From the Two types of Biosynthesis of branched backbones of type 2 polylactosamines
involves reactions catalyzed by
Institute of Biotechnology and Department of
Biosciences,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1,6-GlcNAc transferases (IGnT6)
are involved in in vitro branching of polylactosamines:
dIGnT6 (distally acting), transferring to the penultimate
galactose residue in acceptors like
GlcNAc
1-3Gal
1-4GlcNAc
1-R, and cIGnT6
(centrally acting), transferring to the midchain galactoses
in acceptors of the type (GlcNAc
1-3)Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-R.
The roles of the two transferases in the biosynthesis of
branched polylactosamine backbones have not been clearly elucidated. We
report here that cIGnT6 activity is expressed in human (PA1) and murine
(PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of
the branched pentasaccharide
Gal
1-4GlcNAc
1-3(GlcNAc
1-6)Gal
1-4GlcNAc from the linear
tetrasaccharide Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc. The PA1
cell lysates were shown to also catalyze the formation of the branched
heptasaccharides
Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3(GlcNAc
1-6)Gal
1-4GlcNAc and
Gal
1-4GlcNAc
1-3(GlcNAc
1-6)Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc from the linear hexasaccharide
Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc in
reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was
not detected in the lysates of the two EC cells that were incubated
with UDP-GlcNAc and the acceptor trisaccharide
GlcNAc
1-3Gal
1-4GlcNAc. Hence, it appears likely that cIGnT6,
rather than dIGnT6 is responsible for the synthesis of the
branched polylactosamine chains in these cells.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1,3-N-acetylglucosaminyltransferase (GnT3),1
1,4-galactosyltransferase
(GalT4), and
1,6-N-acetylglucosaminyltransferases (GnT6). However,
the actual pathways leading to in vivo biosynthesis of
branched polylactosamine backbones have not been clearly identified. The branch-forming reactions, in particular, are poorly understood. Two
candidate branching reactions involving distinct
1,6-N-acetylglucosaminyltransferases (IGnT6) have been
described in vitro (1). The "distally acting" dIGnT6
transfers a GlcNAc unit in the
1,6 linkage to the penultimate galactose residue at the growing end of the linear polylactosamine chain (Scheme 1) (2-8). By contrast, the
"centrally acting" cIGnT6 transfers a GlcNAc residue in the
1,6
linkage to midchain galactose units of preformed as well as growing
linear chains (3, 9, 10). The dIGnT6 reactions proceed only with
acceptor chains bearing distal GlcNAc residues, whereas the cIGnT6
works with polylactosamine backbones carrying either a galactose or a
GlcNAc residue at the distal position. There is very little overlapping in the acceptor specificities of the two types of enzymes in
vitro.
View larger version (12K):
[in a new window]
Scheme 1.
Formation of GlcNAc branches at
different positions of a linear polylactosaminoglycan by the two types
of IGnT6s (2-10).
In naturally occurring branched backbones, uniformly short
LacNAc1-6 branches are linked to linear primary chains; this is the
case e.g. in human embryonal carcinoma (EC) cells (11) and adult erythrocyte band 3 (12). It has been suggested that the dIGnT6 is
responsible for the biosynthesis of polylactosamines in these cells (5,
11, 12). However, the suggested role of dIGnT6 in the biosynthesis of
molecules containing exclusively short branches is doubtful because the
extension enzyme, e.g. the GnT3 of human serum elongates
both branches of the hexasaccharide LacNAc
1-3'(LacNAc
1-6')LacNAc (where LacNAc is Gal
1-4GlcNAc) (13), paving routes to the formation of complex as well as short branches (14).
We have recently suggested that midchain branching enzymes similar to
the cIGnT6 activity present in blood serum of mammals may be
responsible for the conversion of linear polylactosamine chains into
branched backbone arrays in vivo (10). The reasoning was
based on data showing that the cIGnT6 activity of rat serum catalyzed the transformation of the linear hexasaccharide
LacNAc1-3LacNAc
1-3LacNAc in two steps into the doubly
branched octasaccharide
LacNAc
1-3'(GlcNAc
1-6')LacNAc
1-3'(GlcNAc
1-6')LacNAc. The latter was
1,4-galactosylated enzymatically into the
mature decasaccharide backbone
LacNAc
1-3'(LacNAc
1-6')LacNAc
1-3'(LacNAc
1-6')LacNAc (10), which strikingly resembles the polylactosamine backbones of human
EC cells of line PA1 (11).
Here, we report experiments involving lysates of human PA1 cells,
exogenous UDP-GlcNAc, and either the tetrasaccharide
LacNAc1-3'LacNAc or the hexasaccharide
LacNAc
1-3'LacNAc
1-3'LacNAc that established the presence of
the cIGnT6 activity in the PA1 cells. In contrast, the dIGnT6 activity
was not detected in experiments where UDP-GlcNAc and
GlcNAc
1-3Gal
1-4GlcNAc were incubated with PA1 cell lysates. Lysates of murine EC cells of line PC13, known to carry large amounts
of branched polylactosamines (15), also expressed the cIGnT6 activity
but not the dIGnT6 activity. The data imply that cIGnT6 rather than
dIGnT6 activity is involved in the biosynthesis of branched
polylactosaminoglycans in human as well as murine EC cells. Hence, it
is suggested that the linear polylactosamine backbones are probably
synthesized first and branched afterward in these cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells-- Mouse embryonal carcinoma cells of line PC13 established from the pluripotent OTT6050 teratocarcinoma tumor (16) were obtained from Dr. C. F. Graham (Department of Zoology, University of Oxford, UK). The human PA1 teratocarcinoma-derived cells (17) were obtained from Dr. Jorma Wartiovaara (Institute of Biotechnology, University of Helsinki, Finland). The cells were maintained in Eagle's minimum essential medium supplemented with 10% fetal calf serum as described (18). For the experiments, the cells were detached from the dishes with 0.02% EDTA in NaCl-P buffer (140 mM NaCl, 10 mM sodium phosphate, pH 7.2) and washed twice in Dulbecco's phosphate-buffered saline, pH 7.2-7.4 (with Ca2+ and Mg2+).
Preparation of Cell Lysates-- Washed human (PA1) and mouse (PC13) EC cell pellets (50-150 µl) were lysed with 200 µl of 0.9% NaCl, 1% TX-100, 1 mM phenylmethylsulfonyl fluoride. In some experiments, more concentrated cell lysates were prepared by suspending the cell pellets in 50 µl of 1.8% NaCl, 2% TX-100, 2 mM phenylmethylsulfonyl fluoride with the cells. Small amounts (0.6 µl) of 40 mM phenylmethylsulfonyl fluoride in ethanol were added to the mixture once each h to a final concentration of 1 mM. In some experiments aprotinin and leupeptin were also added to the lysis buffer to a final concentration of 17 µg/ml and 20 µg/ml, respectively. The lysed cells were kept at 0 °C and homogenized by 5 × 3 strokes in a Potter homogenizer. The lysates were used immediately as the enzyme source in glycosyltransferase reactions.
Acceptor Oligosaccharides--
The acceptor oligosaccharides
(for the structures see also Table I) were synthesized as described:
LacNAc1-3[14C]Gal
1-4GlcNAc (1) (9),
unlabeled 1 (19); two isotopomers of hexasaccharide
3 (LacNAc
1-3[3H]Gal
1-4GlcNAc
1-3'LacNAc and
LacNAc
1-3'LacNAc
1-3[14C]Gal
1-4GlcNAc) (10);
[14C]GlcNAc
1-3'LacNAc (7) (7). A
(1:1)-mixture of [3H]heptasaccharides 4 and
5 was synthesized from [3H]Gal
1-4GlcNAc
1-3'LacNAc
1-3'LacNAc
(3) as described in Leppänen et al.
(10).
Marker Oligosaccharides--
The following radiolabeled marker
oligosaccharides were synthesized as described: 7,
8, and GlcNAc1-6'LacNAc (7); LacNAc
1-3Gal (9);
2 (19); LacNAc
1-3'(LacNAc
1-6')LacNAc (13); a
mixture of the heptasaccharides
[3H]Gal
1-4GlcNAc
1-3LacNAc
1-3'(GlcNAc
1-6')LacNAc
(4) + [3H]Gal
1-4GlcNAc
1-3'(GlcNAc
1-6')LacNAc
1-3'LacNAc
(5) and the octasaccharide
LacNAc
1-3'(GlcNAc
1-6')LacNAc
1-3'(GlcNAc
1-6')LacNAc (10).
[3H]Gal
1-4GlcNAc
1-3'(GlcNAc
1-6')LacNAc
1-3Gal
was obtained by endo-
-galactosidase cleavage of authentic
appropriately radiolabeled heptasaccharide 5 (10). The
octasaccharide 6 (RMP = 0.29, RMH = 0.63; solvent A) was synthesized by
1,4-galactosylating the
hexasaccharide GlcNAc
1-3'(GlcNAc
1-6')LacNAc
1-3'LacNAc
(8).
Glycosyltansferase Reactions--
The cIGnT6 reactions were
performed by incubating the acceptors (3 pmol-100 nmol) and 3.7 µmol
of UDP-GlcNAc with 25 µl of the EC cell lysate for 4 h and in
some cases for 21-23 h in a total volume of 25 µl of 50 mM Tris-HCl buffer, pH 7.5, 8 mM
NaN3, 20 mM EDTA, 0.5 mM ATP, 20 mM D-galactose, 60 mM
-galactonolactone, and 100 mM GlcNAc. EDTA inhibited the
serum GnT3 activity (20), D-galactose and
-galactonolactone were added to inhibit
-galactosidase activity,
and GlcNAc was used to inhibit
-N-acetylhexosaminidase activity. The
dIGnT6 reactions with the teratocarcinoma cell lysates were carried out
essentially as described for hog gastric mucosal microsomes (7), but
incubation times of 4 h were used, and the total volume of the
reaction mixture was 25 µl. All IGnT6 reaction mixtures were passed
through a mixed bed of Dowex AG1 (AcO
) and Dowex AG 50 (H+), and the eluates were lyophilized.
Chromatographic Methods-- Paper chromatographic runs of desalted radiolabeled saccharides were performed on Whatman III Chr paper with the upper phase of 1-butanol/acetic acid/water (4:1:5 v/v; solvent A) or with 1-butanol/ethanol/water (10:1:2 v/v, solvent B). Radioactivity on the chromatograms was monitored as in Leppänen et al. (10) using Optiscint (Wallac, Turku, Finland) as scintillant. Marker lanes of malto-oligosaccharides on both sides of the sample lanes were stained with silver nitrate.
Gel permeation chromatography on a column of Superdex 75 HR 10/30 or Superdex Peptide HR 10/30 (Amersham Pharmacia Biotech) was performed as in Niemelä et al. (19).Degradative Experiments--
Digestions with
endo--galactosidase from Bacteroides fragilis (EC
3.2.1.103) (Boehringer Mannheim) were performed according to
Leppänen et al. (9); parallel control reactions
cleaved over 90% of radiolabeled GlcNAc
1-3Gal
1-4GlcNAc.
1H NMR Experiments-- The 1H NMR experiments were carried out as in Niemelä et al. (19).
Matrix-assisted Laser Desorption/Ionization Mass Spectrometry-- Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed in the positive-ion delayed-extraction mode with a BIFLEXTM mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) using a 337-nm nitrogen laser. 1 µl of sample (10 pmol) and 1.5 µl of 2,5-dihydroxybenzoic acid matrix (10 mg/ml in water) were mixed on the target plate and dried with a gentle stream of air. Dextran standard 5000 from Leuconostoc mesentroides (Fluka Chemica-Biochemica) was used for external calibration.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The structures of key oligosaccharides of the present experiments are shown in Table I and are identified in the text by using appropriate bold face digits.
|
Branching Reactions of Tetrasaccharide 1,
Catalyzed by Lysates of PA1 and PC13 Cells, Gave Pentasaccharide
2--
Incubation of tetrasaccharide
Gal1-4GlcNAc
1-3[14C]Gal
1-4GlcNAc
(1) and UDP-GlcNAc with lysates of human embryonal carcinoma
cells (line PA1) gave an oligosaccharide product that chromatographed
on paper like authentic pentasaccharide 2 marker (peak 1 in
Fig. 1A). Yields of 3-7%
were obtained. The identity of the product as glycan 2 was
established by enzymatic degradation. First, a treatment of the
pentasaccharide with
-galactosidase gave a product
co-chromatographing with authentic GlcNAc
1-3(GlcNAc
1-6)[14C]Gal
1-4GlcNAc
(8) (Fig. 1B). Next, the putative glycan 8 was identified by a partial treatment with
-N-acetylhexosaminidase that gave the trisaccharides
GlcNAc
1-6[14C]Gal
1-4GlcNAc and
GlcNAc
1-3[14C]Gal
1-4GlcNAc as well as the
disaccharide [14C]Gal
1-4GlcNAc (Fig. 1C).
In addition to degradation, the original pentasaccharide product was
subjected to MALDI-TOF mass spectrometry that gave a major signal at
m/z 974.8 (Fig. 1D), assigned to the sodiated molecular ion of Gal2GlcNAc3
(calculated m/z = 974.9). Finally, the
identity of the pentasaccharide product generated by PA1 cell lysates
was confirmed by the 1H NMR spectrum (Fig. 1E,
Table II); the resonances of the
structural reporter groups were practically identical with those of
authentic glycan 2 (23). Some of these resonances probably
would have been different if the GlcNAc branch had been transferred to
C-2 or C-4 of the central galactose unit of glycan 1 (24).
|
|
|
Branching Reactions of Hexasaccharide 3, Catalyzed by
PA1 Cell Lysates Gave Heptasaccharide Isomers 4 and
5--
Two isotopomers of glycan 3 (LacNAc1-3'LacNAc
1-3[14C]Gal
1-4GlcNAc
and LacNAc
1-3[3H]Gal
1-4GlcNAc
1-3'LacNAc)
were synthesized and were separately incubated with PA1 cell
lysates and UDP-GlcNAc to establish whether both the galactose 2 (labeled in the [14C]-acceptor) and the galactose 4 (labeled in the [3H]-acceptor) of glycan 3 serve as independent acceptor sites. A heptasaccharide-like product was
formed from both acceptors. The product chromatographed as a single
peak (peak 1 in Fig.
3A, RMP = 0.49, RMH = 1.04, solvent A), showing the same migration rate as
an unresolved mixture of authentic heptasaccharides 4 and
5 (10). The net yield of the heptasaccharide-like fraction
varied from 3 to 7% in 4-h incubations in several separate experiments; it was not improved in 22-h incubations. To ensure that
peak 1/Fig. 3A represented an authentic product resulting from the transfer of GlcNAc to radiolabeled acceptor 3, an
aliquot of the material was treated with UDP-Gal and GalT4. This
treatment gave 62% [3H]glycans chromatographing like
authentic octasaccharide 6 (Fig. 3B,
RMP = 0.32, RMH = 0.68, solvent A),
establishing the presence of a distal GlcNAc residue in most of the
glycans of peak 1/Fig. 3A. In addition, the data
of Fig. 3B suggest that the glycans of peak
1/Fig. 3A also included the
[3H]hexasaccharide 3 acceptor itself, which
contaminated the heptasaccharide products because of obvious
chromatographic "tailing." Other experiments that are described
below show that the distal GlcNAc units of the heptasaccharide products
of the branching reaction of glycan 3 were
1,6-bonded in
some molecules to the galactose 2 and in others to the galactose 4 of
the acceptor.
|
The Branching Reaction of the Mixed Heptasaccharides 4 and 5, Catalyzed by PA1 Cell Lysates, Gave an
Octasaccharide--
Incubation of an authentic 1:1 mixture of
[3H]-labeled heptasaccharides 4 and
5 with UDP-GlcNAc and PA1 cell lysates gave small amounts
(2.4-4.4%) of a product that chromatographed on paper like the doubly
branched octasaccharide
LacNAc1-3'(GlcNAc
1-6')LacNAc
1-3'(GlcNAc
1-6')LacNAc (not shown; RMP = 0.74, RMH = 1.10). In
the MALDI-TOF mass spectrum the product gave a sodium-containing
molecular ion at m/z 1543.4 (calculated
m/z for Gal3HexNAc5
1543.4). Together with the (M + K)+ signal, this ion
represented 70% of the total polylactosamines in the molecular ion
range (26). A treatment of the octasaccharide concentrate with GalT4
and UDP-Gal gave a crude decasaccharide. In MALDI-TOF mass spectrum of
this product, a major signal at m/z 1867.8 was
observed that was assigned to (M + Na)+ of
Gal5HexNAc5 (calculated
m/z 1867.7). These data suggest that the
octasaccharide formed from the mixture of the heptasaccharides 4 and 5 by the PA1 cell lysate was
[3H]LacNAc
1-3'(GlcNAc
1-6')LacNAc
1-3'(GlcNAc
1-6')LacNAc.
Human (PA1) and Mouse (PC13) Embryonal Carcinoma Cells Did Not
Reveal the Presence of the dIGnT6 Activity--
When the trisaccharide
[14C]GlcNAc1-3Gal
1-4GlcNAc (7) (21 pmol) was incubated with UDP-GlcNAc and human or mouse EC cell lysates,
chromatographically detectable tetrasaccharide-like products were
formed in less than 0.3% yield (not shown), implying that human and
mouse EC cells did not express significant dIGnT6 activities.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present data show that lysates from human embryonal carcinoma
cells of line PA1 contained centrally acting
1,6-N-acetylglucosaminyltransferase activity, which
catalyzed the formation of the branched pentasaccharide 2 from the linear tetrasaccharide 1 in the presence of
exogenous UDP-GlcNAc (For the structures of the saccharides, see Table
I). In addition, heptasaccharides 4 and 5 were
formed from the linear hexasaccharide 3. Evidence was
also provided supporting the notion that a second
1,6-GlcNAc branch
was transferred during incubation of a mixture of the heptasaccharides 4 and 5 with PA1 cell lysates and UDP-GlcNAc. We call the activity responsible for these reactions as cIGnT6 to emphasize the site-specificity of the reaction in the
central area of the acceptor and the formation of precursors
of the blood group I antigen.
Because PA1 cells are known to express branched polylactosamine backbones (11, 27), it is reasonable to assume that the in vitro reactions described in the present experiments are similar to those responsible for the synthesis of the multiply branched polylactosamines in vivo.
Also lysates from murine embryonal carcinoma cells of line PC13 contained cIGnT6 activity. The PC13 cells are also known to express large amounts of branched polylactosamines (15). Hence, the cIGnT6 reactions observed in vitro in the present experiments are likely to occur also in vivo during the synthesis of PC13 glycans.
The action of dIGnT6, too, leads in vitro to the formation
of branched polylactosamines (14), and this enzyme has been suggested to be responsible for the in vivo biosynthesis of branched
polylactosamine backbones (5, 11, 12). However, in the present
experiments we were unable to observe any dIGnT6 activity in lysates of
PA1 cells or PC13 cells that would have converted the linear
trisaccharide GlcNAc1-3Gal
1-4GlcNAc into the branched
tetrasaccharide GlcNAc
1-3(GlcNAc
1-6)Gal
1-4GlcNAc. Taken
together, our present data imply that the cIGnT6 activity rather than
the dIGnT6 activity may be responsible for the in vivo
synthesis of branched polylactosamine backbones in embryonal carcinoma
cells.
The dominant role of cIGnT6 in the branch generation, combined with the data showing that the branches of glycans in PA1 cells are short along the entire backbone chain (11), suggests that the biosynthesis of branched polylactosamine backbones in PA1 cells occurs in rather distinct stages as shown in Scheme 2: First, alternating action of GnT3 and GalT4 elongates the linear backbone chains to their final size. Second, the linear backbones are branched by cIGnT6 at different sites along the chains. Third, the GlcNAc branches are finally galactosylated by GalT4. A process of this kind is likely to produce rather similar branches along the entire primary backbone chain. By contrast, participation of dIGnT6 in the branching process would generate branches in association with chain elongation, probably leading to more complex branches in the proximal than the distal parts of the mature backbones.
|
The concept that linear polylactosamine chains are precursors of the branched backbones is not new. The relationship was proposed already in 1979 when the developmentally regulated expression of small i (linear chains) and big I (branched backbones) as blood group antigens in human and bovine red blood cells was described (28, 29). The present data merely provide the underlying mechanism of the interconversion in EC cells. Scheme 2 suggests also that cIGnT6 is localized in the Golgi compartment of PA1 cells in a more restricted manner than Gal T4 and more distally than GnT3.
The presence of the cIGnT6 activity in the murine EC cell lysates suggests that the polylactosamine backbones of these cells may also consist of primary linear chains that carry short branches. Such arrays may be important scaffolds for "presenting" the binding epitopes of cell adhesion saccharides in multivalent, high affinity modes. This notion is supported by the finding that functionally active sperm receptor saccharides are successfully assembled to ZP3 protein of murine zona pellucida in "appropriately" transfected murine embryonal carcinoma cells but not in a number of other cells similarly transfected (30); the failing cells probably did not express sufficient amounts of enzymes required for synthesis of branched polylactosamines. Recently, a polylactosamine backbone decorated by several sialyl Lewis X-bearing branches has actually proven to be a highly potent antagonist of lymphocyte L-selectin (31).
We note that the cDNA directing the expression of branched polylactosamines has already been isolated from the cDNA expression library from PA1 cells (32). This cDNA probably codes the cIGnT6 observed in the present experiments.
![]() |
FOOTNOTES |
---|
* This work was supported by Academy of Finland Grants 38042 and 40901 and Technology Development Center of Finland Grant TEKES 40057/97.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.
¶ To whom correspondence should be addressed. Tel.: 358-9-708 59375; Fax: 358-9-708 59563; E-mail: ossi.renkonen{at}helsinki.fi.
1
The abbreviations used are: GnT3,
1,3-N-acetylglucosaminyltransferase (GlcNAc to Gal);
GalT4,
1,4-galactosyltransferase (Gal to GlcNAc); cIGnT6, centrally
acting
1,6-N-acetylglucosaminyltransferase (GlcNAc to
Gal); dIGnT6, distally acting
1,6-N-acetylglucosaminyltransferase (GlcNAc to Gal); EC,
embryonal carcinoma; Gal (or G), D-galactose; GlcNAc (or
Gn), N-acetyl-D-glucosamine; Lac, lactose;
LacNAc, N-acetyllactosamine (Gal
1-4GlcNAc); MALDI-TOF
mass spectroscopy, matrix-assisted laser desorption-ionization mass
spectrometry with time-of-flight detection; MH, maltoheptaose
[Glc
1-4(Glc
1-4)5Glc]; MP, maltopentaose
[Glc
1-4(Glc
1-4)3Glc]; MT, maltotriose
(Glc
1-4Glc
1-4Glc); MTet, maltotetraose
[Glc
1-4(Glc
1-4)2Glc].
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|