglucosaminyltransferase that generates branches to polylactosamine backbones (IGnT6) has been cloned previously and sequenced (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The following materials were purchased from indicated sources: Pfu polymerase (Stratagene), oligonucleotides A and B (Amersham Pharmacia Biotech), T4 ligase (Promega), Escherichia coli strains JM 105 and XL-1blue (ATCC), BamHI and EcoRI (Promega), Baculovirus transfer vector pAcSecG2T, linearized BaculoGold DNA, pAcGHLT-XylE control plasmid, and transfection buffers A and B (PharMingen), Sf9 cells (Invitrogen), SF-900 medium, gentamycin, penicillin, streptomycin (all from Life Technologies, Inc.), insect cell lysis buffer, protease inhibitor cocktail, glutathione-agarose beads, phosphate-buffered saline wash buffer, GST elution buffer, glutathione powder (PharMingen), Microcon 30 concentrators (Amicon), plastic ware (Greiner), monoclonal mouse anti-GST (Zymed Laboratories Inc.), ECL reagents (Amersham Pharmacia Biotech). Sf9 cells were grown at 27 °C in SF-900 medium supplemented with 10 μg/ml gentamycin, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum in either 10-cm Petri dishes, six-well platforms, or 75-cm2tissue culture flasks. Restriction endonuclease reactions, DNA ligations, bacterial transfections plasmid isolations, RNA isolations, Northern blots, Western blots, ECL reactions, and Coomassie stainings were performed by standard methods. The truncated segment coding for residues 26–400 of human IGnT6 was synthesized with polymerase chain reaction (PCR) using Pfu polymerase. The IGnT6 cDNA (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar) in pcDNAI vector was used as a template, and oligonucleotides A (5′-CAAGAAGGATCCAATTTTGGGGGAGATCCAAGC) and B (5′-GGATGAATTCCTCAAAAATACCAGCTGGGTTGTATCGC) as primers. Oligonucleotide A created a BamHI site to the 5′ end of the truncated IGnT6 DNA, and oligonucleotide B created an in-frame stop codon and an EcoRI site to its 3′ end. For expression in insect cells as GST fusion protein, the amplified IGnT6 PCR product was subcloned into the plasmid pAcSecG2T (PharMingen) downstream of ATG start site and GST coding region using the BamHI and EcoRI sites. The construct lacks the section of DNA encoding the cytoplasmic N terminus and transmembrane region of IGnT6. Transfer vector pAcSecG2T-IGnT6 (4.4 μg) and BaculoGold Baculovirus-linearized DNA (0.5 μg) were co-transfected into confluent Sf9 cells and incubated for 3 days at 27 °C. GST-IGnT6 virus progeny was isolated using plaque assay and amplified three times. The recombinant virus was stored as a stock solution (4 × 107 plaque-forming units/ml) at 4 °C in SF-900 medium containing supplements and 10% fetal calf serum. For activity assays the recombinant enzyme was stored a few days at −20 °C without loss of activity. Microscale purification was performed: 2.0 × 107 Sf9 insect cells were infected or not infected with recombinant Baculovirus (4 plaque-forming units/cell) and incubated at 27 °C for 3 days. The cells were lysed on ice for 45 min with the lysis buffer containing protease inhibitors and precleared by centrifuging at 40,000 × g for 30 min to pellet the cellular debris. Precleared lysates were loaded into the glutathione bead column after which the column was washed several times with phosphate-buffered saline wash buffer. The fusion protein was eluted with the GST elution buffer containing glutathione (5 mm). Glutathione was removed by dialyzing against 50 mm Tris-HCl (pH 8.0) or by washing the eluates several times in Microcon 30 concentrators. The acceptor oligosaccharides (Table I) were synthesized as described: the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc (23Seppo A. Penttilä L. Makkonen A. Leppänen A. Niemelä R. Jäntti J. Helin J. Renkonen O. Biochem. Cell Biol. 1990; 68: 44-53Crossref PubMed Scopus (27) Google Scholar), the tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc (24Renkonen O. Penttilä L. Niemelä R. Leppänen A. Glycoconj. J. 1991; 8: 376-380Crossref PubMed Scopus (17) Google Scholar), the pentasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc (25Räbinä J. Natunen J. Niemelä R. Salminen H. Ilves K. Aitio O. Maaheimo H. Helin J. Renkonen O. Carbohydr. Res. 1998; 305: 491-499Crossref Scopus (19) Google Scholar), and the octasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc (22Salminen H. Ahokas K. Niemelä R. Penttilä L. Maaheimo H. Helin J. Costello C.E. Renkonen O. FEBS Lett. 1997; 419: 220-226Crossref PubMed Scopus (14) Google Scholar). The marker Galβ1–4GlcNAcβ1–3(GlcNAc1–6)Galβ1–4GlcNAc was synthesized as described in Ref. 17Niemelä R. Rabinä J. Leppänen A. Maaheimo H. Costello C.E. Renkonen O. Carbohydr. Res. 1995; 279: 331-338Crossref PubMed Scopus (18) Google Scholar.Table IStructures of the polylactosamine acceptors 3Gal β1–4GlcNAc GlcNAcβ1/ 3Gal β1–4GlcNAc Galβ1–4GlcNAcβ1/ 3Gal β1–4GlcNAc Galβ1–4GlcNAcβ1/ Fucαl/3 3Gal β1–4GlcNAc 3Gal β1–4GlcNAcβ1/ 3Gal β1–4GlcNAc/ Galβ1–4GlcNAcβ1/ Open table in a new tab The IGnT6 reactions with the purified recombinant enzyme were performed by incubating the acceptor oligosaccharides (1–40 nmol) and UDP-GlcNAc (1.4 μmol) with 1.0 μg of the recombinant enzyme for 120 h in a total volume of 10 μl of a solution containing 200 mm MOPS (pH 7.0), 20 mmEDTA, 0.5 mm ATP, 0.28 mm dithiothreitol, 8 mm NaN3, 10% glycerol, 0.2% bovine serum albumin. The reaction mixtures were passed through a mixed bed of Dowex AG1 (AcO−) and Dowex AG50 (H+), and the eluates were lyophilized. In the IGnT6 reactions performed with the Sf9 cell lysates, the incubation mixtures contained 330 pmol of acceptor (either the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc, the tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, or the pentasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAc), 0.5 mg of UDP-GlcNAc, 2.5 μmol of Tris-HCl (pH 7.5), 0.4 μmol of NaN3, 1.0 μmol of EDTA, 25 nmol of ATP, 3 μmol of galactonolactone, 1 μmol of galactose, and 5 μmol of N-acetylglucosamine and the cell lysate (430 μg total protein) in a total volume of 50 μl. The cell lysate was prepared by incubating equal volumes of the cells and the lysis solution (1.8% NaCl, 1% Triton X-100, and the protease inhibitors). Paper chromatographic runs of desalted radiolabeled saccharides were performed on Whatman III Chr paper with the upper phase of 1-butanol/acetic acid/water (4:1:5 v/v). Radioactivity on the chromatograms was monitored using Opriscint (Wallac, Turku, Finland) as scintillant. Marker lanes of malto-oligosaccharides, lactose, and galactose on both sides of the sample lanes were stained with silver nitrate. Digestions with endo-β-galactosidase from Bacteroides fragilis (EC3.2.1.103; Boehringer Mannheim, Mannheim, Germany) were performed according to Ref. 16Leppänen A. Penttilä L. Niemelä R. Helin J. Seppo A. Lusa S. Renkonen O. Biochemistry. 1991; 30: 9287-9296Crossref PubMed Scopus (31) Google Scholar; parallel control reactions cleaved over 90% of radiolabeled GlcNAcβ1–3Galβ1–4GlcNAc. Digestions with jack bean (exo)-β-galactosidase were performed as described in Ref. 26Renkonen O. Mäkinen P. Hård K. Helin J. Penttilä L. Biochem. Cell Biol. 1988; 66: 449-453Crossref PubMed Scopus (18) Google Scholar. The 1H NMR experiments were carried out as described (17Niemelä R. Rabinä J. Leppänen A. Maaheimo H. Costello C.E. Renkonen O. Carbohydr. Res. 1995; 279: 331-338Crossref PubMed Scopus (18) Google Scholar). IGnT6 is the β1,6-GlcNAc Transferase That Generates Branches to Poly-N-acetyllactosamine backbones in human PA1 cells (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The truncated IGnT6 (amino acids 26–400, Fig. 1 A), encoding for the stem and the Golgi lumenal regions of native IGnT6, was synthesized by PCR. It was inserted downstream of the very late polyhedrin promoter, gp67 signal sequence, and GST coding region of the vector pAcSecG2T, between the BamHI and EcoRI restriction sites in the cloning site to form the transfer vector pAcSecG2T-IGnT6 (Fig. 1 B). Sf9 insect cells were co-transfected with the pAcSecG2T-IGnT6 transfer vector together with the linearized BaculoGold Baculovirus DNA. Northern blot analysis from the infected cells indicated that a new RNA transcript of the size of 2.3 kilobases hybridizing with the full-length IGnT6 cDNA was present (Fig. 2 A). This de novoexpressed transcript was first detected at 48 h, and its level of expression increased up to 72 h postinfection. Expression of the recombinant fusion protein GST-IGnT6 was monitored by Western blot analysis with a monoclonal anti-GST antibody. Proteins in the cell culture media and lysates from both uninfected as well as from infected cells were separated. While no immunoreactive bands were present in the samples from the culture media, two broad bands at 67 and 74 kDa were detected by anti-GST antibody in samples prepared from cell lysates at 48–96 h after infection (Fig. 2 B). The fusion protein GST-IGnT6 has five potentialN-glycosylation sites. To study them we infected Sf9 cells with recombinant virus following by treatment with tunicamycin, an inhibitor of N-glycosylation. After tunicamycin treatment only two bands centered at 67 kDa were detected with the anti-GST antibody in the Western blot (Fig. 3). These data showed that the IGnT6 was N-glycosylated in the Sf9 cells, and the size heterogenicity was at least partially due to differences in N-glycosylation. A one-step purification of the recombinant GST-IGnT6 was achieved by affinity chromatography using glutathione-agarose beads. Samples of the cell lysate and the purified protein were run in SDS-PAGE and stained with Coomassie Blue. A major band was observed at 67 kDa in the lane of the purified protein; minor bands were visible at 58 and 76 kDa (Fig. 4). The yield of the purified fusion protein was typically 750 μg/109 infected Sf9 cells present in 1 liter of the suspension culture. The polylactosamine acceptors used in these experiments are collected in Table I. The functionality of the recombinant GST-IGnT6 was studied first by using Sf9 cell lysates. In a typical experiment, a lysate was incubated with radiolabeled trisaccharide GlcNAcβ1–3[14C]Galβ1–4GlcNAc and UDP-GlcNAc. Neither a tetrasaccharide-like product nor any other product besides the starting trisaccharide was detected by paper chromatography of the neutral oligosaccharides of the incubation mixture (Fig. 5 A). By contrast, similar experiments repeatedly converted significant amounts of the radiolabeled tetrasaccharide [3H]Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc into a product that migrated like a pentasaccharide, suggesting the presence of cIGnT6 activity (data not shown, see below). The functionality of the recombinant enzyme GST-IGnT6 was confirmed by incubating it with UDP-GlcNAc and [3H]Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc. This reaction mixture contained a radiolabeled oligosaccharide product, which co-migrated with the authentic Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc (Fig. 5 B). A partial reaction had taken place, yielding 28% of a pentasaccharide (peak 1) and leaving 72% of the tetrasaccharide acceptor (peak 2) intact. This was confirmed by MALDI-TOF mass spectrometry, performed with a sample from another similar but more exhaustive reaction mixture; the spectrum revealed the presence of 49% of a pentasaccharide Gal2GlcNAc3 in addition to 51% of the acceptor tetrasaccharide (Fig. 6 A). To characterize the pentasaccharide product, the radiolabeled glycan was first incubated with jack bean (exo)-β-galactosidase, which released all tritium label in the form of free [3H]Gal (Fig. 7 A). This implies that the new GlcNAc of the pentasaccharide was not transferred to the distal, tritium-containing galactose residue of the tetrasaccharide acceptor, as this would not have been susceptible to (exo)-β-galactosidase. Hence, the reaction had been different from the β1,6-GlcNAc transfer to the terminal galactose described in other laboratories (12Koenderman A.H.L. Koppen P.L. van den Eijnden D.H. Eur. J. Biochem. 1987; 166: 199-208Crossref PubMed Scopus (51) Google Scholar, 27Fukuda M.N. J. Biol. Chem. 1981; 256: 3900-3905Abstract Full Text PDF PubMed Google Scholar, 28Zdebska E. Krauze R. Koscielak J. Carbohydr. Res. 1983; 120: 113-130Crossref PubMed Scopus (25) Google Scholar, 29Zielenski J. Koscielak J. FEBS Lett. 1983; 163: 114-118Crossref PubMed Scopus (10) Google Scholar, 30Zielenski J. Koscielak J. FEBS Lett. 1983; 158: 164-168Crossref PubMed Scopus (21) Google Scholar). Another enzymatic digestion was performed with endo-β-galactosidase, which cleaves the internal β-galactosidic linkage of the tetrasaccharide acceptor (27Fukuda M.N. J. Biol. Chem. 1981; 256: 3900-3905Abstract Full Text PDF PubMed Google Scholar) and other linear polylactosamines, but does not hydrolyze the branched Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc synthesized by the rat serum cIGnT6 (31Leppänen A. Salminen H. Zhu Y. Maaheimo H. Helin J. Costello C.E. Renkonen O. Biochemistry. 1997; 36: 7026-7036Crossref PubMed Scopus (21) Google Scholar). When the pentasaccharide was incubated with endo-β-galactosidase, no breakdown product was formed (Fig. 7 B). Collectively, these data showed that the pentasaccharide product of the recombinant GST-IGnT6 behaved in several ways like the authentic Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc. However, the possibility still remained that that the new GlcNAc could have been transferred to position 2 or 4 of the internal Gal unit or to either of the two GlcNAc residues of the tetrasaccharide acceptor; in addition, the GlcNAc unit could have become bound to the acceptor with an α-linkage. To address these possibilities, the 1H NMR spectrum of the pentasaccharide product at 500 MHz was obtained (Fig. 8, Table II). The resonances of the structural reporter groups observed were identical with those of the authentic Galβ1–4GlcNAcβ1–3(GlcNAc1–6)Galβ1–4GlcNAc reported in (32Maaheimo H. Räbinä J. Renkonen O. Carbohydr. Res. 1997; 297: 145-151Crossref PubMed Scopus (19) Google Scholar) (see Table II). The NMR data provide evidence for the presence of two β-linked GlcNAc residues in the pentasaccharide product. The similarity of the H1 resonance of the new GlcNAc unit of the pentasaccharide product with the H1 signal of the GlcNAc-5 unit of the authentic marker provides strong support for the notion that the new βGlcNAc residue was 1,6-bonded to the midchain galactose. The similarities of the H4 resonances of the Gal-2 units are also strong indications for the identity of the two pentasaccharides. Bierhuizenet al. (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar) provided methylation data on glycoprotein glycans from appropriately transfected cells, suggesting that the IGnT6 responsible for the polylactosamine branching in PA1 cells converts 3-substituted galactoses to 3,6-disubstituted units. These combined data imply that the pentasaccharide product generated by the recombinant enzyme in the present experiments was almost certainly Galβ1–4GlcNAcβ1–3(GlcNAc1–6)Galβ1–4GlcNAc.Table II1H NMR chemical shifts of structural reporter groups in the pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc at 23 °CReporter groupResidue2-aFor monosaccharide denotation, see Fig. 8.Authentic pentasaccharide synthesized by cIGnT6 of rat serum (32Maaheimo H. Räbinä J. Renkonen O. Carbohydr. Res. 1997; 297: 145-151Crossref PubMed Scopus (19) Google Scholar)Pentasaccharide synthesized by cIGnT6 of PA1 cell lysates (20Leppänen A. Zhu Y. Maaheimo H. Helin J. Lehtonen E. Renkonen O. J. Biol. Chem. 1998; 273: 17399-17405Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar)Pentasaccharide synthesized by recombinant cIGnT6 in a Sf9 cell lysateH-1GlcNAc-1 (α)5.2125.2125.213GlcNAc-1 (β)4.7314.7304.730Gal-24.4544.4534.453GlcNAc-34.701/4.6962-bThe two values given correspond to the two anomers of the pentasaccharide.4.701/4.6964.701/4.696Gal-44.4814.4804.480GlcNAc-54.5852-cI 1,2 8.5 Hz4.5852-dI 1,2 8.5 Hz4.5852-eI 1,2 8.5 HzH-4Gal-24.1494.1484.147NAcGlcNAc-1 (α)2.0562.0552.055GlcNAc-1 (β)2.0712.0712.071GlcNAc-32.0322.0322.032GlcNAc-52.0512.0502.0512-a For monosaccharide denotation, see Fig. 8.2-b The two values given correspond to the two anomers of the pentasaccharide.2-c I 1,2 8.5 Hz2-d I 1,2 8.5 Hz2-e I 1,2 8.5 Hz Open table in a new tab The purified recombinant GST-IGnT6 catalyzed the GlcNAc transfer also to the linear Galβ1–4GlcNAcβ1–3′Galβ1–4GlcNAcβ1–3′Galβ1–4GlcNAcβ1–3′Galβ1–4GlcNAc, generating in a partial reaction nonasaccharides of the type (GlcNAc)1(Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc) and decasaccharides of type (GlcNAc)2(Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc) as shown by MALDI-TOF mass spectrometry (Fig. 6 B). The nona- and decasaccharide products represented mixtures of isomers carrying one and two GlcNAc-branches on the linear octasaccharide acceptor. Hence, it appears likely that the recombinant cIGnT6 shares with the enzymes of mammalian serum (18Leppänen A. Niemelä R. Renkonen O. Biochemistry. 1997; 36: 13729-13735Crossref PubMed Scopus (13) Google Scholar) and hog intestine (46Sakamoto Y. Taguchi T. Tano Y. Ogawa T. Leppänen A. Kinnunen M. Aitio O. Parmanne P. Renkonen O. Taniguchi N. J. Biol. Chem. 1998; 273: 27625-27632Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) the capacity to form multiple branches on long linear polylactosamine acceptors. The recombinant GST-IGnT6 was unable to transfer GlcNAc to the pentasaccharide [3H]Galβ1–4GlcNAcβ1–3Galβ1–4(Fucα-3)GlcNAc, as assessed by paper chromatography, while the control reaction performed with the fucose-free tetrasaccharide [3H]Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc gave a 16% yield of the radiolabeled pentasaccharide product (data not shown). Hence, the recombinant enzyme shares with the rat serum cIGnT6-enzyme (18Leppänen A. Niemelä R. Renkonen O. Biochemistry. 1997; 36: 13729-13735Crossref PubMed Scopus (13) Google Scholar) in the inability to act at the inner galactose of the fucosylated pentasaccharide. Taken together, all properties of the recombinant GST-IGnT6 that were tested in the present experiments were qualitatively similar to those of the cIGnT6 activity of rat serum. The present experiments describe successful functional expression of a truncated form of a human β1,6-GlcNAc transferase as a fusion protein with glutathione S-transferase in Baculovirus-infected insect cells and provide evidence that the purified recombinant enzyme represents a cIGnT6 that catalyzes transfer to centrally located galactose residues of linear polylactosamine chains. The cDNA expressed was originally isolated from human embryonal carcinoma cells of line PA1, where it was shown to code for the enzyme responsible for the biosynthesis of branched polylactosamine backbones (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The present data show that the cDNA does not code for an enzyme that transfers at the distally located galactose units at the nonreducing termini of the acceptor chains. The data show also that the cDNA does not code for dIGnT6, a branching enzyme that acts at peridistal galactoses of polylactosamine chains of the type GlcNAcβ1–3Galβ1–4GlcNAcβ1-OR. Instead, the data imply that the cDNA codes for a branching enzyme that transfers to midchain galactoses of polylactosamines; the presence of at least one complete N-acetyllactosamine unit, bonded to position 3, appears to be necessary for the galactose residues reacting with the purified recombinant cIGnT6. Our recent observations show that PA1 cell lysates contain cIGnT6 rather than dIGnT6 activity (20Leppänen A. Zhu Y. Maaheimo H. Helin J. Lehtonen E. Renkonen O. J. Biol. Chem. 1998; 273: 17399-17405Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Hence, the elimination of the cytoplasmic and the membrane binding segment from the recombinant cIGnT6 of the present experiments was probably not associated with major changes in the substrate specificity. Consequently, it is worth noting that the recombinant cIGnT6 shares several features with the soluble cIGnT6 enzymes present in mammalian serum (18Leppänen A. Niemelä R. Renkonen O. Biochemistry. 1997; 36: 13729-13735Crossref PubMed Scopus (13) Google Scholar, 31Leppänen A. Salminen H. Zhu Y. Maaheimo H. Helin J. Costello C.E. Renkonen O. Biochemistry. 1997; 36: 7026-7036Crossref PubMed Scopus (21) Google Scholar) and with the membrane-bound form of cIGnT6 recently isolated from hog small intestine by Sakamoto et al. (46Sakamoto Y. Taguchi T. Tano Y. Ogawa T. Leppänen A. Kinnunen M. Aitio O. Parmanne P. Renkonen O. Taniguchi N. J. Biol. Chem. 1998; 273: 27625-27632Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The common features include (i) the ability to transfer one or several branches to midchain galactoses of long polylactosamine chains, (ii) the unability to react at peridistal galactose units in acceptors of the type GlcNAcβ1–3Galβ1–4GlcNAcβ1-OR, and (iii) the inability to transfer at midchain galactoses that belong to Lewis x determinants. The present study demonstrates that cIGnT6 adds β1,6-linkedN-acetylglucosamine to a linear poly-N-acetyllactosamine as shown in Fig. 9 A. In this biosynthetic pathway, the addition of I branch does not occur at the termini of elongating poly-N-acetyllactosamine. It is expected that the addition of I branches proceeds randomly along preformed poly-N-acetyllactosamine chains in human and rabbit erythrocytes (33Fukuda M. Dell A. Oates J.E. Fukuda M.N. J. Biol. Chem. 1984; 259: 8260-8273Abstract Full Text PDF PubMed Google Scholar, 34Dabrowski U. Hanfland P. Egge H. Kuhn S. Dabrowski J. J. Biol. Chem. 1984; 259: 7648-7651Abstract Full Text PDF PubMed Google Scholar). Analysis of PA1 cells (7Fukuda M.N. Dell A. Oates J.E. Fukuda M. J. Biol. Chem. 1985; 260: 6623-6631Abstract Full Text PDF PubMed Google Scholar) demonstrated that their poly-N-acetyllactosamine backbones have uniformly short branches, consisting of single N-acetyllactosamine units with or without terminal substituents. Such arrays could result from a relatively late action of cIGnT6 on preformed i-type chains (Fig. 9 A). In contrast, dIGnT6 is expected to form branched poly-N-acetyllactosamine backbones in association of the chain growth, leading occasionally to the formation of branched-branch arrays of N-acetyllactosamine units in the multiply branched polylactosamines (Fig. 9 B). Such structures may be synthesized in gastrointestinal cells and Novikoff cells where dGnT6 activity has been detected (10Piller F. Cartron J.-P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 35van den Eijnden D.H. Winterwerp H. Smeeman P. Schiphorst W.E. J. Biol. Chem. 1983; 258: 3435-3437Abstract Full Text PDF PubMed Google Scholar). Indeed, hog gastric mucosa contains an octadecameric tetra-antennary lipid-bound polylactosamine (45Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Crossref PubMed Scopus (134) Google Scholar) that resembles strikingly the branched branches array of sevenN-acetyllactosamine residues that we have synthesizedin vitro by using dIGnT6 in combination with GnT3 and GalT4 (39Toppila S. Lauronen J. Mattila P. Turunen J.P. Penttilä L. Paavonen T. Renkonen O. Renkonen R. Eur. J. Immunol. 1997; 27: 1360-1365Crossref PubMed Scopus (23) Google Scholar, 41Maaheimo H. Renkonen R. Turunen J. Penttilä L. Renkonen R. Eur. J. Biochem. 1995; 234: 616-625Crossref PubMed Scopus (62) Google Scholar, 42Seppo A. Turunen J.-P. Penttilä L. Keane A. Renkonen O. Renkonen R. Glycobiology. 1996; 6: 65-71Crossref PubMed Scopus (44) Google Scholar). As shown previously, human granulocytes contain heavily fucosylated poly-N-acetyllactosamines such as R1-Galβ1–4(Fucα1–3)GlcNAcβ1-R2, and these side chains do not contain any I branching (36Spooncer E. Fukuda M. Klock J.C. Oates J.E. Dell A. J. Biol. Chem. 1984; 259: 4792-4801Abstract Full Text PDF PubMed Google Scholar, 37Mizoguchi A. Takasaki S. Maeda S. Kobata A. J. Biol. Chem. 1984; 259: 11949-11957Abstract Full Text PDF PubMed Google Scholar). Our present study suggests that the lack of I branching in granulocytes poly-N-acetyllactosamines could be due to the inhibition of cGnT6 by α1,3-fucosyl residues. Alternatively, human granulocytes may not express IGnT6. On the other hand, the termini of I structures such as Fucα1–2Galβ1–4GlcNAc(Fucα1–2Galβ1–4GlcNAcβ1–6)Gal (33Fukuda M. Dell A. Oates J.E. Fukuda M.N. J. Biol. Chem. 1984; 259: 8260-8273Abstract Full Text PDF PubMed Google Scholar, 34Dabrowski U. Hanfland P. Egge H. Kuhn S. Dabrowski J. J. Biol. Chem. 1984; 259: 7648-7651Abstract Full Text PDF PubMed Google Scholar) provide the H antigen or its modifications, the A and B antigens, on two neighboring N-acetyllactosamines. Such bivalent antigenic structures function as much better ligands for anti-ABO antibodies than single antigenic structures (38Romans D.G. Tilley C.A. Dorrington K.J. J. Immunol. 1980; 124: 2807-2811PubMed Google Scholar). In the same vein, it has been demonstrated that multivalent sialyl Lex polylactosamines at very low concentrations can inhibit L-selectin-mediated lymphocyte binding to the endothelium of lymph nodes (39Toppila S. Lauronen J. Mattila P. Turunen J.P. Penttilä L. Paavonen T. Renkonen O. Renkonen R. Eur. J. Immunol. 1997; 27: 1360-1365Crossref PubMed Scopus (23) Google Scholar) and rejecting organ transplants (21Renkonen O. Toppila S. Penttilä L. Salminen H. Helin J. Maaheimo H. Costello C.E. Turunen J.P. Renkonen R. Glycobiology. 1997; 7: 453-461Crossref PubMed Scopus (48) Google Scholar, 40Turunen J.-P. Majuri M.-L. Seppo A. Tiisala S. Paavonen T. Miyasaka M. Lemström K. Penttilä L. Renkonen O. Renkonen R. J. Exp. Med. 1995; 182: 1133-1142Crossref PubMed Scopus (85) Google Scholar, 41Maaheimo H. Renkonen R. Turunen J. Penttilä L. Renkonen R. Eur. J. Biochem. 1995; 234: 616-625Crossref PubMed Scopus (62) Google Scholar, 42Seppo A. Turunen J.-P. Penttilä L. Keane A. Renkonen O. Renkonen R. Glycobiology. 1996; 6: 65-71Crossref PubMed Scopus (44) Google Scholar). In combination with the previously cloned enzymes GalT4 (43Masri K.A. Appert H.E. Fukuda M.N. Biochem. Biophys. Res. Commun. 1988; 157: 657-663Crossref PubMed Scopus (130) Google Scholar, 44Joziasse D.H. Shaper J.H. van den Eijnden D.H. Van Tunen A.J. Shaper N.L. J. Biol. Chem. 1989; 264: 14290-14297Abstract Full Text PDF PubMed Google Scholar) and GnT3 (45Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Crossref PubMed Scopus (134) Google Scholar), the purified recombinant cIGnT6 should allow general polylactosamine synthesis that is totally based on the use of recombinant enzymes. Further studies on synthesis of bioactive poly-N-acetyllactosamines with long, branched backbones are of great interest because such oligosaccharides are expected to be powerful carbohydrate-based antagonists of selectins and other sugar-binding proteins." @default.
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• W2045133714 title "The Centrally Acting β1,6N-Acetylglucosaminyltransferase (GlcNAc to Gal)" @default.
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