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
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ABSTRACT |
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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
Gal Poly-N-acetyllactosamines are unique glycans having
N-acetyllactosamine repeats
(Gal 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
The increased expression of sialyl Lex apparently takes
place in those tumor cells when Previously, we have demonstrated that
poly-N-acetyllactosamines in core 2 branched
O-glycans are synthesized through iGnT and a newly
discovered member of 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
GlcNAc 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 Gal Expression of cDNAs Encoding Synthesis of the Acceptors--
The branched pentasaccharides
GlcNAc
The labeled hexasaccharides
[3H]Gal
Compound 1 (3 mM) was treated with jack bean
Partial 1H NMR results (300 MHz, D2O)
(compounds 1, 2, 13, and 14) are as follows. Compound 1: 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]Gal Poly-N-acetyllactosamine Synthesis by iGnT and
When
[3H]Gal
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
As a third experiment, 0.5 mM
Gal Analysis of Products by Endo- 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,
Gal
To determine which side chain is substituted with
N-acetylglucosamine,
[3H]Gal
To corroborate the above results,
Gal
These results, taken together, indicate that iGnT preferentially acts
on
Gal Poly-N-acetyllactosamine Synthesis on Unbranched or Branched
Acceptor--
As shown previously (30), among different members of
To determine how N-acetyllactosamine repeats are added to
two different side chains,
Gal Poly-N-acetyllactosamine Is Preferentially Added to
Gal
The product containing one N-acetyllactosamine repeat (Fig.
4D, peak 1) was digested with exo-
Similarly, the products containing two N-acetyllactosamine
repeats (Fig. 4D, peak 2) were sequentially digested with
To corroborate the above conclusions,
Gal
These results, taken together, indicate that preferential addition of
N-acetylglucosamine by iGnT to
Gal Galactose Is Preferentially Added to GlcNAc
The above results indicate that galactose is preferentially added to
GlcNAc
Gal
First, the acceptor was incubated with iGnT and
When the same acceptor was incubated with increased amount of iGnT, and
the ratio of iGnT and
In parallel,
GlcNAc
As shown above, galactosylation takes place preferentially on
The present study demonstrates that
Gal 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
Gal It is rather striking that
Gal It is assumed that GnTV adds N-acetylglucosamine as soon as
GlcNAc The present study demonstrated that poly-N-acetyllactosamine
extension takes place more efficiently on
Gal The above results were obtained most likely due to the following
reasons. First, it is almost certain that
Gal1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6 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
Gal
1
4GlcNAc
1
2Man
R side chain than in
Gal
1
4GlcNAc
1
6Man
R, due to preferential action of iGnT
on Gal
1
4GlcNAc
1
2Man
R side chain. On the other hand, galactosylation was much more efficient on
1,6-linked GlcNAc than
1,2-linked GlcNAc, preferentially forming
Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R.
Starting with this preformed acceptor,
N-acetyllactosamine repeats were added almost equally
to Gal
1
4GlcNAc
1
6Man
R and Gal
1
4GlcNAc
1
2Man
R 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
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R structure,
which is consistent with the structures found in nature. The results
also suggest that the addition of Gal
1
4GlcNAc
1
6 side chain
on Gal
1
4GlcNAc
1
2Man
R 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
1
4GlcNAc
1
3)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,
Fuc
1
2Gal
1
4GlcNAc
R (3-5). In human granulocytes, monocytes, and certain T lymphocytes, on the other hand,
poly-N-acetyllactosamines contain
Lex,1
Gal
1
4 (Fuc
1
3)GlcNAc
R, and sialyl Lex,
NeuNAc
2
3Gal
1
4 (Fuc
1
3)GlcNAc
R (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.
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
GlcNAc
1
6 linkage in
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R (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).
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
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.
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 GlcNAc
1
6 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).
1
6Man
R side chain renders the resultant
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R an extremely efficient acceptor for
4Gal-TI and i-extension enzyme. Moreover, we found that the i-extension enzyme prefers
Gal
1
4GlcNAc
1
2Man
R side chain over Gal
1
4
GlcNAc
1
6Man
R
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
GlcNAc
1
6 (GlcNAc
1
2)Man
R structures.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
4Glc
p-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).
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
GlcNAc
p-nitrophenol (Sigma) as an acceptor.
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
O(CH2)7CH3(octyl)
(compound 1) and
Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl (compound 2) were synthesized from octyl
3, 4-di-O-benzyl-
-D-mannopyranosyl(1
6)-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(1
4)-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.
1
4GlcNAc
1
6
(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
(compound 11) and
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
(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.
-N-acetylglucosaminidase (4 units) in 50 mM
sodium acetate buffer, pH 5.5, at 37 °C for 3 days, resulting in the
tetrasaccharide
Gal
1
4GlcNAc
1
2Man
1
6Man
1
octyl
(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
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl (compound 14). Isolation, purification, and characterization of these
compounds was performed as described above. Detailed procedures of
these syntheses will be published
elsewhere.2
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).
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl or
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl.
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.
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).
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
(compound 11, see above) and
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
(compound 12, see above) were used as acceptors, nonradioactive
donor substrates were used.
-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.
1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl
or 0.5 mM
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl 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
Gal
1
4GlcNAc
1
3Gal
1
4Glc. 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.
-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
1
4GlcNAc
1
6Man
R, Gal
1
4GlcNAc
1
2Man
R,
and
Gal
1
4GlcNAc
1
6 (Gal
1
4GlcNAc
1
2)Man
R
were incubated with iGnT. First, iGnT acted in almost identical
efficiency on
Gal
1
4GlcNAc
1
6Man
R
and
Gal
1
4GlcNAc
1
2Man
R (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
4Gal-Ts on the concentration of linear and branched
N-glycan acceptors. A,
Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
(
),
Gal
1
4GlcNAc
1
2Man
1
6Man
1
octyl
(
), or
Gal
1
4GlcNAc
1
6Man
1
6Man
1
octyl
(
) of various concentrations was incubated with iGnT for 1 h.
B,
GlcNAc
1
2Man
1
6Man
1
octyl
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 Gal
1
4Glc
1
p-nitrophenol
(A) or 0.5 mM
GlcNAc
1
p-nitrophenol (B), was present in
those experiments.
Kinetic properties of iGnT
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
Gal 1
4GlcNAc
1
6Man
1
R
(A),
Gal
1
4GlcNAc
1
2Man
1
R
(B), and
Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R
(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]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R.
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
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
1
R.
In E and F, the
mono-N-acetylglucosaminylated products are shown as
dotted lines. Numbers indicate the relative ratio
of these oligosaccharides.
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R was first synthesized with
4Gal-TI and UDP-[3H]Gal
from
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R. 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]Gal
1
4GlcNAc
1
6(GlcNAc
1
2) Man
R (Fig. 2E), indicating that 75% of the products was
[3H] Gal
1
4GlcNAc
1
6(GlcNAc
1
3Gal
1
4GlcNAc
1
2) Man
R,
whereas 25% was
GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R (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- -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.
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
was synthesized from Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
R, and the resultant acceptor was incubated with iGnT and UDP-GlcNAc. The
monosubstituted product obtained was then digested with
endo-
-galactosidase, producing
GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
with 26% yield (Fig. 2F). The results thus indicate
that 26% of the products was
GlcNAc
1
3Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R,
whereas 74% was
Gal
1
4GlcNAc
1
6(GlcNAc
1
4[3H]Gal
1
4GlcNAc
1
2)Man
R
(Fig. 2F, see also Fig. 3B).
1
4GlcNAc
1
2Man
R
side chain over
Gal
1
4GlcNAc
1
6Man
R
in an approximate ratio of 3:1 under the incubation conditions used.
4Gal-Ts
4Gal-TI was most efficient in adding galactose to
GlcNAc
1
6Man
1
6Man
1
octyl. Similarly, we found in the present study that
4Gal-TI is the most
efficient in adding a galactose to
GlcNAc
1
2Man
1
6 Man
1
octyl
(Fig. 1B; see also Table
II).
Kinetic properties of 4Gal-Ts
1
4GlcNAc
1
6Man
R or
Gal
1
4GlcNAc
1
2Man
R
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,
Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl 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
Gal
1
4GlcNAc
1
6
branch to
Gal
1
4GlcNAc
1
2Man
R 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
4Gal-TI.
Gal
1
4GlcNAc
1
6Man
R
(A),
Gal
1
4GlcNAc
1
2Man
R
(B),
Gal
1
4 GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R
(C), or
[3H]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R
(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.
1
4GlcNAc
1
2 Side Chain on
Gal
1
4GlcNAc
1
2(Gal
1
4GlcNAc
1
6)Man
R
Acceptor--
To determine how each side chain of the branched
acceptor was elongated with N-acetyllactosamine repeats,
[3H]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R, 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.
-galactosidase. As
shown in Fig. 5A, 28.6% of
the starting product was recovered as
GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
6(GlcNAc
1
2)-Man
R. The results indicate that 28.6% of the products was
Gal
1
4GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R, whereas 71.4%
was [3H]Gal-
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
2)Man
R, which produced a nonradioactive compound after
-galactosidase treatments.
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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
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
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).
-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]Gal
1
4GlcNAc
1
6[(Gal
1
4GlcNAc
1
3)2GlcNAc
1
2]Man
R (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
(Gal
1
4GlcNAc
1
3)2[3H]Gal
1
4GlcNAc
1
6[(Gal
1
4GlcNAc
1
2)]Man
R (Fig. 6B, left). The rest (9.1% = 35.2 × 0.259) was
Gal
1
4GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
2)Man
R, which contains one N-acetyllactosamine extension in both
branches (Fig. 6B, right). These results indicate that
poly-N-acetyllactosamine is preferentially formed on
Gal
1
4GlcNAc
1
2Man side chain over
Gal
1
4GlcNAc
1
6Man
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]Gal 1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2) Man
1
6Man
1
octyl
and their sequential enzymatic digestion products are shown.
C and D, the products derived from
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
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.
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R,
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
1
4GlcNAc
1
3)2Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
(26.4%),
Gal
1
4GlcNAc
1
6[(Gal
1
4GlcNAc
1
3)2[3H]Gal
1
4GlcNAc
1
2]Man
R
(63.6%), and
Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
2)Man
R
(10%) (see Fig. 6D).
1
4GlcNAc
1
2Man
R side chain leads into preferential formation of
poly-N-acetyllactosamine in
Gal
1
4GlcNAc
1
2Man
R branch.
1
6Man
R
Branch Over GlcNAc
1
2Man
R Branch--
The next question we
asked is how the first galactosylation influences the formation of
poly-N-acetyllactosamines.
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R or
Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
R was incubated with
4Gal-TI and UDP-[3H]Gal. As shown
in Fig. 7A,
4Gal-TI adds a
galactose to
GlcNAc
1
6Man
R branch much more efficiently than the
GlcNAc
1
2Man
R 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
4Gal-TI or iGnT on the concentration of
acceptors.
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
(
),
Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl
(
), and
GlcNAc
1
6(GlcNAc
1
2)Man
1
6(Man
1
octyl
(
) of various concentrations were incubated with
4Gal-TI and
UDP-[3H]Gal (A) or iGnT and
UDP-[3H]GlcNAc (B).
Kinetic properties of 4Gal-TI
1
6Man
R
branch, forming
Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
R.
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
Gal
1
4GlcNAc
1
6 (GlcNAc
1
2)Man
R
and
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R (Fig. 7B and Table I).
1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl was then incubated with iGnT,
4Gal-TI,
UDP-[3H]GlcNAc, and UDP-[3H]Gal under
conditions where only one N-acetyllactosamine extension takes place. As products,
[3H] Gal
1
4[3H]GlcNAc
1
3Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
and
Gal
1
4GlcNAc
1
6([3H]Gal
1
4[3H]GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
2)Man
R
were expected (Fig. 8, A and
B, dotted line). When these products are digested by
endo-
-galactosidase, a radioactive product would be derived only
from
[3H]Gal
1
4[3H]GlcNAc
1
3Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R,
producing
GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R.
Gal
1
4GlcNAc
1
6([3H]Gal
1
4[3H]GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
2)Man
R
would lose radioactivity after endo-
-galactosidase digestion (the
endo-
-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,
Gal 1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl
was incubated with iGnT and
4Gal-TI in a ratio of 1:4 (A)
or 1:2 (B) and radioactive donor substrates. C
and D,
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
was incubated with iGnT and
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-
-galactosidase digestion.
Numbers indicate the relative ratio of the radioactivity.
The specific activity of UDP-[3H]Gal and
UDP-[3H]GlcNAc was the same.
View larger version (15K):
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Fig. 9.
Schematic representation of
poly-N-acetyllactosaminyl products derived from
asymmetrically galactosylated acceptors. Man ( ), GlcNAc (
),
and Gal (
) 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.
4Gal-TI in a ratio
of 1:4, as done for all the other experiments. The products obtained
from this reaction resulted in
GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
with a 1.0:9.3 yield after endo-
-galactosidase digestion. The
results indicate that the molar ratio between
Gal
1
4GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
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
Gal
1
4GlcNAc
1
6Man
R
and GlcNAc
1
2Man
vR in the acceptor in a ratio of 1:2.1.
4Gal-TI was 1:2, 1.4/7.2 of the radioactivity
was recovered as
GlcNAc
1
6([3H]Gal
1
4GlcNAc
1
2)Man
R
after endo-
-galactosidase digestion (Fig. 8B). The
results indicate that Nacetyllactosamine repeat was
added to
Gal
1
4GlcNAc
1
6Man
branch 1.4 times more than to
GlcNAc
1
2Man branch.
These results indicate that both branches were almost equally elongated by N-acetyllactosamine repeats when iGnT and
4Gal-TI was
in a ratio of 1:2 under the incubation conditions employed (Fig.
9A).
1
6(Gal
1
4GlcNAc
1
2)Man
1
6Man
1
octyl
was incubated with iGnT,
4Gal-TI, and radioactive donor substrates
under the same conditions described above. In these experiments,
[3H]Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
R should be produced only from
[3H]Gal
1
4GlcNAc
1
6([3H]Gal
1
4[3H]GlcNAc
1
3 Gal
1
4GlcNAc
1
2)Man
R
after endo-
-galactosidase digestion (the
endo-
-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-
-galactosidase digestion was found to be 10.5:2.5 in both
conditions. These results indicate that the products contained
[3H]Gal
1
4GlcNAc
1
6([3H]Gal
1
4[3H]GlcNAc
1
3 Gal
1
4GlcNAc
1
2)Man
R
and
[3H]Gal
1
4[3H]GlcNAc
1
3[3H]Gal
1
4GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R in a ratio of 2.5:1.0. The results indicate that
poly-N-acetyllactosamine extension preferentially takes
place on
Gal
1
4GlcNAc
1
2Man
R side chain when the reaction was started from
GlcNAc
1
6(Gal
1
4GlcNAc
1
2)Man
R, regardless of the ratio of iGnT and
4Gal-TI.
1,6-linked GlcNAc in
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl. These combined results thus indicate that
poly-N-acetyllactosamine extension almost equally takes
place on both
GlcNAc
1
6Man and
GlcNAc
1
2Man side
chains after the formation of
Gal
1
4GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
4GlcNAc
1
2Man
R
antenna is extended by N-acetyllactosamine repeats as much
as does
Gal
1
4GlcNAc
1
6Man
R
antenna (Figs. 1 and 2). When both galactosylated side chains are
present in a branched acceptor,
Gal
1
4GlcNAc
1
2Man
R
is utilized more efficiently to add N-acetyllactosamine
repeats than
Gal
1
4GlcNAc
1
6Man
R (Figs. 4-6). This is mainly because iGnT preferentially acts on Gal
1
4GlcNAc
1
2Man
side chain (Figs. 2 and 3). Similarly, it was reported that iGnT from
Novikoff hepatoma acts 1.6 times more efficiently on
Gal
1
4GlcNAc
1
2Man
R side chain than on
Gal
1
4GlcNAc
1
6Man
R
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.
1
4GlcNAc
1
6 side chain on
Gal
1
4GlcNAc
1
2Man
R
side chain, or vice versa, must change the conformation of
Gal
1
4GlcNAc
1
2Man
R and possibly that of
Gal
1
4GlcNAc
1
6Man
R.
As a result, both side chains become more favorable for iGnT and
4Gal-TI to act. This enhancement can be observed even when first galactosylation takes place on
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
1
octyl
(see Tables II and III). In this context, it is noteworthy that GnTV
adds N-acetylglucosamine only when Man
1
6Man is in a
gauche-gauche conformation (45). This result indicates that
the action of GnTV restricts the conformation of resultant branched
oligosaccharide,
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R. This conclusion is consistent with the results in the recent report indicating that Gal
1
4GlcNAc
1
6 side chain may be extended
toward the proximal region of N-glycans (46). Addition of
GlcNAc
1
6 by chemical synthesis may also bring some conformational
change in both side chains, which is favorable for the actions by iGnT and
4Gal-TI.
1
4GlcNAc
1
2Man
1
6Man
R
is as good as
Gal
1
4GlcNAc
1
6Man
1
6Man
R
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
Gal
1
4GlcNAc
1
6Man
1
6Man
R side chain yet contains very extended
poly-N-acetyllactosamines. It is also noteworthy that a side
chain extending from Man
1
6Man
R contains more
poly-N-acetyllactosamines than that extending from Man
1
3Man
R in human red cell band 3 (18, 47).
1
2Man
1
6Man
R
is formed (48). The resultant oligosaccharide,
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R,
is most likely an acceptor for
4Gal-TI. The present study
demonstrated that GlcNAc
1
6 residue is much more favored by
4Gal-TI over GlcNAc
1
2 residue, although iGnT does not have such a preference (Fig. 7). This finding is similar to those obtained on galactosylation of
GlcNAc
1
6(GlcNAc
1
3)Gal, I-branch precursor structure. In that particular study, almost 95% of
the product was
Gal
1
4GlcNAc
1
6(GlcNAc
1
3)Gal (49). Such a preferential galactosylation of GlcNAc
1
6 takes place when both branches are terminated with GlcNAc residues.
1
4GlcNAc
1
2Man
R side chain than
Gal
1
4GlcNAc
1
6Man
R
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
Gal
1
4 GlcNAc
1
6(GlcNAc
1
2)Man
R 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
Gal
1
4GlcNAc
1
6Man
R and
Gal
1
4GlcNAc
1
2Man
R
side chains (52-54). These results, as a whole, strongly suggest that
the concentration of iGnT relative to that of
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
4Gal-TI
(see also Ref. 55).
1
4GlcNAc
1
6(GlcNAc
1
2)Man
R is formed before the formation of
GlcNAc
1
6(Gal
1
4GlcNAc
1
2) Man
R,
considering that
4Gal-TI greatly prefers
1,6-linked GlcNAc over
1,2-linked GlcNAc residue (Fig.
10B). This allows poly-N-acetyllactosamine extension in
Gal
1
4GlcNAc
1
6Man side chain before initiation of poly-N-acetyllactosamine
synthesis in
GlcNAc
1
2Man side
chain (Fig. 10, B--E). In contrast, iGnT
preferentially acts on
Gal
1
4GlcNAc
1
2Man
R side chain once this side chain is galactosylated. Such a branch specificity of iGnT compensates the inefficient galactosylation of
GlcNAc
1
2Man
R
branch, leading into N-acetyllactosamine extension in
Gal
1
4GlcNAc
1
2Man
R
branch (Fig. 10I). These results indicate that branch
specificity of
4Gal-TI and iGnT has the opposite effect on
poly-N-acetyllactosamine extension in the
GlcNAc
1
6Man
R
branch versus the
GlcNAc
1
2Man
R branch. Such complemental branch specificities of
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
GlcNAc 1
6(GlcNAc
1
2)Man
1
6Man
R.
1,6-linked N-acetylglucosamine is first added by GnTV,
forming
GlcNAc
1
6(GlcNAc
1
2)Man
1
6Man
R
(A). This is followed mainly by galactosylation of
1,6-linked GlcNAc (B), addition of
1,3-linked GlcNAc
and
1,4-linked galactose (C and D) and
galactosylation of
GlcNAc
1
2Man
R
side chain (E), forming poly-N-acetyllactosamine
in the GlcNAc
1
6 branch. As a minor biosynthetic pathway,
B is converted to G by galactosylation of
GlcNAc
1
2Man
R
side chain in B. This is followed by addition of
N-acetylglucosamine (H) and galactose
(I) forming poly-N-acetyllactosamine extension on
Gal
1
4GlcNAc
1
2Man
R
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
Gal
1
4GlcNAc
1
6
and
Gal
1
4GlcNAc
1
2
branches. This biosynthetic pathway is based on the results obtained in
the present study.
In summary, the present study demonstrated that
GlcNAc1
6Man
R
side chain itself is not a preferential site for
poly-N-acetyllactosamine formation. Rather, addition of this side chain on
GlcNAc
1
2Man
R
side chain by GnTV, forming
GlcNAc
1
6(GlcNAc
1
2)Man
1
Man
R, converts the acceptor extremely favorable for
poly-N-acetyllactosamine formation (Fig. 4). Moreover,
we found that the branched acceptor formed is first galactosylated at
GlcNAc
1
6Man side,
which is a key step to add N-acetyllactosamine extension equally in both
GlcNAc
1
6Man
R and
GlcNAc
1
2Man
R 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.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Henrik Clausen for providing the
samples of 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.
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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.
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.
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ABBREVIATIONS |
---|
The abbreviations used are:
Lex, Lewisx;
GnTV, N-acetylglucosaminyltransferase V;
iGnT, i-extension 1,3-N-acetylglucosaminyltransferase;
4Gal-T,
1,4-galactosyltransferase;
HPLC, high performance liquid
chromatography.
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REFERENCES |
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