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INTRODUCTION |
Poly-N-acetyllactosamines are unique glycans having
N-acetyllactosamine repeats,
(Gal
1
4GlcNAc
1
3)n (1).
Poly-N-acetyllactosamines are attached to
N-glycans (2-5), O-glycans (6, 7), and glycolipids (8-10) and can be digested by endo-
-galactosidase (11).
Poly-N-acetyllactosamines are often modified to express
differentiation antigens and functional oligosaccharides. Among those oligosaccharides, sialyl Lewisx
(Lex)1 and its
sulfated forms are ligands for E-, P-, and L-selectin (12-16). 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. L-selectin on lymphocytes, on the other hand, recognizes
sulfated sialyl Lex expressed in high endothelial venules
of blood vessels (15, 16). This L-selectin/counterreceptor interaction
allows lymphocytes to migrate into lymphoid system, allowing
lymphocytes to circulate fully in the body.
In human granulocytes, monocytes, and certain T lymphocytes,
poly-N-acetyllactosamines contain Lex,
Gal
1
4(Fuc
1
3)GlcNAc
R, and sialyl
Lex,
NeuNAc
2
3Gal
1
4(Fuc
1
3)GlcNAc
R (17, 18). In
contrast, poly-N-acetyllactosamines in human
erythrocytes contain ABO blood group antigens, synthesized from the
precursor structure, Fuc
1
2Gal
1
4GlcNAc
R (5, 19, 20).
In addition, poly-N-acetyllactosamines can contain
I-branches,
Gal
1
4GlcNAc
1
3(Gal
1
4GlcNAc
1
6)Gal
R. During
development of human erythrocytes, the linear i antigen represented by
Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
R is converted to those
containing I-branches (21). In early mouse embryonic development, embryos express I antigen, which is gradually replaced with i antigen
during development (2, 22).
The acquisition of I-branches is important, since two of the
N-acetyllactosamine side chains can have functional terminal structures. It has been demonstrated that multivalent sialyl
Lex poly-N-acetyllactosamines inhibit
L-selectin-mediated binding and the rejection of organ
transplants with much better efficacy than monovalent sialyl
Lex poly-N-acetyllactosamines (23, 24).
Similarly, blood group H antigens present at both termini in branched
poly-N-acetyllactosamines were shown to have much better
avidity to anti-ABO antibodies than linear
poly-N-acetyllactosamines containing single antigenic structures (25). It was suggested that expression of i antigen in fetal
erythrocytes minimizes a detrimental immune response when mother and
fetus have incompatible blood group antigens (25).
These results, as a whole, indicate the significance of understanding
how linear and I-branched poly-N-acetyllactosamines are
synthesized. To this end, we have cloned cDNAs encoding
1,3-N-acetylglucosaminyltransferase (iGnT) that forms
linear poly-N-acetyllactosamines (26) and
1,6-N-acetylglucosaminyltransferase (IGnT) that forms
I-branches (27). The IGnT cloned was found to add
1,6-N-acetylglucosamine at the central galactose
(underlined) of
Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
R, thus
termed as centrally acting IGnT (cIGnT) (28, 29). In addition, another
IGnT, distally acting IGnT (dIGnT), was found to add
1,6-N-acetylglucosamine to peridistal galactose
(underlined) of GlcNAc
1
3Gal
1
4GlcNAc
R
(29-33). No studies have been reported, however, to determine how
I-branched poly-N-acetyllactosamine is synthesized by iGnT,
IGnT, and
4Gal-T.
In the present study, we first describe how the IGnT cloned from PA-1
cells (27) adds I-branches to linear
poly-N-acetyllactosamines containing multiple acceptor
sites. We then demonstrate that
4Gal-TI is responsible for
galactosylation in the synthesis of both linear and branched
poly-N-acetyllactosamines. Finally, we
reconstituted the synthesis of I-branched
poly-N-acetyllactosamine, the structure of which resembles
that present in human erythrocytes (5), PA-1 human embryonic carcinoma
cells (34), and rabbit erythrocytes (10). The results demonstrate an
intricate interaction between acceptor substrates and these glycosyltransferases.
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EXPERIMENTAL PROCEDURES |
Isolation of cDNA Encoding iGnT and IGnT--
cDNA
encoding iGnT was cloned into pcDNA3.1, resulting in
pcDNA3.1-iGnT, as described previously (26). 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.
cDNA encoding IGnT was cloned from a cDNA library constructed
from human PA-1 embryonic carcinoma cells, resulting in pcDNAI-IGnT, as
described previously (27). A catalytic domain of IGnT was prepared by
polymerase chain reaction using pcDNAI-IGnT as a template. 5'- and
3'-primers for this polymerase chain reaction were
5'-GCGGATCCAAGCTTCCAAAGGCTA-3' and
5'-GGCTCGAGCTCAAAAATACCAGCTGGGT-3'
(BamHI and XhoI sites are underlined). The
polymerase chain reaction product encoding amino acid residues 30-400
of the IGnT was digested with BamHI and XhoI and
then cloned into the same sites of pcDNAI-A, resulting in pcDNAI-A·IGnT.
Expression of the Protein A-iGnT and Protein A-IGnT Fusion
Protein--
pcDNAI-A, pcDNAI-A·iGnT, and
pcDNAI-A·IGnT were separately transfected with Lipofectamine Plus
(Life Technologies) 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 enzymes to IgG-Sepharose 6FF (Amersham
Pharmacia Biotech) as described previously (37). Alternatively, the
culture medium was concentrated 100-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, since
IgG-Sepharose-bound enzymes had a low activity as seen for other
glycosyltransferases (38, 39). Typically, the activities of iGnT and
IGnT in the incubation mixture were 38.0 nmol/h/ml using 0.5 mM Gal
1
4Glc
p-nitrophenol (Toronto
Research Chemicals) and 40.0 nmol/h/ml using 0.5 mM
Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl (see below) as acceptors, respectively.
The medium from mock-transfected COS-1 cells contained less than
of iGnT activity as described (26) or less than
of IGnT activity compared with that derived from pcDNAI-A·iGnT-
or pcDNAI-A·IGnT-transfected COS-1 cells.
Expression of cDNAs Encoding
4Gal-TII, -TIII, -TIV, and
-TV--
Isolation of cDNAs encoding
4Gal-TII, -TIII, and -TIV
was described previously (40, 41).
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 (36,
41). Human milk
4Gal-T preparation (Sigma) was directly used as
4Gal-TI (36).
4Gal-TV (42) was cloned and expressed in COS-1 cells as described
previously (36). The supernatant from the transfected COS-1 cells was
concentrated 100-fold as described above and used as an enzyme source.
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.
Oligosaccharides--
(Gal
1
4GlcNAc
1
3)nGal
1
4GlcNAc
1
6Man
1
6Man
O(CH2)7CH3(octyl),
where n = 0, 1, and 2, were synthesized, starting from the derivatives of Gal
1
4GlcNAc and
Man
1
6Man
octyl, as described previously (36).
GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl and
GlcNAc
1
3Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl were prepared by Escherichia coli
-galactosidase
treatment of Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl
and
Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl, respectively, as described previously (36).
Lacto-N-neo-tetraose
(Gal
1
4GlcNAc
1
3Gal
1
4Glc) and
lacto-N-neo-hexaose
(Gal
1
4GlcNAc
1
3(Gal
1
4GlcNAc
1
6)Gal
1
4Glc) were pyridylaminated, as described previously (31, 43). Purification of
the product was achieved by HPLC on a TSK gel ODS-80TS column (4.6 × 250 mm; TOSOH) equipped with a Gilson 306 HPLC apparatus. The column
was equilibrated with 20 mM ammonium acetate buffer, pH
4.0, and eluted with the same buffer at a flow rate of 1.0 ml/min.
Fluorescence was detected with a fluorescent spectrophotometer (Shimadzu, model RF-535) with excitation and emission wavelengths of
320 and 400 nm, respectively. The concentration of PA-oligosaccharides was estimated by comparing fluorescent intensity of synthesized PA-oligosaccharides and standard PA-glucose purchased from Takara Shuzo
(PanVera, Madison, WI). Lacto-N-neo-tetraose,
lacto-N-neo-hexaose, and
lacto-N-hexaose
(Gal
1
3GlcNAc
1
3(Gal
1
4GlcNAc
1
6)Gal
1
4Glc) were purchased from Oxford GlycoSystems.
Addition of N-Acetylglucosamine by iGnT and IGnT--
To assay
the transfer of N-acetylglucosamine residues by the iGnT,
the reaction mixture was exactly the same as described previously (36).
As acceptors, lacto-N-neo-tetraose,
lacto-N-neo-hexaose, lacto-N-hexaose,
pyridylaminated lacto-N-neo-hexaose
(PA-lacto-N-neo-hexaose), and
lacto-N-neo-tetraose
(PA-lacto-N-neo-tetraose) were used. The assay
products derived from the above oligosaccharides were purified by
QAE-Sephadex A-25 gel and subjected to Bio-Gel P-4 gel filtration as
described previously (36). The radioactivity of the aliquots was
determined by a scintillation counter. The products derived from the
PA-oligosaccharides were filtered through Ultrafree-MC (10-kDa cut;
Millipore Corp.) and applied to the same ODS-80TS column and eluted as
described above. Since PA-oligosaccharides can be detected by
fluorescence, nonradioactive UDP-GlcNAc was used as a donor for the
experiments using PA-oligosaccharides.
To assay the transfer of N-acetylglucosamine residues by
IGnT, the reaction mixture contained 5 mM
UDP-[3H]GlcNAc (2 × 104 cpm/nmol; NEN
Life Science Products), 10 mM EDTA, 20 µl of IGnT preparation as described above, 10 mM
N-acetylglucosamino-1,5-lactone (Toronto Research
Chemicals), 10 mM galactono-1,5-lactone, and various
concentrations of an acceptor in 50 µl (final volume) of 100 mM cacodylate buffer, pH 7.0, modified from the previously described protocol (31). As acceptors,
GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl, Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl,
GlcNAc
1
3Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
1
6Man
1
6Man
octyl, and
(Gal
1
4GlcNAc
1
3)2Gal
1
4GlcNAc
1
6Man
1
6Man
octyl
were used. The incubation mixture was applied to a C18 reverse
phase Sep-Pak column (Waters), and the product was eluted as described previously (36). The product was then analyzed by HPLC using NH2-bonded silica column (Varian Micropak AX-5) as
described previously (36). The radioactivity of aliquots was
determined. 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.
Substrate Specificity of
4Gal-TI, -TII, -TIII, -TIV, and
-TV--
Assays of
4Gal-Ts were performed exactly as described
previously (36). As acceptors, GlcNAc
1
3Gal
1
4Glc
PA
and Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA were used.
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA
was synthesized by incubating 5 mM
Gal
1
4GlcNAc
1
3Gal
1
4Glc
PA with the recombinant IGnT
and 5 mM UDP-GlcNAc for 10 h in 100 µl of the
reaction mixture as described above. The product was purified by HPLC
using the same ODS column as described above and used as an acceptor.
Poly-N-Acetyllactosamine Formation in I-branched
Oligosaccharide--
To assay poly-N-acetyllactosamine
formation, 0.5 mM
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA was
incubated with
4Gal-TI (152 nmol/h/ml), iGnT (38 nmol/h/ml), 0.5 mM UDP-GlcNAc, and 0.5 mM UDP-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 mixture was filtered and subjected to HPLC
as described above. In these experiments, the incubation condition was
first determined using Gal
1
4GlcNAc
1
3Gal
1
4Glc, where only one N-acetyllactosamine unit can be added. In
addition,
4Gal-TI was 4-fold in excess over iGnT, the same ratio as
present in HL-60 cells (44).
Analysis of Products by Endo-
-Galactosidase
Digestion--
Products were digested with Escherichia
freundii endo-
-galactosidase for 18 h at 37 °C (11).
The digestion condition used allowed the cleavage of galactose linkage,
where no
-1,6-linked N-acetylglucosamine is attached (11,
45). The digests were subjected to HPLC using AX-5 column or TSK gel
ODS-80TS.
 |
RESULTS |
Addition of I-branch to Various Poly-N-Acetyllactosaminyl Side
Chains--
Recently, it was reported that the IGnT cloned from PA-1
cells exclusively adds
1,6-linked N-acetylglucosamine to
a galactose residue in a central position as seen in
Gal
1
4GlcNAc
1
3Gal
1
4Glc(NAc)
R (the
underlined galactose is the accepting galactose (28)). To determine if
the IGnT can add N-acetylglucosamine residues far from
nonreducing terminals, acceptors with various numbers of
N-acetyllactosamine repeats were used. As shown in Fig.
1, B and D, the
oligosaccharide containing two potential acceptor sites,
(Gal
1
4GlcNAc
1
3)2Gal
1
4GlcNAc
1
6Man
R,
incorporated slightly more N-acetylglucosamine than that
containing only one acceptor site. When the products obtained from
(Gal
1
4GlcNAc
1
3)2Gal
1
4GlcNAc
1
6Man
R were analyzed by endo-
-galactosidase digestion, the majority of
singly branched products contained
1,6-linked GlcNAc close to the
nonreducing terminal (Fig. 1F and Fig.
2, compound e). These results indicate that the IGnT displays a preference for a
galactose residue separated by one N-acetyllactosamine unit from the nonreducing terminal (Fig. 2, compounds
b, e, and g). The results shown in
Fig. 1, C and D, also indicate that the addition of two I-branches to neighboring galactose residues
(compounds d and g in Fig. 2) occurs
less efficiently than the addition of one branch.

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Fig. 1.
Analysis of the products after incubation of
poly-N-acetyllactosamine acceptors with IGnT.
A, B, C, and D, HPLC
analysis of the IGnT products derived from
GlcNAc 1 3Gal 1 4GlcNAc 1 6Man 1 R (A),
Gal 1 4GlcNAc 1 3Gal 1 4GlcNAc 1 6Man 1 R
(B),
GlcNAc 1 3Gal 1 4GlcNAc 1 3Gal 1 4GlcNAc 1 6Man 1 R
(C), and
Gal 1 4GlcNAc 1 3Gal 1 4GlcNAc 1 3Gal 1 4GlcNAc 1 6Man 1 R
(D). The first and second
peaks in C and D are the
oligosaccharides containing one and two 1,6-linked GlcNAc residues,
respectively. E and F, HPLC analysis of
endo- -galactosidase digestion of singly branched products from
GlcNAc 1 3Gal 1 4GlcNAc 1 3Gal 1 4GlcNAc 1 6Man 1 R
shown in C (E) and
(Gal 1 4GlcNAc 1 3)2Gal 1 4GlcNAc 1 6Man 1 R
shown in D (F). The compounds eluted at
fractions 28 and 45 in E and F are
GlcNAc 1 3([3H]GlcNAc 1 6)Gal 1 4GlcNAc 1 6Man 1 6Man octyl
and
Gal 1 4GlcNAc 1 3([3H]GlcNAc 1 6)Gal 1 4GlcNAc 1 3Gal,
respectively. The numbers indicate the relative molar
ratio of the IGnT products (A-D) or
endo- -galactosidase-digested products (E, F).
In this HPLC, amino-bonded AX-5 column was used. The structures of the
IGnT products are shown in Fig. 2.
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Fig. 2.
Schematic representation of IGnT
oligosaccharide products. Gal ( ), GlcNAc ( ), Man ( ), and
1,6-linked GlcNAc in I-branch ( ) are denoted. The
numbers indicate the relative molar ratio of the products.
The vertical lines indicate the positions where
endo- -galactosidase cleaves.
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Fig. 1, A and C, illustrate that oligosaccharides
containing N-acetylglucosamine at nonreducing terminals can
also serve as acceptors. In particular, the results shown in Fig.
1A demonstrate that the IGnT can add
1,6-linked GlcNAc to
a peridistal galactose (Fig. 2, compound a), thus
containing dIGnT activity. This finding is consistent with the results
reported in our recent study (29). When two galactoses are available in
the oligosaccharide containing nonreducing terminal
N-acetylglucosamine, all of the singly branched product(s)
remained radioactive after endo-
-galactosidase digestion (Fig.
1E), indicating its structure as
GlcNAc
1
3Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4GlcNAc
1
6Man
R (Fig. 2, compound c). Peridistal galactose was
utilized only when the central galactose was utilized (Fig. 2,
compound d). These results indicate that a
peridistal galactose is the least favored by the cIGnT cloned from PA-1
cells. When the preferred galactose is missed by the IGnT, the enzyme
can still add an I-branch to that galactose after one
N-acetylglucosamine (Fig. 2, compounds c and d) or N-acetyllactosamine is
added (Fig. 2, compounds f and g).
The above results were obtained using the recombinant IGnT bound to
IgG-Sepharose, the activity of which was 3.6 nmol/h/ml. Almost
identical results were obtained when a concentrated culture medium from
IGnT-transfected cells, of which activity was 40.0 nmol/h/ml, was used.
4Gal-TI Is Responsible for Galactosylation of Linear and
Branched Poly-N-acetyllactosamine Synthesis--
In our previous
study, we demonstrated that
4Gal-TI is involved in
N-acetyllactosamine formation in N-glycans, while
4Gal-TIV forms N-acetyllactosamine in core 2 branched
O-glycans (36). To determine which
4Gal-T is involved in
galactosylation of I-branched oligosaccharides,
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA was enzymatically synthesized using the recombinant IGnT and
Gal
1
4GlcNAc
1
3Gal
1
4Glc
PA and used as an
acceptor. As shown in Fig. 3A,
4Gal-TI transfers a galactose most efficiently to this acceptor,
whereas
4Gal-TII, -TIII, and -TV exhibit substrate inhibition at
higher concentrations of the acceptor substrate, probably because
-galactose in the acceptor competes with UDP-Gal (Fig.
3A). Although
4Gal-TIV does not exhibit substrate
inhibition, it is less efficient than
4Gal-TI (Table
I).

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Fig. 3.
Dependence of iGnT and
4Gal-T activity on the concentration of linear and
branched poly-N-acetyllactosamine acceptors.
A and B,
Gal 1 4GlcNAc 1 3(GlcNAc 1 6)Gal 1 4Glc PA
(A) and GlcNAc 1 3Gal 1 4Glc PA
(B) of various concentrations were incubated with 4Gal-TI
( ), -TII ( ), -TIII ( ), -TIV ( ), and -TV ( ). C
and D,
Gal 1 4GlcNAc 1 3(GlcNAc 1 6)Gal 1 4Glc PA
(C) and Gal 1 4GlcNAc 1 3Gal 1 4Glc PA
(D) of various concentrations were incubated with the iGnT.
The same amount of the enzyme, 38.0 nmol/h/ml, determined using
0.5 mM GlcNAc p-nitrophenol (A,
B) or 0.5 mM
Gal 1 4Glc p-nitrophenol (C,
D), was present in these experiments.
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It is also evident that
4Gal-TI, among these
4Gal-Ts, acts
most efficiently on a linear poly-N-acetyllactosamine
acceptor, GlcNAc
1
3Gal
1
4Glc
PA (Fig. 3B, Table
I). In both experiments, similar results were obtained when the
concentration of these enzymes was increased 5-fold or decreased
5-fold. These results indicate that
4Gal-TI is mostly responsible
for galactosylation of both branched and linear
poly-N-acetyllactosamines.
Addition of N-Acetylglucosamine to I-branched Acceptors--
To
determine how iGnT adds N-acetylglucosamine residues to
I-branched oligosaccharides, the incorporation of
N-acetylglucosamine was compared between different acceptors
including linear and branched oligosaccharides. The results shown in
Fig. 4 demonstrate that
lacto-N-neo-hexaose did not incorporate twice the
amount of N-acetylglucosamine compared with
lacto-N-neo-tetraose, despite the fact that the
former contains two acceptor sites (A and B). Moreover, lacto-N-hexaose containing only one acceptor
galactose in the I-branch incorporated N-acetylglucosamine
as much as lacto-N-neo-hexaose (Fig.
4C). The results strongly suggest that the addition of
N-acetylglucosamine to one side chain precludes the addition
of another GlcNAc to the other side chain in branched structures.

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Fig. 4.
iGnT activity on linear and branched
N-acetyllactosaminyl oligosaccharides.
Gal 1 4GlcNAc 1 3Gal 1 4Glc (A),
Gal 1 4GlcNAc 1 3(Gal 1 4GlcNAc 1 6)Gal 1 4Glc
(B), and
Gal 1 3GlcNAc 1 3(Gal 1 4GlcNAc 1 6)Gal 1 4Glc
(C) were incubated with the iGnT and
UDP-[3H]GlcNAc for 10 h. The products were analyzed
by Bio-Gel P-4 gel filtration, and numbers shown in the
upper right indicate the relative amount of
incorporated radioactivity. A peak eluted at fraction 51 was due to a
contaminant derived from UDP-[3H]GlcNAc.
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To determine the structures of products, the enzymatic reaction
products derived from PA-lacto-N-neo-hexaose were subjected to HPLC using an ODS column. Two products, peak A and B, were then
separately digested with endo-
-galactosidase followed by exo-
-N-acetylglucosaminidase treatment (Fig.
5). The results indicate that peak A, the
major product (75% of the total), contains N-acetylglucosamine in the I-branch, while peak B contains
N-acetylglucosamine at the linear side chain (Fig.
6). These results indicate that iGnT
prefers the Gal
1
4GlcNAc
1
6 branch over
Gal
1
4GlcNAc
1
3, which was originally part of linear
poly-N-acetyllactosamine. More importantly, no product
containing two N-acetylglucosamines at both terminals was
formed, supporting the above conclusion that
N-acetylglucosamine can be added to only one of the
terminals. This result indicates that a branched acceptor probably
becomes a competitive inhibitor for iGnT as soon as one
N-acetylglucosamine is added.

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Fig. 5.
HPLC analysis of the products after
incubation of PA-lacto-N-neo-hexaose
with iGnT. The products obtained after incubation of
Gal 1 4GlcNAc 1 3(Gal 1 4GlcNAc 1 6)Gal 1 4Glc PA
with the iGnT and UDP-GlcNAc were applied to HPLC (A,
E). Peak A (B-D) and
peak B (F-H) were purified
(B and F) and then sequentially digested with
endo- -galactosidase (C and G) and
exo- -N-acetylglucosaminidase (D and
H) and analyzed by the same HPLC. Peak
S corresponds to the starting material. ODS-80TS column was
used in this HPLC.
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Fig. 6.
Structures of the iGnT products from
PA-lacto-N-neo-hexaose. The
products after incubation of
PA-lacto-N-neo-hexaose with the iGnT are shown.
The products, obtained after sequential treatment of
endo- -galactosidase and -N-acetylglucosaminidase, are
also shown. Peaks A and B correspond
to A and B in Fig. 5, respectively.
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Elongation of N-Acetyllactosamine Units on Branched
Poly-N-acetyllactosamine--
To determine how
N-acetyllactosamine elongation takes place after the IGnT
adds a
1,6-linked N-acetylglucosamine,
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA was
incubated with a mixture of
4Gal-TI and the iGnT, and the products
were separated by reverse phase ODS column as shown in Fig.
7.

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Fig. 7.
HPLC analysis of the products derived from
Gal 1 4GlcNAc 1 3(GlcNAc 1 6)Gal 1 4Glc PA
after incubation with iGnT and 4Gal-TI.
Gal 1 4GlcNAc 1 3(GlcNAc 1 6)Gal 1 4Glc PA
was incubated with the iGnT, 4Gal-TI, UDP-GlcNAc, and UDP-Gal
(A). Peak E was purified
(B) and then sequentially digested with
endo- -galactosidase (C) and
exo- -N-acetylglucosaminidase (D).
Peak D was eluted at the same position as
Gal 1 4GlcNAc 1 6Gal 1 4Glc PA shown in Fig.
5H. Peaks S and F
correspond to the starting material and
PA-lacto-N-neo-hexaose, respectively.
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The minor peak of the products, peak F, eluted shortly after the
starting material and corresponds to
Gal
1
4GlcNAc
1
3(Gal
1
4GlcNAc
1
6)Gal
1
4Glc
PA, which represents the addition of one galactose to the I-branch in the
acceptor substrate. This structure was further confirmed by
endo-
-galactosidase digestion and exoglycosidase digestion (data not
shown). The major peak (84% of the total products), peak E, was
digested by endo-
-galactosidase, followed by
-N-acetylglucosaminidase (Fig. 7, C and
D). This digested material was eluted at the position corresponding to Gal
1
4GlcNAc
1
6Gal
1
4Glc
PA (Fig.
7D), which was also obtained in the above experiment (Fig.
5H). These results indicate that the products are either
those containing one N-acetyllactosamine extension at
Gal
1
4GlcNAc
1
3Gal
1
4Glc side chain (compound E in Fig. 8) or
lacto-N-neo-hexaose (compound
F in Fig. 8).

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Fig. 8.
Proposed biosynthetic steps of
I-branched poly-N-acetyllactosamine. 1,6-Linked
N-acetylglucosamine is first added to a central galactose by
cIGnT (B). This is followed by the addition of
1,3-linked N-acetylglucosamine by iGnT
(C) and 1,4-linked galactose by 4Gal-TI
(D), adding N-acetyllactosamine to the
linear poly-N-acetyllactosamine side chain. This is followed
by galactosylation of 1,6-linked N-acetylglucosamine,
forming I-branch (E). As a minor biosynthetic pathway,
galactosylation of I-branch may take place as soon as 1,6-linked
N-acetylglucosamine is added (F). If
compound F is formed, 1,3-linked
N-acetylglucosamine is preferentially added to I-branch by
iGnT, potentially leading to more complex
poly-N-acetyllactosamines. E and F
correspond to peaks E and F in Fig. 7,
respectively. This biosynthetic pathway is based on the results
obtained in the present study.
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Since the majority of the product was compound E, the extension of
N-acetyllactosamine units along the linear
poly-N-acetyllactosamine side is favored over
galactosylation of the I-branch forming compound F. These results are
entirely consistent with the previous findings that I-branches are
usually composed of only one N-acetyllactosamine unit in
erythrocytes (5) and human PA-1 embryonic carcinoma cells (34) from
which the cIGnT was cloned.
Galactosylation of
1,6-GlcNAc Branch Is a Rate-limiting
Step--
The above results demonstrate that I-branch formed by cIGnT
is not extended further and that N-acetyllactosamine
extension takes place preferentially at linear
poly-N-acetyllactosamine side chain. To understand how this
is achieved, Km and Vmax
values of
4Gal-TI and iGnT were obtained for linear and branched
oligosaccharide acceptors. As shown in Table I,
4Gal-TI exhibits
much lower affinity toward the branched acceptor
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA
(Km = 2.49 mM) than its linear
counterpart GlcNAc
1
3Gal
1
4Glc
PA (Km = 0.31 mM). In contrast, iGnT
exhibits higher affinity toward the branched acceptor
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA (Km = 0.52 mM) than the linear acceptor
Gal
1
4GlcNAc
1
3Gal
1
4Glc
PA (Km = 1.09 mM) (Fig. 3, C and D, Table
II).
These results indicate that
1,3-linked GlcNAc is added to the
Gal
1
4GlcNAc
1
3Gal side chain before galactosylation of
1,6-linked GlcNAc branch (Fig. 8C). This reaction is,
most likely, immediately followed by galactosylation of the
GlcNAc
1
3Gal
1
4GlcNAc
1
3Gal moiety (Fig. 8D),
considering that the additions of GlcNAc and Gal to
Gal
1
4GlcNAc
1
3Gal are favored by these two enzymes
(Tables I and II). As the last step, galactosylation of I-branch
takes place (Fig. 8E). Only as a minor biosynthetic pathway,
galactosylation of I-branch precedes other reactions forming
compound F in Fig. 8, which may lead to more
complex poly-N-acetyllactosamines.
 |
DISCUSSION |
The present study demonstrated that I-branch formation takes place
more efficiently at sites closer to nonreducing termini than at
internal sites, suggesting that I-branch is formed preferentially at
the end of elongating poly-N-acetyllactosamine (Figs. 1 and 2). If this potential site is missed, the IGnT still utilizes the site
with a lower efficiency after N-acetylglucosamine (Fig. 2,
c and d) or N-acetyllactosamine is
added (Fig. 2, f and g). It is noteworthy that
the addition of I-branches is not facilitated by having two potential
acceptor sites. Rather, the total amount of
N-acetylglucosamine transferred is close to that in the
compound containing one acceptor site (Fig. 2, b and
e-g). The addition of one branch appears to inhibit the
addition of another branch on the same side chain. These results
strongly suggest that the second branch is usually formed when two
acceptor sites are separated by more than one
N-acetyllactosamine unit.
These results are consistent with the structures of branched
poly-N-acetyllactosamines determined on human adult band 3 and PA-1 cells (5, 34). First, the majority of nonreducing termini contain I-branches. Second, two I-branches are mostly located more than
one N-acetyllactosaminyl unit apart. Similar results were
obtained on the ceramide pentadecasaccharide from rabbit erythrocytes
(10). This spacing may be necessary for other modifications, since
I-branch and A-blood group terminal structure are separated by two
N-acetyllactosamine units in Ad glycolipid from
human erythrocytes (8). The IGnT was cloned from a cDNA library of
human PA-1 embryonic carcinoma cells (27). These results suggest that
human and rabbit erythroid precursor cells contain IGnT that is the
same as or similar to the IGnT cloned from PA-1 cells. Hog small
intestine may have similar poly-N-acetyllactosamine structures as seen in erythrocytes and PA-1 cells, since a cIGnT was
purified from this tissue (46).
The present study also demonstrated that intrinsic properties of
4Gal-TI and iGnT are critical in forming short I-branches. The
addition of a galactose residue to
1,6-linked
N-acetylglucosamine branch is a much slower process than the
addition of N-acetylglucosamine or galactose to an
elongating Gal
1
4GlcNAc
1
3 side chain, as demonstrated by
kinetic data (Table I and II). The addition of
1,3-linked
N-acetylglucosamine to the Gal
1
4GlcNAc
1
3 side chain is even more efficient when this acceptor has a
1,6-linked GlcNAc branch (Table II). Combined together, these results indicate that galactosylation of a GlcNAc
1
6 branch takes place as a
rate-limiting step in the synthesis of branched
poly-N-acetyllactosamines. This is most likely the reason
why no elongation of I-branch was observed in the present study when
the acceptor containing
1,6-linked GlcNAc was incubated with a
mixture of iGnT and
4Gal-TI (Figs. 7 and 8).
These results are entirely consistent with the structures determined on
poly-N-acetyllactosamines from human erythrocyte band 3 (5),
human PA-1 embryonic carcinoma cells (34), and rabbit erythrocytes
(10). Only one N-acetyllactosamine unit is present in each
I-branch of these glycans.
The results obtained in the present study predict that it takes longer
for additional modifications of formed I-branch than the extension of
Gal
1
4GlcNAc
1
3 side chain. If this is the case, I-branches
in internal positions contain fewer modifications than I-branches at
nonreducing termini. Similarly, additional modification at elongating
linear poly-N-acetyllactosamines should take place faster
than at I-branches even at nonreducing termini. In fact, more
1,2-fucosylated I-branch was found in those branches at nonreducing
termini than those in internal positions in human band 3 (5). Moreover,
Fuc
1
2Gal
1
4GlcNAc
1
3(Gal
1
4GlcNAc
1
6)Gal
1
R but not
Gal
1
4GlcNAc
1
3(Fuc
1
2Gal
1
4GlcNAc
1
6)Gal
1
R
was found in monofucosylated termini of human band 3 (5). Thus, all of
these structural data are consistent with the results predicted from
the present study, demonstrating that galactosylation of I-branch is a
rate-limiting step.
It is noteworthy that the iGnT can act very efficiently on
Gal
1
4GlcNAc
1
3(GlcNAc
1
6)Gal
1
4Glc
PA
(Fig. 3C and Table II) but not on
Gal
1
4GlcNAc
1
3(GlcNAc
1
3Gal
1
4GlcNAc
1
6)Gal
1
4Glc
PA (Fig. 5, peak A). This result suggests that short
GlcNAc
1
6 branch may not be recognized by the iGnT,
while the terminal GlcNAc residue in the extended
GlcNAc
1
3Gal
1
4GlcNAc
1
6 branch may be
recognized by the iGnT, preventing the addition of GlcNAc to the other
side chain due to a substrate inhibition. It is thus tempting to
speculate that the short GlcNAc
1
6 branch attached to linear
poly-N-acetyllactosamines may be difficult for
4Gal-TI to
recognize because of its conformation. In fact, NMR studies on the
pentadecaglycolipid indicate that the anomeric proton of
1,6-GlcNAc
linked to the internal galactose is not detected, suggesting that it is
conformationally inaccessible (10). These results, combined together,
indicate that intricate interaction between these glycosyltransferases
and I-branched acceptors play a critical role in the synthesis of
I-branched poly-N-acetyllactosamines.
Previously, it was determined that core 2 branch
GlcNAc
1
6(Gal
1
3)GalNAc
R is galactosylated most
efficiently by
4Gal-TIV (36). Other galactosyltransferases such as
4Gal-TI, -TII, -TIII, and -TV exhibit a substrate inhibition toward
the core 2 acceptor, probably because of competition between
-galactose in the acceptor and the donor substrate, UDP-Gal.
Similarly,
4Gal-TII, -TIII, and -TV exhibited a substrate inhibition
toward the I-branched acceptor (Fig. 3A).
4Gal-TI and
4Gal-TIV did not show a substrate inhibition toward the I-branched
acceptor. However, the kinetic efficiency
(Vmax/Km) for
4Gal-TIV is
less than a half of that for
4Gal-TI (Table I), indicating that
4Gal-TI is probably dominant in I-branch formation.
4Gal-TI is
most efficient in galactosylation of a linear
poly-N-acetyllactosamine as well (Fig. 3B and
Table I). Moreover,
4Gal-TI is most efficient in the synthesis of
N-glycan poly-N-acetyllactosamine as shown in the previous study (36). Overall, these results indicate that
4Gal-TI plays a major role in poly-N-acetyllactosamine extension and
I-branch formation in N-glycans. It is noteworthy that
4Gal-TI knock-out mice survive during development (47), and those
mutant mice express polysialic acid (35) and the HNK-1 carbohydrate
epitope (48) in brain glycoproteins (49). These results suggest that a
4Gal-T other than
4Gal-TI partly compensates for the loss of
4Gal-TI in the knock-out mice and is possibly involved in N-acetyllactosamine synthesis under normal conditions as well.
The experiments carried out in the present study were designed to mimic
cellular biosynthetic pathways. The biosynthetic oligosaccharide products are also a result of the balance between the amount of glycosyltransferases present and the movement of glycoproteins in the
Golgi apparatus during biosynthesis (50). For large scale synthesis of
oligosaccharides in vitro, however, enzymatic synthesis can
be achieved despite the fact that such a reaction is unlikely in
vivo. For example, Renkonen et al. (51) synthesized
highly branched poly-N-acetyllactosaminyl oligosaccharides
containing four sialyl Lex termini using the cIGnT. In this
oligosaccharide, every possible acceptor site was occupied by I-branch
and all of the I-branches contained sialyl Lex. It
was also reported that galactosylation of core 2 branch
GlcNAc
1
6(Gal
1
3)GalNAc could be achieved using an excess
amount of
4Gal-TI (36, 52), although
4Gal-TI is unlikely to be
involved in its galactosylation in vivo. These results
strongly suggest that the results obtained by in vitro
studies need to be evaluated regarding how these findings reflect the
biosynthesis taking place in cells.
The present study reveals the biosynthetic pathway involving the cIGnT
that adds
1,6-linked GlcNAc to central galactose residues. It has
been demonstrated that there is an additional IGnT, dIGnT, which adds
1,6-linked GlcNAc at peridistal galactose residues, forming
GlcNAc
1
3(GlcNAc
1
6)Gal
R at nonreducing termini
(30-33). In this situation, galactosylation at I-branch may not be a
rate-limiting step, since no substrate inhibition takes place.
Recently, we have cloned a novel
1,6-N-acetylglucosaminyltransferase that has more dIGnT
activity than cIGnT activity (29). Future studies will be of
significance to determine the structures of I-branched poly-N-acetyllactosamines synthesized by this newly cloned enzyme.