Synthesis of Poly- N -acetyllactosamine in Core 2 Branched O -Glycans THE REQUIREMENT OF NOVEL b -1,4-GALACTOSYLTRANSFERASE IV AND b -1,3- N -ACETYLGLUCOSAMINYLTRANSFERASE*

Poly- N -acetyllactosamine is a unique carbohydrate composed of N -acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Le x . Poly- N -acetyllactosamines in mucin-type O -glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 b -1,6- N -acetylglucosaminyltransferase.Usinga b -1,4-galactosyltransferase ( b 4Gal-TI) present in milk and the recently cloned b -1,3- N -acetylglucosami-nyltransferase, the formation of poly- N -acetyllac-tosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAc b 1 3 6-(Gal b 1 3 3)GalNAc a 3 R. Since the majority of synthesized oligosaccharides contained N -acetylglucosa-mine at the nonreducing ends, galactosylation was jud-ged to be inefficient, prompting us to test novel members of the b 4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant b 4Gal-Ts, b 4Gal-TIV the enzymatic activity of different b 4Gal-T samples, each enzyme preparation was first calibrated using 0.5 m M GlcNAc b 3 p - nitrophenol (Sigma) as an acceptor. This assay showed that the samples of b 4Gal-TI, -TII, -TIII, -TIV, and -TV had the activity of 1.90, 1.36, 1.90, 1.27, and 0.962 (all expressed in m mol/h/ml), respectively. The final concentration of each enzyme was adjusted to 38.0 nmol/h/ml in all experiments.

Mucin-type O-glycans are present in a wide variety of cells and play various roles in different cells. Mucin-type glycoproteins are also present in the plasma membrane, and they are often involved in cell-cell interaction (1). For example, O-glycans present in eggs were shown to be a receptor for both mouse and sea urchin (2,3). In granulocytes, monocytes, and certain T lymphocytes, mucin-type O-glycans can carry sialyl Le x , NeuNAc␣233Gal␤134(Fuc␣133)GlcNAc3 R, at their termini (4 -6). Sialyl Le x and its sulfated form are ligands for E-, P-, and L-selectin (7)(8)(9)(10)(11). Importantly, these selectins, in particular P-and L-selectin, preferentially bind to sialyl Le x in a limited number of mucin-type glycoproteins such as PSGL-1 (for P-selectin) and GlyCAM-1 and CD34 (for L-selectin) (12)(13)(14). As shown previously, sialyl Le x and its derivatives of O-glycans in blood cells can be only formed on core 2 branches, Gal␤134GlcNAc␤136(Gal␤133)GalNAc␣3 R (4,5). Recent studies demonstrate that sialyl Le x and sialyl Le a in core 2 branches are highly correlated to tumor invasion and vessel invasion of colon carcinomas (15), probably because tumor cells utilize selectin-carbohydrate interaction for their adhesion.
In patients with immunodeficiency such as Wiskott-Aldrich syndrome, AIDS, and leukemia, leukocytes in the peripheral blood express a substantial amount of core 2 branched oligosaccharides, while leukocytes of normal individuals do not express them (16 -19). Most recent studies employing transgenic mice demonstrated that such an overexpression of core 2 oligosaccharides weakens the interactions between T lymphocytes and antigen-presenting cells or B lymphocytes, resulting in reduced immune responses such as delayed type hypersensitivity and immunoglobulin isotype switching (20, 21). It has also been shown that AIDS patients produce antibodies against leukosialin expressing core 2 branched oligosaccharides, possibly causing T lymphocyte depletion in those patients (22,23). Moreover, the overexpression of a mucin-type glycoprotein carrying those oligosaccharides was shown to interfere with cell adhesion (24), while knockout of the leukosialin gene in mice resulted in hyperimmune responses (25). It has been also shown that poly-N-acetyllactosamine can be extended from core 2 branches, forming poly-N-acetyllactosaminyl O-glycans (4 -6, 26). Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine repeats (Gal␤134GlcNAc-␤133) n . Poly-N-acetyllactosamines are susceptible to endo-␤galactosidase and larger than typical N-glycans or O-glycans containing only one N-acetyllactosamine in a side chain. Poly-N-acetyllactosamines provide the backbone structure for additional modifications, which are often cell type-specific oligosaccharides, such as sialyl Le x (1). These results, as a whole, indicate that core 2 branched oligosaccharides play critical roles in cell-cell interaction.
When we tried to synthesize poly-N-acetyllactosamine on core 2 branched oligosaccharides, iGnT and milk ␤4Gal-T (␤4Gal-TI) failed to form poly-N-acetyllactosamines. Since the majority of the products contained N-acetylglucosamine at the nonreducing ends, inefficient galactosylation by ␤4Gal-TI was a likely cause for the lack of poly-N-acetyllactosamine synthesis in core 2 branched oligosaccharides. These unexpected results prompted us to test if any of the new members of ␤4Gal-Ts, which have been identified recently (29 -33), is responsible for poly-N-acetyllactosaminyl extension in core 2 branched oligosaccharides. In this report, we summarize these findings and demonstrate that ␤4Gal-TIV (33) is the enzyme involved in poly-N-acetyllactosamine extension of core 2 branched oligosaccharides. Moreover, we show that ␤4Gal-TIV is a rate-limiting factor and responsible for short poly-N-acetyllactosamine extension in core 2 branched O-glycans.

EXPERIMENTAL PROCEDURES
Isolation of cDNA Encoding iGnT and iGnTc-cDNA encoding the iGnT was isolated, as described previously (28). The cDNA insert in the cloned pAMo vector was digested with HindIII and XmnI and cloned into the HindIII and EcoRV sites of pcDNA3.1 (Invitrogen), resulting in pcDNA3.1-iGnT as described previously (28). pcDNAI-A, harboring cDNA encoding a signal peptide sequence, and the IgG binding domain of Staphylococcus aureus protein A, was constructed as described before (28). The catalytic domain of iGnT was cloned into this vector, resulting in pcDNAI-A⅐iGnTc (28).
Expression of the Protein A-iGnT Fusion Vector-pcDNAI-A⅐iGnTc and pcDNAI-A were separately transfected with Lipofectamine (Life Technologies, Inc.) into COS-1 cells as described previously (28), and 48 h after the transfection the medium was replaced with serum-free medium, Opti-MEM (Life Technologies) and cultured for an additional 24 h. The chimeric iGnT secreted into the Opti-MEM was adsorbed into IgG-Sepharose 6FF (Amersham Pharmacia Biotech), and the enzyme bound to the beads was used as an enzyme source (34). Alternatively, the culture medium was concentrated 100-fold (for iGnT alone) or 1000-fold (for poly-N-acetyllactosamine synthesis) by a Centricon 10 concentrator (Amicon) and directly used as an enzyme source. In most of the studies, the concentrated culture medium was used, since IgG-Sepharose-bound iGnT had a low activity as seen for other glycosyltransferases (35,36). For poly-N-acetyllactosamine synthesis, the activity of iGnT in the incubation mixtures was 380 nmol/h/ml using 0.5 mM Gal␤134Glc␤3pNP as an acceptor substrate. As described previously (28), the supernatant from mock-transfected COS-1 cells contained less than one-fifth of the activity compared with that derived from pcDNAI-A⅐iGnTc-transfected COS-1 cells.
Isolation and Expression of cDNAs Encoding ␤4Gal-TII, -TIII, -TIV, and -TV-Isolation of the cDNAs encoding ␤4Gal-TII, -TIII, and -TIV has been described previously (29,33). Based on the nucleotide sequences of these cDNAs, cDNAs encoding catalytic domains of ␤4Gal-TII and -TIII have been prepared using RT-PCR as described before (29). The catalytic domain of ␤4Gal-TIV was prepared by PCR using the expressed sequence tag sequence (expressed sequence tag 489768) as a template as described previously (33). These cDNAs were cloned into pAcGP67 (Pharmingen) and expressed in insect cells as described before (33). The supernatants from these transfected insect cells were used as an enzyme source.
␤4Gal-TV was cloned by PCR based on the published nucleotide sequence (31). The cDNA encoding a soluble form of ␤4Gal-TV was prepared by PCR using the obtained cDNA as a template. 5Ј-and 3Ј-primers for this PCR were 5Ј-CCGGATCCCCAAGGCATTCTGATC-CGGGAC-3Ј (BamHI site is underlined) and 5Ј-CCCTCGAGTCAG-TACTCGTTCACCTGAGCCAG-3Ј (XhoI site is underlined). The resultant cDNA encoding codon 49 to the stop codon was cloned into BamHI and XhoI sites of pcDNAI-A, resulting in pcDNAI-A⅐␤4Gal-TVc. pcDNAI-A⅐␤Gal-TVc was transiently expressed in COS-1 cells as described above for pcDNAI-A⅐iGnTc. The supernatant from the transfected COS-1 cells was then concentrated 100-fold as described above and used as an enzyme source.
The acceptors (Gal␤134GlcNAc␤133) n Gal␤134GlcNAc␤136 Man␣136Man␤13 O(CH 2 ) 7 CH 3 (octyl), where n ϭ 0, 1, and 2, were synthesized from octyl 2,3, (38). Thus, the acceptor compound 1 was glycosylated with the donor compound 2 (1.2 eq) in dichloromethane at Ϫ40°C with catalytic triethylsilyl trifluoromethanesulfonate to give the derivative of tetrasaccharide (compound 3). Selective removal of the chloroacetyl groups of compound 3 was effected with thiourea and 2,6-lutidine in ethanol, the product of which was then glycosylated with compound 2 as before to give the hexasaccharide (compound 4). Repetition of this cycle of chloroacetyl group removal, followed by glycosylation with compound 2, gave the octasaccharide (compound 5). Deprotection of compounds 3-5 was achieved using published conditions (38). The crude products, compounds 6, 7, and 8, were isolated on C18 reverse-phase silica cartridges and purified on LH-20 Sephadex. The products were characterized by 1 H NMR spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Compounds 6, 7, and 8 contain one, two, and three N-acetyllactosamines, corresponding to those where n ϭ 0, 1, and 2, respectively, in the above structure.
As acceptors, the following oligosaccharides were used: GlcNAc-␤136(Gal␤133)GalNAc␣3pNP for galactosylation of core 2 oligosaccharide and GlcNAc␤136Man␣136Man␤3octyl, GlcNAc␤133Gal-␤134GlcNAc␤136Man␣136Man␤3octyl, and GlcNAc␤133Gal-␤134GlcNAc␤133Gal␤134GlcNAc␤136Man␣136Man␤3octyl for galactosylation of an antennary extended from GlcNAc␤136Man␣136 branch, which is formed by N-acetylglucosaminyltransferase V. After incubation for 1 h at 37°C, the reaction mixture was applied to a Sep-Pak column, and the amount of the product was determined by measuring the radioactivity of the eluted product as described above. To compare the enzymatic activity of different ␤4Gal-T samples, each enzyme preparation was first calibrated using 0.5 mM GlcNAc␤3pnitrophenol (Sigma) as an acceptor. This assay showed that the samples of ␤4Gal-TI, -TII, -TIII, -TIV, and -TV had the activity of 1.90, 1.36, 1.90, 1.27, and 0.962 (all expressed in mol/h/ml), respectively. The final concentration of each enzyme was adjusted to 38.0 nmol/h/ml in all experiments.
After incubation for 10 h at 37°C, the product was purified by a Sep-Pak column as described above. The sample was then lyophilized and subjected to HPLC using a column (4 ϫ 300 mm) of NH 2 (27). As a source for C2GnTc, pcDNAI-A⅐C2GnTc (27) was transiently expressed in COS-1 cells, and the spent medium obtained was used. After incubation, unreactive UDP-[ 3 H]GlcNAc was removed by QAE-Sephadex gel as described (41). [ 3 H]GlcNAc␤136(Gal␤133)GalNAc was then isolated after applying the sample to Bio-Gel P-4 gel filtration as described (28). The synthesized [ 3 H]GlcNAc␤136(Gal␤133)GalNAc (0.5 mM) was incubated with ␤4Gal-TI or ␤4Gal-TIV and nonradioactive UDP-Gal under the same incubation mixture as described above, except that the final concentration of ␤4Gal-TI or ␤4Gal-TIV was 80.0 nmol/ h/ml as assayed using 0.5 mM GlcNAc as an acceptor. After the incubation, UDP-Gal was removed by QAE-Sephadex gel (41), and the products were analyzed by HPLC using the same conditions described above.
␤4Gal-TIV Is Responsible for Galactosylation of Core 2 Branches-Recently, it has been demonstrated from several laboratories that there exist at least five ␤4Gal-Ts in addition to ␤4Gal-TI (29 -33, 49). The above results suggest that one of these newly identified ␤4Gal-Ts might be involved in galactosylation of core 2 branches. To determine if any of these new members of the ␤4Gal-T family can form galactosylated core 2 branch, GlcNAc␤136(Gal␤133)GalNAc␣3pNP was incubated with each of these new members of the ␤4Gal-T family. As shown in Fig. 3A, ␤4Gal-TII, -TIII, and -TV exhibited substrate inhibition toward this substrate as did ␤4Gal-TI. Similar results were obtained when the concentration of these enzymes was increased 5-fold or decreased 5-fold. This substrate inhibition is most likely the reason why the amount of the final product was very low in ␤4Gal-TI-directed reaction (see also Fig. 1D). This substrate inhibition occurs probably because ␤-galactose in the acceptor competes with UDP-Gal.
In contrast, the core 2 branch was efficiently galactosylated by ␤4Gal-TIV with a K m of 0.29 mM (Fig. 3B; also see Table I). The identical K m was obtained when the concentration of ␤4Gal-TIV was decreased 5-fold as expected. These results indicate that ␤4Gal-TIV is most likely responsible for galactosylation of core 2 branched oligosaccharides. ␤4Gal-TVI was not included in the studies, since this enzyme is the least related to ␤4Gal-TI and shown to synthesize Gal␤134Glc␤3 ceramide from Glc␤3ceramide (49).
To address the question of whether the nature of the aglycon attached to the core 2 branched acceptor influences the galactosylation, [ 3 H]GlcNAc␤136(Gal␤133)GalNAc was enzymatically synthesized using recombinant soluble C2GnT (27) Fig. 4, C and D, clearly demonstrated that the reaction by ␤4Gal-TI was still incomplete, since the majority of the acceptor was not galactosylated (see the peak at fraction 33). In contrast, ␤4Gal-TIV completely galactosylated the acceptor. These results indicate that ␤4Gal-TIV is the enzyme responsible for galactosylation of core 2 branches and that such a synthesis is not influenced by the nature of aglycons.
It is noteworthy that the size of poly-N-acetyllactosamine in core 2 branched oligosaccharides shown in Fig. 5B was much smaller than that in N-glycan acceptor shown in Fig. 1A, when the same amount of the enzymes was used in both experiments. While four and possibly five N-acetyllactosamine units were added to Gal␤134GlcNAc␤136Man␣136Man␤3octyl (Fig.  1A), only two N-acetyllactosamine units were added as a maximum to Gal␤134GlcNAc␤136(Gal␤133)GalNAc␣3pNP (Fig. 5B).
These results are consistent with the facts that poly-Nacetyllactosamines on core 2 branched oligosaccharides are shorter than those in N-glycans and that the majority of poly-N-acetyllactosaminyl core 2 O-glycans contains only two Nacetyllactosamine repeats in many cells (4 -6, 48).
␤4Gal-TIV Is Responsible for Short Poly-N-acetyllactosamines in Core 2 Branched O-Glycans-The above results suggest that ␤4Gal-TI and ␤4Gal-TIV may differ in the efficiency for adding a galactose to acceptors containing poly-Nacetyllactosamines. We were particularly interested in deter- GlcNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-6Man␣1-6Man␤-octyl 1.08 103 4.98 5 a These parameters could not be obtained due to substrate inhibition. b V max for ␤4Gal-TI and ␤4Gal-TIV is compared with the V max (98.6 pmol/min) obtained for ␤4Gal-TI using GlcNAc␤1-6Man␣1-6Man␤-octyl as an acceptor. mining if the inefficiency in galactosylation by ␤4Gal-TIV can be universally observed in various acceptors containing poly-N-acetyllactosamines. To determine whether this is the case, (GlcNAc␤133Gal␤134) n GlcNAc␤136Man␣136Man␤3octyl, where n ϭ 0, 1, or 2, were used as acceptors for ␤4Gal-TI or ␤4Gal-TIV. Fig. 6B demonstrates that ␤4Gal-TIV substantially decreased the efficiency of the enzymatic reaction as the acceptor became longer (Table I). Such a dramatic decrease was not, however, observed for galactosylation by ␤4Gal-TI ( Fig. 6A and Table I). These results, combined together, indicate that the intrinsic nature of ␤4Gal-TIV is a likely cause for shorter poly-N-acetyllactosamines in core 2 branched oligosaccharides. DISCUSSION In the present study, we found that ␤4Gal-TI, abundantly present in milk, cannot efficiently add a galactose residue to a core 2 branched oligosaccharide, GlcNAc␤136(Gal␤133)Ga-lNAc ( Figs. 1 and 2). This unexpected finding led us to discover that a novel member of the ␤4Gal-T family, ␤4Gal-TIV, is responsible for galactosylation of core 2 branched oligosaccharides and, together with iGnT, can form poly-N-acetyllactosaminyl core 2 branched O-glycans (Figs. [3][4][5]. After cloning ␤4Gal-TI from bovine and human milk (50 -52), ␤4Gal-TI has been the only ␤4Gal-T recognized until recently. The presence of additional members of ␤4Gal-TI was, however, suggested from the knockout of the ␤4Gal-TI gene in mouse, since the mice deficient in ␤Gal-TI survived during embryonic development (53). More recent accumulation in the expressed sequence tag data base, PCR homology cloning, and purification and cloning of lactosylceramide synthase allowed several laboratories to isolate novel members of the ␤4Gal-T family, ␤4Gal-TII to ␤4Gal-TVI (29,(31)(32)(33)49). There appear to be overlapping but different acceptor specificities in ␤4Gal-TI, -TII, and -TIII, and all of them are capable of adding a galactose residue to form N-acetyllactosamine in both glycoproteins and glycolipids. Except for ␤4Gal-TVI, which is mainly responsible for lactosylceramide synthesis from glucosylceramide, however, the roles of the novel ␤4Gal-Ts have been elusive. For example, ␤4Gal-TV, which is remotely related to ␤4Gal-TI, was reported to be inactive toward a glycolipid or glycoprotein acceptor (31).
To our knowledge, the present study is the first report among novel members of ␤4Gal-Ts that a particular ␤4Gal-T, ␤4Gal-TIV, is exclusively responsible for forming specific structures. ␤4Gal-TIV is the most recently added member of the ␤4Gal-T gene family (33). In the present study, we demonstrated that ␤4Gal-TIV is very efficient in adding galactose to the core 2 branched oligosaccharide but inefficient in adding to N-glycanrelated acceptors such as GlcNAc␤136Man␣136Man␤3octyl. This finding is consistent with the previous report that ␤4Gal-TIV adds very little galactose to asialoagalactotransferrin, which contains only N-glycans (33). Our results are also consistent with the report that ␤4Gal-TIV acts inefficiently on asialoagalactofetuin, since O-glycans in fetuin lack core 2 branches (54). It was shown in the previous report that ␤4Gal-TIV can act with reasonable efficiency on glycolipid acceptors (33). We were thus concerned about whether the hydrophobic nature of the aglycon, p-nitrophenol in acceptors used, might affect the enzymatic reactions. We thus used a core 2 branched acceptor without an aglycon and found that ␤4Gal-TIV acts on this acceptor much better than ␤4Gal-TI, (Fig. 4, C and D), demonstrating that ␤4Gal-TIV efficiently acts on core 2 branched oligosaccharide regardless of whether or not a hydrophobic aglycon is attached. These results, taken together, support our conclusion that ␤4Gal-TIV and iGnT cooperatively form poly-N-acetyllactosamines on core 2 branched oligosaccharides initiated by C2GnT.
The present study also demonstrated that poly-N-acetyllactosamines formed in core 2 branched oligosaccharides were shorter than those formed in N-glycan acceptors when both were synthesized under the same conditions (see Fig. 1A and Fig. 5B). This finding is consistent with the fact that core 2 branched O-glycans are mostly composed of those containing one or two N-acetyllactosamine units, while poly-N-acetyllactosaminyl N-glycans contain three or more N-acetyllactosamine units (4 -6, 42-46). Moreover, the amount of poly-Nacetyllactosaminylated O-glycans is much less than that in N-glycans when N-glycans and O-glycans were analyzed in the same lamp molecules (55) or the same CHO cells (46,48,55). The present study also demonstrated that ␤4Gal-TIV, but not ␤4Gal-TI, drastically reduces its efficiency as acceptors become longer (Fig. 6, Table I). ␤4Gal-TIV was also shown to act less efficiently on longer lacto-series glycolipids than shorter ones (33). These results, combined together, indicate that ␤4Gal-TIV acts less efficiently on those containing N-acetyllactosamine repeats, synthesizing shorter poly-N-acetyllactosamines in core 2 branched oligosaccharides than poly-N-acetyllactosamines in N-glycans synthesized by ␤4Gal-TI.
The formation of core 2 branched oligosaccharides has been found to be critical in many biological processes, as described in the Introduction. Further studies for understanding the biosynthesis of core 2 branches and its N-acetyllactosamine extension are expected to provide important insights into the physiological roles of core 2 branched oligosaccharides.