J Biol Chem, Vol. 274, Issue 24, 16717-16726, June 11, 1999

Poly-N-acetyllactosamine Synthesis in Branched N-Glycans Is Controlled by Complemental Branch Specificity of i-Extension Enzyme and 1,4-Galactosyltransferase I^*

Minoru Ujita, Joseph McAuliffe, Ole Hindsgaul, Katsutoshi Sasaki^§, Michiko N. Fukuda, and Minoru Fukuda^¶

From The Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and ^§ Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Machida, Tokyo 194, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Gal14GlcNAc16(Gal14GlcNAc12)Man16 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and 1,4galactosyltransferase I (4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Gal14GlcNAc12ManR side chain than in Gal14GlcNAc16ManR, due to preferential action of iGnT on Gal14GlcNAc12ManR side chain. On the other hand, galactosylation was much more efficient on 1,6-linked GlcNAc than 1,2-linked GlcNAc, preferentially forming Gal14GlcNAc16(GlcNAc12)Man16Man R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Gal14GlcNAc16ManR and Gal14GlcNAc12ManR side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and 4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAc16(GlcNAc12)Man16ManR structure, which is consistent with the structures found in nature. The results also suggest that the addition of Gal14GlcNAc16 side chain on Gal14GlcNAc12ManR side chain converts the acceptor to one that is much more favorable for iGnT and 4Gal-TI.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Poly-N-acetyllactosamines are unique glycans having N-acetyllactosamine repeats (Gal14GlcNAc13)_n and can be digested by endo--galactosidase (1, 2). Poly-N-acetyllactosamines are often modified to express differentiation antigens and functional oligosaccharides. Poly-N-acetyllactosamines in human erythrocytes contain ABO blood group antigens, synthesized from a precursor structure, Fuc12Gal14GlcNAcR (3-5). In human granulocytes, monocytes, and certain T lymphocytes, on the other hand, poly-N-acetyllactosamines contain Le^x,¹ Gal14 (Fuc13)GlcNAcR, and sialyl Le^x, NeuNAc23Gal14 (Fuc13)GlcNAcR (6-9). Sialyl Le^x and its sulfated forms are ligands for E-, P- and L-selectin (10-12). During inflammation, E- and P-selectin expressed on activated endothelial cells bind to sialyl Le^x oligosaccharides present on granulocytes, and such initial binding leads to the extravasation of granulocytes (10-12). L-selectin on lymphocytes, on the other hand, recognizes sulfated sialyl Le^x expressed in high endothelial venules of blood vessels (13-15). This L-selectin-counter-receptor interaction enables lymphocytes to migrate into lymphoid system, allowing lymphocytes to circulate fully in the body.

Poly-N-acetyllactosamines are attached to N-glycans (3-7, 16-18), O-glycans (8, 9, 19), and glycolipids (20-22). Poly-N-acetyllactosamines are synthesized through alternate actions of 1,3-N-acetylglucosaminyltransferase, i-extension enzyme (iGnT), and 1,4-galactosyltransferase (4Gal-T). In N-glycans, poly-N-acetyllactosamines are found more often in tetraantennary and triantennary N-glycans that contain a side chain linked to 1,6-linked mannose through a GlcNAc16 linkage in GlcNAc16(GlcNAc12)Man16ManR (23-27). This side chain is formed by N-acetylglucosaminyltransferase V (GnTV). Significantly, the amount of GnTV is increased in various tumors including colonic carcinoma cells and those transformed with oncogenes (23-27). Overexpression of GnTV in cultured cells was reported to result in acquiring the capability of growth in soft agar and tumor formation after subcutaneous injection of the transfected cells (28).

The increased expression of sialyl Le^x apparently takes place in those tumor cells when 1,3-fucosyltransferase is also present. In fact, highly metastatic colonic carcinoma cells express more sialyl Le^x in poly-N-acetyllactosamines than poorly metastatic counterparts (27). Moreover, our recent studies demonstrated that B16 mouse melanoma cells produced many more lung tumor foci after the cells were transfected with 1,3-fucosyltransferase to form sialyl Le^x in poly-N-acetyllactosamines (29). These results, as a whole, indicate that the formation of poly-N-acetyllactosamine plays a critical role for carbohydrate recognition in cell-cell interaction.

Previously, we have demonstrated that poly-N-acetyllactosamines in core 2 branched O-glycans are synthesized through iGnT and a newly discovered member of 1,4-galactosyltransferase, 4Gal-TIV (30). We have also found that 4Gal-TI, iGnT, and I-branching enzyme are involved in the synthesis of I-branched poly-N-acetyllactosamines (31). In the same study, we found that the addition of N-acetyllactosamine repeats to linear poly-N-acetyllactosamines is preferred over the extension of N-acetyllactosamine to I-branches, mainly because the first galactosylation of a GlcNAc16 branch is inefficient. These studies thus demonstrate intricate interaction between a specific glycosyltransferase and an acceptor molecule, which largely contributes to the control of poly-N-acetyllactosamine synthesis. In these studies, we utilized molecular tools that have recently become available, such as cDNAs encoding i-extension enzyme (32), novel members of 4Gal-T (33), and I-branching enzyme (34).

These results prompted us in the present study to determine how poly-N-acetyllactosamine is formed in branched N-glycans. Our results demonstrated that the addition of GlcNAc16ManR side chain renders the resultant GlcNAc16(GlcNAc12)Man16ManR an extremely efficient acceptor for 4Gal-TI and i-extension enzyme. Moreover, we found that the i-extension enzyme prefers Gal14GlcNAc12ManR side chain over Gal14 GlcNAc16ManR side chain, whereas the opposite is true for 4Gal-TI. Such complementary effects apparently result in forming similar size and abundance of poly-N-acetyllactosamines in both chains of GlcNAc16 (GlcNAc12)ManR structures.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Expression of cDNA Encoding iGnT-- cDNA encoding iGnT was cloned into pcDNA3.1, resulting in pcDNA3.1-iGnT as described previously (32). pcDNAI-A, harboring cDNA encoding a signal sequence and an IgG binding domain of Staphylococcus aureus protein A, was constructed as described before (35). The catalytic domain of iGnT was cloned into this vector, resulting in pcDNAI-A·iGnT (32).

pcDNAI-A and pcDNAI-A·iGnT were separately transfected with LipofectAMINE Plus (Life Technologies, Inc.) into COS-1 cells as described previously (36). The chimeric enzyme released into serum-free OPTI-MEM was used after adsorbing the protein A chimeric enzyme to IgG-Sepharose 6FF (Amersham Pharmacia Biotech) as described previously (36). Alternatively, the culture medium was concentrated 100-fold or 1000-fold by a Centricon 10 concentrator (Amicon) and directly used as an enzyme source. In most of the studies, the concentrated culture medium was used for iGnT because IgG-Sepharose bound enzymes had a low activity, as seen for other glycosyltransferases (37, 38). Typically, the activities of iGnT in the incubation mixture was 38.0 nmol/h/ml (for addition of one GlcNAc) or 380 nmol/h/ml (for poly-N-acetyllactosamine synthesis), using 0.5 mM Gal14Glcp-nitrophenol (Toronto Research Chemicals) as an acceptor. The medium from mock-transfected COS-1 cells contained less than one-fifth of iGnT activity compared with that derived from pcDNAI-A·iGnT-transfected COS-1 cells, as described (32).

Expression of cDNAs Encoding 4Gal-TII, -TIII, -TIV, and -TV-- 4Gal-TII, -TIII, and -TIV were expressed in insect cells, and the supernatants from these transfected insect cells were used as an enzyme source as described previously (30, 33). Human milk 4Gal-T preparation (Sigma) was directly used as 4Gal-TI (30). 4Gal-TV (39) was cloned and expressed in COS-1 cells as described previously (30). For comparing the enzymatic activities of different 4Gal-T samples, the final concentration of 4Gal-TI, -TII, -TIII, -TIV, and -TV was adjusted to 38.0 nmol/h/ml as measured using 0.5 mM GlcNAcp-nitrophenol (Sigma) as an acceptor.

Synthesis of the Acceptors-- The branched pentasaccharides GlcNAc16(Gal14GlcNAc12)Man16Man1O(CH₂)₇CH₃(octyl) (compound 1) and Gal14GlcNAc16(GlcNAc12)Man16Man1octyl (compound 2) were synthesized from octyl 3, 4-di-O-benzyl--D-mannopyranosyl(16)-2,3,4-tri-O-benzyl--Dmannopyranoside (compound 3) and the donors 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido--D-glucopyranosyl chloride (compound 4), the corresponding trichloroacetimidate (compound 5), or 2,3,4,6-tetra-Oacetyl--D-galactopyranosyl(14)-3,6-di-O-benzyl-2-deoxy-2-phthalimido--D-glucopyranosyl trichloroacetimidate (compound 6). All glycosylations were performed under nitrogen in the presence of 4 Å molecular sieves and monitored by TLC. Thus, the acceptor compound 3 was glycosylated with the donor 4 (1.5 eq) in dichloromethane at 20 °C with a mixture of silver trifluoromethanesulfonate and silver carbonate (1:3) to give a trisaccharide (compound 7). Compound 7 was then glycosylated with the donor compound 6 (1.2 eq) in dichloromethane at 40 °C with catalytic triethyl trifluoromethanesulfonate to give a pentasaccharide (compound 8). Conversely, the 2'-O-p-methoxybenzyl derivative of the acceptor compound 3 was glycosylated with the donor compound 6 (1.3 eq) in dichloromethane at 40 °C with catalytic triethyl trifluoromethanesulfonate to give a tetrasaccharide (compound 9). Compound 9 was then treated with 80% acetic acid at 50 °C to remove the 2'-O-p-methoxybenzyl group and subsequently glycosylated with the donor compound 5 (2 eq) in dichloromethane at 10 °C with catalytic triethyl trifluoromethanesulfonate to give a pentasaccharide (compound 10). Deprotection of compounds 8 and 10 was performed using published conditions as detailed previously (40). The crude products, compounds 1 and 2 respectively, were isolated on C₁₈ reverse-phase Sep-Pak cartridges as described previously (30) (Waters Associates) and purified on LH-20 Sephadex eluted with water. The products were characterized by ¹H NMR spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The labeled hexasaccharides [³H]Gal14GlcNAc16 (Gal1 4GlcNAc12)Man16Man1octyl (compound 11) and Gal14GlcNAc16([³H]Gal14GlcNAc12)Man16Man1octyl (compound 12) were synthesized from compounds 1 and 2, respectively, through treatment with 50 milliunits of bovine milk 4Gal -TI (Sigma) and UDP-[³H]Gal (10 µCi/µmol acceptor) in 50 mM HEPES buffer (pH 7.4) containing 10 mM MnCl₂ at 37 °C. Thus, UDP-[³H]Gal (10 µCi/µmol acceptor) was added to the reaction medium containing either compound 1 or 2 (1 mM), along with UDP-galactose (0.5 mM). After 12 h, additional UDP-galactose (1.5 mM) was added, and incubation was continued for another 12 h. TLC of the products indicated absence of the starting material. Compounds 11 and 12 were isolated as described above and determined to have specific activities of ~8.5 Ci/mol.

Compound 1 (3 mM) was treated with jack bean -N-acetylglucosaminidase (4 units) in 50 mM sodium acetate buffer, pH 5.5, at 37 °C for 3 days, resulting in the tetrasaccharide Gal14GlcNAc1 2Man16Man1octyl (compound 13). Similarly, compound 1 (5 mM) was treated with Escherichia coli -galactosidase (20 units) in 50 mM Tris-HCl buffer, pH 7.4, at 37 °C overnight, resulting in the tetrasaccharide GlcNAc16(GlcNAc12)Man16Man1octyl (compound 14). Isolation, purification, and characterization of these compounds was performed as described above. Detailed procedures of these syntheses will be published elsewhere.²

Partial ¹H NMR results (300 MHz, D₂O) (compounds 1, 2, 13, and 14) are as follows. Compound 1:  4.90 (s, H-1'), 4.68 (s, H-1), 4.63 (d, J = 7.8 Hz, 1H), 4.56 (d, J = 7.7 Hz, 1H), 4.48 (d, J = 8.3 Hz, 1H), 2.09, 2.05 (2 s, 6H, NHAc). Compound 2:  4.88 (s, H-1'), 4.66 (s, H-1), 4.60-4.55 (m, 2H), 4.47 (d, J = 8.3 Hz, 1H), 2.06, 2.03 (2 s, 6H, NHAc). Compound 13:  4.94 (s, H-1'), 4.67 (s, H-1), 4.63-4.60 (m, 1H), 4.48 (d, J = 7.6 Hz, 1H), 2.06 (s, 3H, NHAc). Compound 14:  4.87 (s, H-1'), 4.66 (s, H-1), 4.58 (d, J = 8.1 Hz, 1H), 4.54 (d, J = 8.1 Hz, 1H) 2.05, 2.03 (2 s, 6H, NHAc).

The Addition of N-Acetylglucosamine by iGnT-- To assay the transfer of N-acetylglucosamine residues by the iGnT, the reaction mixture was exactly the same as described previously (30, 32). The incubation mixture was applied to a C₁₈-reverse phase Sep-Pak cartridge column (Waters), and the product was eluted as described previously (30). The product was then analyzed by HPLC using NH₂-bonded silica column (Varian Micropak AX-5) as described previously (30). The radioactivity of aliquots in the effluent was determined. The iGnT was also incubated with [³H]Gal14GlcNAc16(Gal14GlcNAc12)Man16Man1octyl or Gal14GlcNAc16([³H]Gal14GlcNAc12)Man16Man1octyl. In these experiments, nonradioactive 5 mM UDP-GlcNAc was used. In all of the above reactions, the reaction mixture was incubated for 10 h to analyze the products or for 1 h to obtain kinetic parameters.

Poly-N-acetyllactosamine Synthesis by iGnT and 4Gal-TI-- To assay poly-N-acetyllactosamine formation, 0.5 mM acceptor was incubated with 4Gal-TI (760.0 nmol/h/ml) and iGnT (380.0 nmol/h/ml) under the conditions described previously (30, 32).

When [³H]Gal14GlcNAc16(Gal14GlcNAc12)Man1 6Man1octyl (compound 11, see above) and Gal14GlcNAc16([³H]Gal14GlcNAc12)Man16Man1octyl (compound 12, see above) were used as acceptors, nonradioactive donor substrates were used.

The products were purified by HPLC using the same NH₂-bonded silica column as described above. A peak containing one N-acetyllactosamine repeat was digested with diplococcal -galactosidase (41), and the digest was purified by a Sep-Pak column and then analyzed by HPLC as described above. A peak containing two N-acetyllactosamine repeats was sequentially digested with diplococcal -galactosidase, jack bean -N-acetylglucosaminidase, and diplococcal -galactosidase. After each digestion, the digest was analyzed by HPLC using the same NH₂-bonded column as described above. Aliquots were taken for determining radioactivity, obtaining the ratio of the radioactivity in the product and starting material.

As a third experiment, 0.5 mM Gal14GlcNAc16(GlcNAc12)Man16Man1octyl or 0.5 mM GlcNAc16(Gal14GlcNAc12)Man16Man1octyl was incubated with 4Gal-TI (152.0 nmol/h/ml), iGnT (38.0 or 76.0 nmol/h/ml), 0.5 mM UDP-[³H]GlcNAc, and 0.5 mM UDP-[³H]Gal in 50 µl of 100 mM cacodylate buffer, pH 7.0, containing 20 mM MnCl₂ and 10 mM each of GlcNAc-1,5-lactone and Gal-1,5-lactone. After incubation at 37 °C for 4 h, the reaction products were purified by a Sep-Pak column and subjected to HPLC as described above. In these experiments, the incubation conditions were first determined where only one N-acetyllactosamine unit can be added to Gal14GlcNAc13Gal14Glc. In addition, 4Gal-TI was 4-fold in excess over iGnT, the same ratio as in HL-60 cells (42). In certain experiments, 4Gal-TI was 2-fold in excess over iGnT.

Analysis of Products by Endo--galactosidase Digestion-- Products were digested with Escherichia freundii endo--galactosidase for 18 h at 37 °C (43). The digestion conditions used allowed the cleavage of galactose linkage, where no 1,6-linked N-acetylglucosamine is attached (2, 43). The digests were subjected to HPLC using AX-5 column.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Addition of N-Acetylglucosamine to N-Glycan Acceptor by i-Extension Enzyme-- To determine whether iGnT has a preference for the addition of N-acetylglucosamine to one of N-glycan acceptors, Gal14GlcNAc16ManR, Gal1 4GlcNAc12ManR, and Gal14GlcNAc16 (Gal14GlcNAc12)ManR were incubated with iGnT. First, iGnT acted in almost identical efficiency on Gal1 4GlcNAc16ManR and Gal14GlcNAc1 2ManR (Figs. 1A and 2, A and B), indicating that the enzyme does not prefer one acceptor over the other. Second, the branched acceptor has a better affinity and higher V_max for addition of one N-acetylglucosamine (Fig. 1A and Table I). When the products from the 10-h incubation of the branched acceptor was analyzed, the majority of the products contained only one N-acetylglucosamine residue, and a mere 5.7% (in molar ratio) of the whole products contained two N-acetylglucosamine residues (Fig. 2C).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Dependence of iGnT and 4Gal-Ts on the concentration of linear and branched N-glycan acceptors. A, Gal14GlcNAc16(Gal14GlcNAc12)Man16Man1octyl (), Gal 14GlcNAc12Man16Man1octyl (), or Gal14GlcNAc16Man16Man1octyl () of various concentrations was incubated with iGnT for 1 h. B, GlcNAc12Man16Man1octyl of various concentrations was incubated with 4Gal-TI (), -TII (), -TIII (), -TIV (), and -TV () for 1 h. The same amount of the enzyme, 38.0 nmol/h/ml, determined using 0.5 mM Gal14Glc1p-nitrophenol (A) or 0.5 mM GlcNAc1p-nitrophenol (B), was present in those experiments.

View this table: [in this window] [in a new window]   Table I Kinetic properties of iGnT Arrows indicate where 1,3-linked N-acetylglucosamine is added.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Analysis of the products after 10 h-incubation of N-glycan acceptors with iGnT. A-C, HPLC analysis of the iGnT products derived from Gal14GlcNAc16Man1R (A), Gal1 4GlcNAc12Man1R (B), and Gal14GlcNAc16(Gal14GlcNAc12)ManR (C). Numbers indicate the relative ratio of incorporated radioactivity. Peaks at Fraction 34 and 37 in C correspond to the products containing one and two GlcNAc residues, respectively. D, HPLC analysis of the iGnT product from [3H]Gal14GlcNAc16(Gal14GlcNAc12)ManR. Peaks at Fractions 31 and 35 correspond to the starting material and the product containing one GlcNAc residue, respectively. E, HPLC analysis of the product substituted with one N-acetylglucosamine, shown in D (Fraction 35) after endo--galactosidase digestion (solid line). F, HPLC analysis after endo--galactosidase digestion of the product substituted with one N-acetylglucosamine (solid line), which was derived from Gal14GlcNAc16([3H]Gal14GlcNAc12)Man1R. In E and F, the mono-N-acetylglucosaminylated products are shown as dotted lines. Numbers indicate the relative ratio of these oligosaccharides.

To determine which side chain is substituted with N-acetylglucosamine, [³H]Gal14GlcNAc16(Gal14GlcNAc12)ManR was first synthesized with 4Gal-TI and UDP-[³H]Gal from GlcNAc16(Gal14GlcNAc12)ManR. The radioactively labeled acceptor was then incubated with iGnT and unlabeled UDP-GlcNAc. As shown in Fig. 2D, the product containing one GlcNAc addition (Fraction 35) was obtained together with the starting material (Fraction 31). The product substituted with one N-acetylglucosamine was digested with endo--galactosidase, and the digest was subjected to the same HPLC. Seventy-five % of the radioactivity was recovered as [³H]Gal14GlcNAc16(GlcNAc12) ManR (Fig. 2E), indicating that 75% of the products was [³H] Gal14GlcNAc16(GlcNAc13Gal14GlcNAc12) ManR, whereas 25% was GlcNAc13[³H]Gal14GlcNAc16(Gal14GlcNAc12)ManR (see Fig. 3A).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Structures of the radioactively labeled acceptors and the iGnT products. Galactose was asymmetrically labeled in two different positions (A and B), and the resultant product was used as an acceptor. After endo--galactosidase digestion, those side chains elongated by iGnT lost radioactivity. Numbers indicate the molar ratio calculated from the results obtained in Fig. 2, E and F.

To corroborate the above results, Gal14GlcNAc16([³H]Gal14GlcNAc12)ManR was synthesized from Gal14GlcNAc16(GlcNAc12)ManR, and the resultant acceptor was incubated with iGnT and UDP-GlcNAc. The monosubstituted product obtained was then digested with endo--galactosidase, producing GlcNAc16([³H]Gal14GlcNAc12)ManR with 26% yield (Fig. 2F). The results thus indicate that 26% of the products was GlcNAc13Gal14GlcNAc16([³H]Gal14GlcNAc12)Man R, whereas 74% was Gal14GlcNAc16(GlcNAc14[³H]Gal14GlcNAc12)ManR (Fig. 2F, see also Fig. 3B).

These results, taken together, indicate that iGnT preferentially acts on Gal14GlcNAc12ManR side chain over Gal14GlcNAc16ManR in an approximate ratio of 3:1 under the incubation conditions used.

Poly-N-acetyllactosamine Synthesis on Unbranched or Branched Acceptor-- As shown previously (30), among different members of 4Gal-Ts 4Gal-TI was most efficient in adding galactose to GlcNAc16Man16Man1octyl. Similarly, we found in the present study that 4Gal-TI is the most efficient in adding a galactose to GlcNAc12Man16 Man1octyl (Fig. 1B; see also Table II).

View this table: [in this window] [in a new window]   Table II Kinetic properties of 4Gal-Ts Arrows indicate where galactose is added.

To determine how N-acetyllactosamine repeats are added to two different side chains, Gal14GlcNAc16ManR or Gal14GlcNAc12ManR was incubated with iGnT, 4Gal-TI, UDP-[³H]Gal and UDP-[³H]GlcNAc. Fig. 4, A and B, illustrates that poly-N-acetyllactosamines were almost equally formed from these two acceptors. We then incubated a branched acceptor, Gal14GlcNAc16(Gal14GlcNAc12)Man16Man1octyl with i-GnT, 4Gal-TI, and radioactive donor substrates. First, the branched acceptor incorporated 8.4 times more [³H]Gal and [³H]GlcNAc than the unbranched acceptors when incubated under the same conditions (Fig. 4C). The results also demonstrated that the branched acceptor contained more N-acetyllactosamine units, and the product containing three N-acetyllactosamine units constituted the major product (Fig. 4C). In contrast, the major product obtained from the unbranched acceptors contained one N-acetyllactosamine unit (Fig. 4, A and B). These results indicate that the addition of Gal14GlcNAc16 branch to Gal14GlcNAc12ManR in the acceptor has a synergistic effect on the branched acceptor, forming many more N-acetyllactosamine repeats than a summation of N-acetyllactosamine repeats formed when each branch was individually assayed. Moreover, such an effect extends to the formation of longer poly-N-acetyllactosamines.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   HPLC analysis of the products after incubation of N-glycan acceptors with iGnT and 4Gal-TI. Gal14GlcNAc16ManR (A), Gal14GlcNAc12ManR (B), Gal14 GlcNAc16(Gal14GlcNAc12)ManR (C), or [3H]Gal 14GlcNAc16(Gal14GlcNAc12)ManR (D) was incubated with iGnT, Gal-TI, and radioactive (A-C) or nonradioactive (D) donor substrates, and the products were separated by HPLC. In each chromatography, peaks represent the products containing one (1), two (2), three, and four N-acetyllactosamine repeats, which are depicted in D. In A-C, numbers indicate the relative ratio of the incorporated radioactivity.

Poly-N-acetyllactosamine Is Preferentially Added to Gal14GlcNAc12 Side Chain on Gal14GlcNAc12(Gal14GlcNAc16)ManR Acceptor-- To determine how each side chain of the branched acceptor was elongated with N-acetyllactosamine repeats, [³H]Gal14GlcNAc16(Gal14GlcNAc12)ManR, synthesized as described above, was incubated with iGnT, 4Gal-TI, and nonradioactive donor substrates. The products obtained exhibited an elution profile similar to that obtained in the above experiments (Fig. 4D). The difference between Fig. 4C and Fig. 4D was due to the fact that larger products were more visible in Fig. 4C because those products contained a greater amount of [³H]GlcNAc and [³H]Gal than smaller products.

The product containing one N-acetyllactosamine repeat (Fig. 4D, peak 1) was digested with exo--galactosidase. As shown in Fig. 5A, 28.6% of the starting product was recovered as GlcNAc13[³H]Gal14GlcNAc16(GlcNAc12)-ManR. The results indicate that 28.6% of the products was Gal14GlcNAc13[³H]Gal14GlcNAc16(Gal1 4GlcNAc12)ManR, whereas 71.4% was [³H]Gal-14GlcNAc16(Gal14GlcNAc13Gal14GlcNAc12)ManR, which produced a nonradioactive compound after -galactosidase treatments.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Analysis of the poly-N-acetyllactosaminyl products derived from asymmetrically labeled acceptors. A, -galactosidase digest of peak 1 in Fig. 4D. B-D, sequential digestion of peak 2 in Fig. 4D by -galactosidase (B), -N-acetylhexosaminidase (C), and -galactosidase (D). E-H, the products derived from Gal14GlcNAc16([3H]Gal14GlcNAc12)ManR were separated by HPLC and those containing one N-acetyllactosamine repeat (E) and two Nacetyllactosamine repeats (F-H) were digested with -galactosidase (E) or sequentially digested with -galactosidase (F), followed by -N-acetylhexosaminidase (G) and -galactosidase (H), and subjected to HPLC. Numbers indicate the relative ratio of the radioactivity of the digested product (solid line) and the starting material (dotted line) (see Fig. 6).

Similarly, the products containing two N-acetyllactosamine repeats (Fig. 4D, peak 2) were sequentially digested with -galactosidase, -N-acetylglucosaminidase, and -galactosidase. After first -galactosidase digestion, 35.2% of the products was recovered as radioactive oligosaccharides, indicating that 64.8% of the starting products was [³H]Gal14GlcNAc16[(Gal14GlcNAc13)₂GlcNAc12]ManR (Fig. 5B, see also Fig. 6B, middle). The additional digestion of the above -galactosidase digest with -N-acetylglucosaminidase resulted in no release of [³H]galactose as expected (Fig. 5C). The above digested products were finally digested again with -galactosidase, which recovered 74.1% of the radioactivity (Fig. 5D). This result indicates that 26.1% (= 35.2 × 0.741) of the starting material was (Gal14GlcNAc13)₂[³H]Gal 14GlcNAc16[(Gal14GlcNAc12)]ManR (Fig. 6B, left). The rest (9.1% = 35.2 × 0.259) was Gal14GlcNAc 13[³H]Gal14GlcNAc16(Gal14GlcNAc13Gal 14GlcNAc12)ManR, which contains one N-acetyllactosamine extension in both branches (Fig. 6B, right). These results indicate that poly-N-acetyllactosamine is preferentially formed on Gal14GlcNAc12Man side chain over Gal14GlcNAc16Man side chain under these conditions.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Schematic representation of the poly-N-acetyllactosaminyl products derived from asymmetrically labeled acceptors. A and B, the products containing one (A) and two (B) N-acetyllactosamine repeats derived from [3H]Gal14GlcNAc16(Gal14GlcNAc12) Man16Man1octyl and their sequential enzymatic digestion products are shown. C and D, the products derived from Gal14GlcNAc16([3H]Gal14GlcNAc12)Man16Man1octyl were analyzed after sequential exoglycosidase digestions. Numbers in parentheses denote the relative molar ratio of the products formed. The results shown in Fig. 5, A-D, correspond to Fig. 6, A and B, whereas the results shown in Fig. 5, E-H, correspond to Fig. 6, C and D. Man (), GlcNAc (), galactose (), and radioactive galactose () are denoted.

To corroborate the above conclusions, Gal14GlcNAc16([³H]Gal14GlcNAc12)ManR, synthesized as described above, was incubated under the same conditions. The results shown in Fig. 5, E-H, are mirror images of those described in Fig. 5, A-D. The results thus indicate that the products with two N-acetyllactosamine repeats were a mixture of (Gal 14GlcNAc13)₂Gal14GlcNAc16([³H]Gal1