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

Minoru UjitaDagger , 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6 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 beta 1,4galactosyltransferase I (beta 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain than in Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR, due to preferential action of iGnT on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain. On the other hand, galactosylation was much more efficient on beta 1,6-linked GlcNAc than beta 1,2-linked GlcNAc, preferentially forming Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta 4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 side chain on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manright-arrowR side chain converts the acceptor to one that is much more favorable for iGnT and beta 4Gal-TI.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Poly-N-acetyllactosamines are unique glycans having N-acetyllactosamine repeats (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)n and can be digested by endo-beta -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, Fucalpha 1right-arrow2Galbeta 1right-arrow4GlcNAcright-arrowR (3-5). In human granulocytes, monocytes, and certain T lymphocytes, on the other hand, poly-N-acetyllactosamines contain Lex,1 Galbeta 1right-arrow4 (Fucalpha 1right-arrow3)GlcNAcright-arrowR, and sialyl Lex, NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4 (Fucalpha 1right-arrow3)GlcNAcright-arrowR (6-9). Sialyl Lex 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 Lex 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 Lex 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 beta 1,3-N-acetylglucosaminyltransferase, i-extension enzyme (iGnT), and beta 1,4-galactosyltransferase (beta 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 alpha 1,6-linked mannose through a GlcNAcbeta 1right-arrow6 linkage in GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR (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 Lex apparently takes place in those tumor cells when alpha 1,3-fucosyltransferase is also present. In fact, highly metastatic colonic carcinoma cells express more sialyl Lex 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 alpha 1,3-fucosyltransferase to form sialyl Lex 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 beta 1,4-galactosyltransferase, beta 4Gal-TIV (30). We have also found that beta 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 GlcNAcbeta 1right-arrow6 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 beta 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 GlcNAcbeta 1right-arrow6Manalpha right-arrowR side chain renders the resultant GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR an extremely efficient acceptor for beta 4Gal-TI and i-extension enzyme. Moreover, we found that the i-extension enzyme prefers Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain over Galbeta 1right-arrow4 GlcNAcbeta 1right-arrow6Manalpha right-arrowR side chain, whereas the opposite is true for beta 4Gal-TI. Such complementary effects apparently result in forming similar size and abundance of poly-N-acetyllactosamines in both chains of GlcNAcbeta 1right-arrow6 (GlcNAcbeta 1right-arrow2)Manalpha right-arrowR 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 Galbeta 1right-arrow4Glcbeta right-arrowp-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 beta 4Gal-TII, -TIII, -TIV, and -TV-- beta 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 beta 4Gal-T preparation (Sigma) was directly used as beta 4Gal-TI (30). beta 4Gal-TV (39) was cloned and expressed in COS-1 cells as described previously (30). For comparing the enzymatic activities of different beta 4Gal-T samples, the final concentration of beta 4Gal-TI, -TII, -TIII, -TIV, and -TV was adjusted to 38.0 nmol/h/ml as measured using 0.5 mM GlcNAcbeta right-arrowp-nitrophenol (Sigma) as an acceptor.

Synthesis of the Acceptors-- The branched pentasaccharides GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowO(CH2)7CH3(octyl) (compound 1) and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (compound 2) were synthesized from octyl 3, 4-di-O-benzyl-alpha -D-mannopyranosyl(1right-arrow6)-2,3,4-tri-O-benzyl-beta -Dmannopyranoside (compound 3) and the donors 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta -D-glucopyranosyl chloride (compound 4), the corresponding trichloroacetimidate (compound 5), or 2,3,4,6-tetra-Oacetyl-beta -D-galactopyranosyl(1right-arrow4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta -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]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 (Galbeta 1 right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (compound 11) and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (compound 12) were synthesized from compounds 1 and 2, respectively, through treatment with 50 milliunits of bovine milk beta 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 beta -N-acetylglucosaminidase (4 units) in 50 mM sodium acetate buffer, pH 5.5, at 37 °C for 3 days, resulting in the tetrasaccharide Galbeta 1right-arrow4GlcNAcbeta 1right-arrow 2Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (compound 13). Similarly, compound 1 (5 mM) was treated with Escherichia coli beta -galactosidase (20 units) in 50 mM Tris-HCl buffer, pH 7.4, at 37 °C overnight, resulting in the tetrasaccharide GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (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: delta  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: delta  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: delta  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: delta  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]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl or Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl. 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 beta 4Gal-TI-- To assay poly-N-acetyllactosamine formation, 0.5 mM acceptor was incubated with beta 4Gal-TI (760.0 nmol/h/ml) and iGnT (380.0 nmol/h/ml) under the conditions described previously (30, 32).

When [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1 right-arrow6Manbeta 1right-arrowoctyl (compound 11, see above) and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (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 beta -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 beta -galactosidase, jack bean beta -N-acetylglucosaminidase, and diplococcal beta -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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl or 0.5 mM GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl was incubated with beta 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc. In addition, beta 4Gal-TI was 4-fold in excess over iGnT, the same ratio as in HL-60 cells (42). In certain experiments, beta 4Gal-TI was 2-fold in excess over iGnT.

Analysis of Products by Endo-beta -galactosidase Digestion-- Products were digested with Escherichia freundii endo-beta -galactosidase for 18 h at 37 °C (43). The digestion conditions used allowed the cleavage of galactose linkage, where no beta 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, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR, Galbeta 1 right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR, and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR were incubated with iGnT. First, iGnT acted in almost identical efficiency on Galbeta 1right-arrow 4GlcNAcbeta 1right-arrow6Manalpha right-arrowR and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow 2Manalpha right-arrowR (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 Vmax 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).


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Fig. 1.   Dependence of iGnT and beta 4Gal-Ts on the concentration of linear and branched N-glycan acceptors. A, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (black-square), Gal beta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha 1right-arrow6Manbeta 1right-arrowoctyl (open circle ), or Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta 1right-arrowoctyl () of various concentrations was incubated with iGnT for 1 h. B, GlcNAcbeta 1right-arrow2Manalpha 1right-arrow6Manbeta 1right-arrowoctyl of various concentrations was incubated with beta 4Gal-TI (), -TII (triangle ), -TIII (), -TIV (open circle ), and -TV (black-square) for 1 h. The same amount of the enzyme, 38.0 nmol/h/ml, determined using 0.5 mM Galbeta 1right-arrow4Glcbeta 1right-arrowp-nitrophenol (A) or 0.5 mM GlcNAcbeta 1right-arrowp-nitrophenol (B), was present in those experiments.

                              
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Table I
Kinetic properties of iGnT
Arrows indicate where beta 1,3-linked N-acetylglucosamine is added.


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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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR (A), Galbeta 1right-arrow 4GlcNAcbeta 1right-arrow2Manalpha 1right-arrowR (B), and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (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]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR. 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-beta -galactosidase digestion (solid line). F, HPLC analysis after endo-beta -galactosidase digestion of the product substituted with one N-acetylglucosamine (solid line), which was derived from Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrowR. 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]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR was first synthesized with beta 4Gal-TI and UDP-[3H]Gal from GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR. 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-beta -galactosidase, and the digest was subjected to the same HPLC. Seventy-five % of the radioactivity was recovered as [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2) Manalpha right-arrowR (Fig. 2E), indicating that 75% of the products was [3H] Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2) Manalpha right-arrowR, whereas 25% was GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (see Fig. 3A).


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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-beta -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, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR was synthesized from Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, and the resultant acceptor was incubated with iGnT and UDP-GlcNAc. The monosubstituted product obtained was then digested with endo-beta -galactosidase, producing GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manright-arrowR with 26% yield (Fig. 2F). The results thus indicate that 26% of the products was GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Man right-arrowR, whereas 74% was Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow4[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (Fig. 2F, see also Fig. 3B).

These results, taken together, indicate that iGnT preferentially acts on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain over Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR 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 beta 4Gal-Ts beta 4Gal-TI was most efficient in adding galactose to GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta 1right-arrowoctyl. Similarly, we found in the present study that beta 4Gal-TI is the most efficient in adding a galactose to GlcNAcbeta 1right-arrow2Manalpha 1right-arrow6 Manbeta 1right-arrowoctyl (Fig. 1B; see also Table II).

                              
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Table II
Kinetic properties of beta 4Gal-Ts
Arrows indicate where galactose is added.

To determine how N-acetyllactosamine repeats are added to two different side chains, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR or Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR was incubated with iGnT, beta 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, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl with i-GnT, beta 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 branch to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR 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.


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Fig. 4.   HPLC analysis of the products after incubation of N-glycan acceptors with iGnT and beta 4Gal-TI. Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR (A), Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR (B), Galbeta 1right-arrow4 GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (C), or [3H]Gal beta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (D) was incubated with iGnT, beta 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2 Side Chain on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2(Galbeta 1right-arrow4GlcNAcalpha 1right-arrow6)Manalpha right-arrowR Acceptor-- To determine how each side chain of the branched acceptor was elongated with N-acetyllactosamine repeats, [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, synthesized as described above, was incubated with iGnT, beta 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-beta -galactosidase. As shown in Fig. 5A, 28.6% of the starting product was recovered as GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)-Manalpha right-arrowR. The results indicate that 28.6% of the products was Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1 right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, whereas 71.4% was [3H]Gal-beta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, which produced a nonradioactive compound after beta -galactosidase treatments.


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Fig. 5.   Analysis of the poly-N-acetyllactosaminyl products derived from asymmetrically labeled acceptors. A, beta -galactosidase digest of peak 1 in Fig. 4D. B-D, sequential digestion of peak 2 in Fig. 4D by beta -galactosidase (B), beta -N-acetylhexosaminidase (C), and beta -galactosidase (D). E-H, the products derived from Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR were separated by HPLC and those containing one N-acetyllactosamine repeat (E) and two Nacetyllactosamine repeats (F-H) were digested with beta -galactosidase (E) or sequentially digested with beta -galactosidase (F), followed by beta -N-acetylhexosaminidase (G) and beta -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 beta -galactosidase, beta -N-acetylglucosaminidase, and beta -galactosidase. After first beta -galactosidase digestion, 35.2% of the products was recovered as radioactive oligosaccharides, indicating that 64.8% of the starting products was [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6[(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2GlcNAcbeta 1right-arrow2]Manright-arrowR (Fig. 5B, see also Fig. 6B, middle). The additional digestion of the above beta -galactosidase digest with beta -N-acetylglucosaminidase resulted in no release of [3H]galactose as expected (Fig. 5C). The above digested products were finally digested again with beta -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 (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2[3H]Gal beta 1right-arrow4GlcNAcbeta 1right-arrow6[(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)]Manright-arrowR (Fig. 6B, left). The rest (9.1% = 35.2 × 0.259) was Galbeta 1right-arrow4GlcNAc beta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Gal beta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, which contains one N-acetyllactosamine extension in both branches (Fig. 6B, right). These results indicate that poly-N-acetyllactosamine is preferentially formed on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Man side chain over Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Man side chain under these conditions.


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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]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2) Manalpha 1right-arrow6Manbeta 1right-arrowoctyl and their sequential enzymatic digestion products are shown. C and D, the products derived from Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl 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 (open circle ), and radioactive galactose (oplus ) are denoted.

To corroborate the above conclusions, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, 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 beta 1right-arrow4GlcNAcbeta 1right-arrow3)2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow 4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (26.4%), Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6[(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2[3H]Galbeta 1right-arrow4GlcNAcbeta 1 right-arrow2]Manalpha right-arrowR (63.6%), and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (10%) (see Fig. 6D).

These results, taken together, indicate that preferential addition of N-acetylglucosamine by iGnT to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain leads into preferential formation of poly-N-acetyllactosamine in Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR branch.

Galactose Is Preferentially Added to GlcNAcbeta 1right-arrow6Manalpha right-arrowR Branch Over GlcNAcbeta 1right-arrow2Manalpha right-arrowR Branch-- The next question we asked is how the first galactosylation influences the formation of poly-N-acetyllactosamines. GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR or Galbeta 1right-arrow4GlcNAcbeta 1right-arrow 6(GlcNAcbeta 1right-arrow2)Manalpha right-arrowR was incubated with beta 4Gal-TI and UDP-[3H]Gal. As shown in Fig. 7A, beta 4Gal-TI adds a galactose to GlcNAcbeta 1right-arrow6Manalpha right-arrowR branch much more efficiently than the GlcNAcbeta 1right-arrow2Manalpha right-arrowR branch. The kinetic efficiency (Vmax/Km) exhibited a 2.5-fold difference between these two acceptors (Table III).


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Fig. 7.   Dependence of beta 4Gal-TI or iGnT on the concentration of acceptors. GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Man beta 1right-arrowoctyl (), Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Man beta 1right-arrowoctyl (open circle ), and GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6(Man beta 1right-arrowoctyl (black-square) of various concentrations were incubated with beta 4Gal-TI and UDP-[3H]Gal (A) or iGnT and UDP-[3H]GlcNAc (B).

                              
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Table III
Kinetic properties of beta 4Gal-TI
Incubation conditions were exactly the same as Table II. Arrows indicate where galactose is added (see Fig. 7A).

The above results indicate that galactose is preferentially added to GlcNAcbeta 1right-arrow6Manalpha right-arrowR branch, forming Galbeta 1right-arrow 4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha right-arrowR. We then tested how N-acetyllactosamine repeats are added on these preformed acceptors. First, the addition of N-acetylglucosamine by iGnT was found to be almost equal between Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 (GlcNAcbeta 1right-arrow2)Manalpha right-arrowR and GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR (Fig. 7B and Table I).

Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1 right-arrowoctyl was then incubated with iGnT, beta 4Gal-TI, UDP-[3H]GlcNAc, and UDP-[3H]Gal under conditions where only one N-acetyllactosamine extension takes place. As products, [3H] Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manright-arrowR and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manright-arrowR were expected (Fig. 8, A and B, dotted line). When these products are digested by endo-beta -galactosidase, a radioactive product would be derived only from [3H]Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, producing GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR. Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR would lose radioactivity after endo-beta -galactosidase digestion (the endo-beta -galactosidase-susceptible galactose is underlined in both structures) (see Fig. 9A).


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Fig. 8.   Poly-N-acetyllactosamine synthesis on asymmetrically galactosylated acceptors. A and B, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl was incubated with iGnT and beta 4Gal-TI in a ratio of 1:4 (A) or 1:2 (B) and radioactive donor substrates. C and D, GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl was incubated with iGnT and beta 4Gal-TI in a ratio of 1:4 (C) or 1:2 (D), and radioactive donor substrates. The products were analyzed by HPLC before (dotted line) and after (solid line) endo-beta -galactosidase digestion. Numbers indicate the relative ratio of the radioactivity. The specific activity of UDP-[3H]Gal and UDP-[3H]GlcNAc was the same.


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Fig. 9.   Schematic representation of poly-N-acetyllactosaminyl products derived from asymmetrically galactosylated acceptors. Man (), GlcNAc (), and Gal (open circle ) are denoted. The radioactive galactose and N-acetylglucosamine incorporated are indicated by asterisks. Numbers indicate the relative molar ratio of the products, calculated from the results shown in Fig. 8.

First, the acceptor was incubated with iGnT and beta 4Gal-TI in a ratio of 1:4, as done for all the other experiments. The products obtained from this reaction resulted in GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manright-arrowR with a 1.0:9.3 yield after endo-beta -galactosidase digestion. The results indicate that the molar ratio between Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manright-arrowR and the whole products was 1.0:3.1, because each initial product contained three radioactive sugars (Fig. 8A; see also Fig. 9A). This can be translated into the conclusion that the N-acetyllactosamine repeat was formed on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR and GlcNAcbeta 1right-arrow2Manalpha vR in the acceptor in a ratio of 1:2.1.

When the same acceptor was incubated with increased amount of iGnT, and the ratio of iGnT and beta 4Gal-TI was 1:2, 1.4/7.2 of the radioactivity was recovered as GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR after endo-beta -galactosidase digestion (Fig. 8B). The results indicate that Nacetyllactosamine repeat was added to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Man branch 1.4 times more than to GlcNAcbeta 1right-arrow2Man branch. These results indicate that both branches were almost equally elongated by N-acetyllactosamine repeats when iGnT and beta 4Gal-TI was in a ratio of 1:2 under the incubation conditions employed (Fig. 9A).

In parallel, GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl was incubated with iGnT, beta 4Gal-TI, and radioactive donor substrates under the same conditions described above. In these experiments, [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha right-arrowR should be produced only from [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR after endo-beta -galactosidase digestion (the endo-beta -galactosidase-susceptible galactose is underlined; see Fig. 9B). As shown in Fig. 8, C and D, the ratio between the oligosaccharide synthesized and product after endo-beta -galactosidase digestion was found to be 10.5:2.5 in both conditions. These results indicate that the products contained [3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6([3H]Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR and [3H]Galbeta 1right-arrow4[3H]GlcNAcbeta 1right-arrow3[3H]Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR in a ratio of 2.5:1.0. The results indicate that poly-N-acetyllactosamine extension preferentially takes place on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain when the reaction was started from GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2)Manalpha right-arrowR, regardless of the ratio of iGnT and beta 4Gal-TI.

As shown above, galactosylation takes place preferentially on beta 1,6-linked GlcNAc in GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta 1right-arrowoctyl. These combined results thus indicate that poly-N-acetyllactosamine extension almost equally takes place on both GlcNAcbeta 1right-arrow6Man and GlcNAcbeta 1right-arrow2Man side chains after the formation of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1 right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manright-arrowR antenna is extended by N-acetyllactosamine repeats as much as does Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manright-arrowR antenna (Figs. 1 and 2). When both galactosylated side chains are present in a branched acceptor, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manright-arrowR is utilized more efficiently to add N-acetyllactosamine repeats than Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manright-arrowR (Figs. 4-6). This is mainly because iGnT preferentially acts on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Man side chain (Figs. 2 and 3). Similarly, it was reported that iGnT from Novikoff hepatoma acts 1.6 times more efficiently on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain than on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR side chain in a branched acceptor (44). The results suggest that iGnT present in Novikoff hepatoma is probably the same as the cloned iGnT used in the present study.

More importantly, the amount of poly-N-acetyllactosamine formed in the branched acceptor was much more than the summation of two separate reactions using either one of these side chains (Fig. 4). These results strongly suggest that addition of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 side chain on Galbeta 1right-arrow4GlcNAc beta 1right-arrow2Manalpha right-arrowR side chain, or vice versa, must change the conformation of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR and possibly that of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR. As a result, both side chains become more favorable for iGnT and beta 4Gal-TI to act. This enhancement can be observed even when first galactosylation takes place on GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Man alpha 1right-arrow6Manbeta 1right-arrowoctyl (see Tables II and III). In this context, it is noteworthy that GnTV adds N-acetylglucosamine only when Manalpha 1right-arrow6Man is in a gauche-gauche conformation (45). This result indicates that the action of GnTV restricts the conformation of resultant branched oligosaccharide, GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR. This conclusion is consistent with the results in the recent report indicating that Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 side chain may be extended toward the proximal region of N-glycans (46). Addition of GlcNAcbeta 1right-arrow6 by chemical synthesis may also bring some conformational change in both side chains, which is favorable for the actions by iGnT and beta 4Gal-TI.

It is rather striking that Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Man alpha 1right-arrow6Manbeta right-arrowR is as good as Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Man alpha 1right-arrow6Manbeta right-arrowR as an acceptor for iGnT (Figs. 1 and 2 and Table I). The results obtained in the present study are, however, consistent with the structural data on human red cell band 3 (18, 47). Human red cell band 3 does not contain Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowR side chain yet contains very extended poly-N-acetyllactosamines. It is also noteworthy that a side chain extending from Manalpha 1right-arrow6Manbeta right-arrowR contains more poly-N-acetyllactosamines than that extending from Manalpha 1right-arrow3Manbeta right-arrowR in human red cell band 3 (18, 47).

It is assumed that GnTV adds N-acetylglucosamine as soon as GlcNAcbeta 1right-arrow2Manalpha 1right-arrow6Manbeta right-arrowR is formed (48). The resultant oligosaccharide, GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Man alpha 1right-arrow6Manbeta right-arrowR, is most likely an acceptor for beta 4Gal-TI. The present study demonstrated that GlcNAcbeta 1right-arrow6 residue is much more favored by beta 4Gal-TI over GlcNAcbeta 1right-arrow2 residue, although iGnT does not have such a preference (Fig. 7). This finding is similar to those obtained on galactosylation of GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow3)Gal, I-branch precursor structure. In that particular study, almost 95% of the product was Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow3)Gal (49). Such a preferential galactosylation of GlcNAcbeta 1right-arrow6 takes place when both branches are terminated with GlcNAc residues.

The present study demonstrated that poly-N-acetyllactosamine extension takes place more efficiently on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain than Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR side chain when a fully galactosylated branch ed acceptor was utilized (Figs. 4 and 5). In contrast, poly-Nacetyllactosamine extension took place almost equally between two branches when increased amount of iGnT and Galbeta 1right-arrow4 GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha right-arrowR as an acceptor were used (Figs. 8 and 9). The results obtained in the latter experiments are consistent with those obtained on the structural analysis of glycoproteins containing poly-N-acetyllactosamines from granulocytes (6, 7) and glycoproteins, such as human erythropoietin produced in Chinese hamster ovary cells (50-54). In particular, NMR studies of the N-glycans isolated from the recombinant erythropoietin demonstrated that Nacetyllactosamine extension takes place almost equally in Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha right-arrowR and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chains (52-54). These results, as a whole, strongly suggest that the concentration of iGnT relative to that of beta 4Gal-TI may be more than that estimated from the activities in total cell lysates. Further studies will be of significance to determine whether iGnT is more enriched in narrower compartments of the Golgi than beta 4Gal-TI (see also Ref. 55).

The above results were obtained most likely due to the following reasons. First, it is almost certain that Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha right-arrowR is formed before the formation of GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2) Manalpha right-arrowR, considering that beta 4Gal-TI greatly prefers beta 1,6-linked GlcNAc over beta 1,2-linked GlcNAc residue (Fig. 10B). This allows poly-N-acetyllactosamine extension in Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Man side chain before initiation of poly-N-acetyllactosamine synthesis in GlcNAcbeta 1right-arrow2Man side chain (Fig. 10, B--E). In contrast, iGnT preferentially acts on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain once this side chain is galactosylated. Such a branch specificity of iGnT compensates the inefficient galactosylation of GlcNAcbeta 1right-arrow2Manalpha right-arrowR branch, leading into N-acetyllactosamine extension in Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR branch (Fig. 10I). These results indicate that branch specificity of beta 4Gal-TI and iGnT has the opposite effect on poly-N-acetyllactosamine extension in the GlcNAcbeta 1right-arrow6Manalpha right-arrowR branch versus the GlcNAcbeta 1right-arrow2Manalpha right-arrowR branch. Such complemental branch specificities of beta 4Gal-TI and iGnT is a likely cause for almost equal distribution of poly-N-acetyllactosamine extension in two side chains in nature. Recently, a new member of iGnT was reported (56). However, no studies on branch specificity of this enzyme was carried out, and it is not known whether this additional iGnT may be responsible for poly-N-acetyllactosamine formation in certain cells.


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Fig. 10.   Proposed biosynthetic steps of poly-N-acetyllactosamines in dibranched GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR. beta 1,6-linked N-acetylglucosamine is first added by GnTV, forming GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrow6Manbeta right-arrowR (A). This is followed mainly by galactosylation of beta 1,6-linked GlcNAc (B), addition of beta 1,3-linked GlcNAc and beta 1,4-linked galactose (C and D) and galactosylation of GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain (E), forming poly-N-acetyllactosamine in the GlcNAcbeta 1right-arrow6 branch. As a minor biosynthetic pathway, B is converted to G by galactosylation of GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain in B. This is followed by addition of N-acetylglucosamine (H) and galactose (I) forming poly-N-acetyllactosamine extension on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain (I). As a minor biosynthetic pathway, G is also formed from A through F, leading to the formation of I. Although it is not shown in the present study, E and I are most likely converted to those containing N-acetyllactosamine repeats in both Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow2 branches. This biosynthetic pathway is based on the results obtained in the present study.

In summary, the present study demonstrated that GlcNAcbeta 1right-arrow6Manalpha right-arrowR side chain itself is not a preferential site for poly-N-acetyllactosamine formation. Rather, addition of this side chain on GlcNAcbeta 1right-arrow2Manalpha right-arrowR side chain by GnTV, forming GlcNAcbeta 1right-arrow6(GlcNAcbeta 1right-arrow2)Manalpha 1right-arrowManbeta right-arrowR, converts the acceptor extremely favorable for poly-N-acetyllactosamine formation (Fig. 4). Moreover, we found that the branched acceptor formed is first galactosylated at GlcNAcbeta 1right-arrow6Man side, which is a key step to add N-acetyllactosamine extension equally in both GlcNAcbeta 1right-arrow6Manright-arrowR and GlcNAc beta 1right-arrow2Manright-arrowR side chains (Fig. 10). The present study, however, did not address why poly-N-acetyllactosamines are added more readily on membrane proteins than secretory proteins (57). Because the iGnT has a unique transmembrane domain (32), further studies will be of significance to address this point in relation to the actions of membrane-bound iGnT.

    ACKNOWLEDGEMENTS

We thank Dr. Henrik Clausen for providing the samples of beta 4Gal-TII, -TIII, and -TIV; Dr. Kiyohiko Angata for pcDNAI-A vector; Dr. Edgar Ong for critical reading of the manuscript; and Susan Greaney and Sanae Ujita for organizing the manuscript.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health, Grants RO1 CA48737 and PO1 CA71932.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Toyobo Biotechnology Fellowship.

To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3144; Fax: 619-646-3193; E-mail: minoru{at}burnham-inst.org.

2 J. McAuliffe, M. Ujita, M. Fukuda, and O. Hindsgaul, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: Lex, Lewisx; GnTV, N-acetylglucosaminyltransferase V; iGnT, i-extension beta 1,3-N-acetylglucosaminyltransferase; beta 4Gal-T, beta 1,4-galactosyltransferase; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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