The Centrally Acting b1,6N-Acetylglucosaminyltransferase (GlcNAc to Gal) FUNCTIONAL EXPRESSION, PURIFICATION, AND ACCEPTOR SPECIFICITY OF A HUMAN ENZYME INVOLVED IN MIDCHAIN BRANCHING OF LINEAR POLY-N-ACETYLLACTOSAMINES*

In the present experiments the cDNA coding for a truncated form of the b1,6N-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 mg 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 Galb1– 4GlcNAcb1–3Galb1– 4GlcNAc, converting the acceptor to the branched pentasaccharide Galb1–4GlcNAcb1–3(GlcNAcb1–6)Galb1–4GlcNAc as shown by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry, degradative experiments, and H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcb1–3Galb1–4GlcNAc. Accordingly, we call the recombinant b1,6-GlcNAc transferase cIGnT6 to emphasize its action at central 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, b1,4galactosyltransferase and b1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

The cDNA coding for the enzyme responsible for the key reaction in the biosynthesis of the branched polylactosamine backbones has been isolated (19), 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 glutathione S-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 vitro synthesis of a type of multivalent sialyl Lewis x glycans (21,22) that are high affinity inhibitors of lymphocyte L-selectin.
Construction of the Baculovirus Transfer Vector pAcSecG2T-IGnT6 -The truncated segment coding for residues 26 -400 of human IGnT6 was synthesized with polymerase chain reaction (PCR) using Pfu polymerase. The IGnT6 cDNA (19) in pcDNAI vector was used as a template, and oligonucleotides A (5Ј-CAAGAAGGATCCAATTTTGG-GGGAGATCCAAGC) and B (5Ј-GGATGAATTCCTCAAAAATACCAG-CTGGGTTGTATCGC) as primers. Oligonucleotide A created a BamHI site to the 5Ј end of the truncated IGnT6 DNA, and oligonucleotide B created an in-frame stop codon and an EcoRI site to its 3Ј 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.
Expression of GST-IGnT6 in the Baculovirus/Insect Cell System-Transfer vector pAcSecG2T-IGnT6 (4.4 g) and BaculoGold Baculovirus-linearized DNA (0.5 g) 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 ϫ 10 7 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 Ϫ20°C without loss of activity.
Purification of GST-IGnT6 -Microscale purification was performed: 2.0 ϫ 10 7 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 ϫ 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.
Glycosyltransferase Reactions-The IGnT6 reactions with the purified recombinant enzyme were performed by incubating the acceptor oligosaccharides (1-40 nmol) and UDP-GlcNAc (1.4 mol) with 1.0 g of the recombinant enzyme for 120 h in a total volume of 10 l of a solution containing 200 mM MOPS (pH 7.0), 20 mM EDTA, 0.5 mM ATP, 0.28 mM dithiothreitol, 8 mM NaN 3 , 10% glycerol, 0.2% bovine serum albumin. The reaction mixtures were passed through a mixed bed of Dowex AG1 (AcO Ϫ ) and Dowex AG50 (H ϩ ), and the eluates were lyophilized.
Chromatographic Methods-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). Radio-activity 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.

RESULTS
Construction of pAcSecG2T-IGnT6 -IGnT6 is the ␤1,6-Glc-NAc Transferase That Generates Branches to Poly-N-acetyllactosamine backbones in human PA1 cells (19). The truncated IGnT6 (amino acids 26 -400, Fig. 1A), 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. 1B).
Expression of Human GST-IGnT6 in Sf9 Insect Cells-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.  2A). This de novo expressed 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. 2B).
The fusion protein GST-IGnT6 has five potential N-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.
Purification of Recombinant GST-IGnT6 -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 g/10 9 infected Sf9 cells present in 1 liter of the suspension culture.
Characterization of the GlcNAc Transferase Activity of the Recombinant GST-IGnT6 -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 GlcNAc␤1-3[ 14 C]-Gal␤1-4GlcNAc and UDP-GlcNAc. Neither a tetrasaccharidelike product nor any other product besides the starting trisaccharide was detected by paper chromatography of the neutral oligosaccharides of the incubation mixture (Fig. 5A). By contrast, similar experiments repeatedly converted significant amounts of the radiolabeled tetrasaccharide [ 3 H]Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc into a product that migrated like a pentasaccharide, suggesting the presence of cIGnT6 activity (data not shown, see below).
To characterize the pentasaccharide product, the radiolabeled glycan was first incubated with jack bean (exo)-␤-galactosidase, which released all tritium label in the form of free [ 3 H]Gal (Fig. 7A). This implies that the new GlcNAc of the pentasaccharide was not transferred to the distal, tritiumcontaining galactose residue of the tetrasaccharide acceptor, as this would not have been susceptible to (exo)-␤-galactosidase. Hence, the reaction had been different from the ␤1,6-GlcNAc transfer to the terminal galactose described in other laboratories (12, 27-30). Another enzymatic digestion was performed with endo-␤-galactosidase, which cleaves the internal ␤-galactosidic linkage of the tetrasaccharide acceptor (27) and other linear polylactosamines, but does not hydrolyze the branched Gal␤1-4GlcNAc␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc synthesized by the rat serum cIGnT6 (31). When the pentasaccharide was incubated with endo-␤-galactosidase, no breakdown product was formed (Fig. 7B). Collectively, these data showed that the pentasaccharide product of the recombinant GST-IGnT6 behaved in several ways like the authentic Gal␤1-4GlcNAc␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc. 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 ␣-linkage.
4GlcNAc␤1-3(GlcNAc1-6)Gal␤1-4GlcNAc reported in (32) (see Table II). The NMR data provide evidence for the presence of two ␤-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 Glc-NAc-5 unit of the authentic marker provides strong support for the notion that the new ␤GlcNAc 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. Bierhuizen et al. (19) 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 Gal␤1-4GlcNAc␤1-3(GlcNAc1-6)Gal␤1-4GlcNAc.
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.

DISCUSSION
The present experiments describe successful functional expression of a truncated form of a human ␤1,6-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 (19). 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 GlcNAc␤1-3Gal␤1-4GlcNAc␤1-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 (20). 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 (18,31) and with the membrane-bound form of cIGnT6 recently isolated from hog small intestine by Sakamoto et al. (46). 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 GlcNAc␤1-3Gal␤1-4GlcNAc␤1-OR, and (iii) the inability to transfer at midchain galactoses that belong to Lewis x determinants.
The present study demonstrates that cIGnT6 adds ␤1,6linked N-acetylglucosamine to a linear poly-N-acetyllactosamine as shown in Fig. 9A. 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 (33,34). Analysis of PA1 cells (7) demonstrated that their poly-Nacetyllactosamine 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.  9A). In contrast, dIGnT6 is expected to form branched poly-Nacetyllactosamine backbones in association of the chain growth, leading occasionally to the formation of branchedbranch arrays of N-acetyllactosamine units in the multiply branched polylactosamines (Fig. 9B). Such structures may be synthesized in gastrointestinal cells and Novikoff cells where dGnT6 activity has been detected (10,35). Indeed, hog gastric mucosa contains an octadecameric tetra-antennary lipid-bound polylactosamine (45) that resembles strikingly the branched branches array of seven N-acetyllactosamine residues that we have synthesized in vitro by using dIGnT6 in combination with GnT3 and GalT4 (39,41,42).
As shown previously, human granulocytes contain heavily fucosylated poly-N-acetyllactosamines such as R 1 -Gal␤1-4(Fuc␣1-3)GlcNAc␤1-R 2 , and these side chains do not contain any I branching (36,37). Our present study suggests that the lack of I branching in granulocytes poly-N-acetyllactosamines could be due to the inhibition of cGnT6 by ␣1,3-fucosyl residues. Alternatively, human granulocytes may not express IGnT6. On the other hand, the termini of I structures such as Fuc␣1-2Gal␤1-4GlcNAc(Fuc␣1-2Gal␤1-4GlcNAc␤1-6)Gal (33,34) 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 (38). 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 (39) and rejecting organ transplants (21, 40 -42).
In combination with the previously cloned enzymes GalT4 (43,44) and GnT3 (45), 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.
FIG. 9. The differences of acceptor and site specificities of the cIGnT6 and dIGnT6 enzymes. Open symbols represent galactose and closed symbols N-acetylglucosamine residues.