From the
The accompanying article (Manzi, A., Salimath, P. V., Spiro, R.
C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163) reported the complete structure of a novel
[Abstract/Full Text]
molecule made by human melanoma cells incubated with 1 m
M
4-methylumbelliferyl-
In the previous paper
(1) we showed that human melanoma
cells make a novel pentasaccharide when incubated with
Xyl
Materials C-18 cartridges were from Analtech, Inc. The Helix pomatia agglutinin-agarose beads were from EY Laboratories. Bovine
testicular
We have shown that a novel structure can be made on different
The
The reason for the preferential synthesis of the sialylated molecule
is unclear, but we speculate that the co-localization of the first
galactosyl (GalT I) and a sialyltransferases accounts for these
results. Several studies have suggested that some galactosyl and
sialyltransferases are co-localized in the same Golgi compartments
(21, 22, 23, 24) , and we recently found
that the first GAG-core galactosyltransferase is substantially
co-localized with an
The addition of the
The significance of
Effects of various enzyme digestions on the binding of anionic
[
Each
cell line was incubated for 6 h in 1 m
M Xyl
Each of
the cell lines were incubated for 6 h with 1 m
M Xyl
Each sample described in Table
III with one negative charge was digested with a single or combination
of enzymatic digestions to determine the terminal sugar residue(s). The
amount neutralized by each digestion is expressed as a percentage of
total with one charge. The size of the neutralized product was
determined by amine adsorption chromatography on an AX-5 column as
described under ``Experimental Procedures.''
Xyl (Xyl
MU). The product resembles a
common pentasaccharide core region found in chondroitin/dermatan
sulfate glycosaminoglycans, except that a terminal
-GalNAc residue
is found in a location normally occupied by
-GalNAc in these
chains or
-GlcNAc in heparan sulfate chains. In this paper we show
that several other human cancer cell lines and Chinese hamster ovary
cells also make
-GalNAc-capped xylosides. The
[6-
H]galactose-labeled Xyl
MU product binds
to immobilized
-GalNAc-specific lectin from Helix pomatia and the binding is competed by GalNAc, but not by Glc. Binding to
the lectin is destroyed by digestion with
- N-acetylgalactosaminidase, but not
-hexosaminidase.
The nature of the aglycone influences the amount and relative
proportion of this material made, with
p-nitrophenyl-
-xyloside being a better promoter of
-GalNAc-terminated product than Xyl
MU. This novel
oligosaccharide accounts for 45-65% of xyloside-based products
made by both human melanoma and Chinese hamster ovary cells when they
are incubated with 30 µ
M Xyl
MU, but at 1 m
M
both the total amount and the proportion decreases to only 5-10%.
In both cell lines this product is replaced by a corresponding amount
of Sia
2,3Gal
4Xyl
MU. Preferential synthesis of the
-GalNAc-capped material at very low xyloside concentration argues
that it is a normal biosynthetic product and not an experimental
artifact. This pentasaccharide may be a previously unrecognized
intermediate in glycosaminoglycan chain biosynthesis. Since this
-GalNAc residue occurs at a position that determines whether
chondroitin or heparan chains are added to the acceptor, it may
influence the timing, type, and extent of further chain elongation.
MU.
(
)
This novel structure contains a
terminal
-GalNAc residue at the nonreducing end of an otherwise
typical GAG core tetrasaccharide. In typical GAG core structures, this
position is normally occupied by
-GalNAc in chondroitin or
dermatan sulfate, or
-GlcNAc in heparin or heparan sulfate
(2) . However, terminal
-GalNAc residues have been reported
to occur in blood group A-containing oligosaccharides and in some
glycolipids
(3, 4) . Since several studies have shown
that
-xylosides can serve as acceptors for the synthesis of small
oligosaccharides which are not part of the well established GAG chain
core
(5, 6, 7) , an important question is
whether the
-GalNAc-terminated structure is an artifact of the
incubation. Here we present evidence that several types of cells can
synthesize this molecule when incubated with Xyl
MU. Significantly,
the
-GalNAc-terminated pentasaccharide is the preferred product
when cells are incubated with very low concentration of acceptor. This
result argues that it is a natural product rather than an experimental
artifact.
-glucuronidase was kindly provided by Dr. Philip Stahl,
Washington University School of Medicine, St. Louis, MO.
Arthrobacter ureafaciens sialidase, chicken liver
-galactosidase,
- N-acetylgalactosaminidase, and jack
bean
-hexosaminidase were all from Oxford Glycosystems. Human
placental
-hexosaminidase A was gift from Dr. Don Mahuran,
Hospital for Sick Children, Toronto. Human melanoma cells were provided
by Dr. J. M. Trent, University of Michigan, Ann Arbor, MI. Human
leukemia cell line HL60, and monoblast line U937 were supplied by Dr.
Minoru Fukuda, La Jolla Cancer Research Foundation. Chinese hamster
ovary cells were provided by Dr. Jeff Esko, University of Alabama,
Birmingham, and the neuroblastoma cell line IMR32 was obtained from
American Type Culture Collection. Tissue culture medium was purchased
from Life Technologies, Inc. and [6-
H]galactose
(20 Ci/mmol) was purchased from American Radiochemical, St. Louis, MO. Methods
Labeling of Cells with Xyl
Confluent cells were washed twice with PBS and
incubated in XylMU and
Xyl
pNP
MU or Xyl
pNP at 1 m
M in serum-free
Dulbecco's modified Eagle's medium containing 0.1 mg/ml
glucose. [
H]Gal was added (100 µCi to UACC;
10 µCi to IMR) and incubated at 37 °C for 6 h. At the end of
the incubation period, medium was centrifuged to remove debris and
passed over a 400-mg C-18 SPICE cartridge equilibrated in 0.15
M NaCl. It was washed 5 times with 1.5 ml of 1
M NaCl
and then 1
1.5 ml with water to remove the salt. Inclusion of
NaCl during the wash improves binding of the xyloside-based products
which are then eluted with 5
1.5 ml of 40% MeOH.
QAE-Sephadex Chromatography
2-cm columns of
QAE-Sephadex in 2 m
M Tris base were prepared in glass
wool-plugged Pasteur pipettes and the sample applied in 1.5 ml of
water. The column was washed with 4 1.5 ml of water; 4
1.5 ml of 100 m
M NaCl in 2 m
M Tris base; and, a 2
1.5-ml wash of 1000 m
M NaCl.
C-18 Cartridge Chromatography
Samples from the
cell labelings were purified on C-18 SPICE as described above and the
fractions for analysis were eluted in 40% MeOH. Cartridges were reused
many times and regenerated with 10-20 ml of 100% MeOH followed by
20 ml of HO. HPLC Analysis
Anion Exchange Chromatography
Anionic
oligosaccharides (about 1000 cpm) were analyzed on an AX-5 column
(Varian Instruments) starting with water for 5 min, followed by a
linear gradient of 10-50 m
M
NaHPO
, pH 4.3, for 30 min, and a 5-min wash in
100 m
M Na
HPO
. 1 ml/min fractions were
collected and counted
(6) .
Size Analysis of Neutral Oligosaccharides
Neutral
oligosaccharides were analyzed by amine adsorption on a Varian AX-5
anion exchange column using a linear gradient of 80-65%
acetonitrile in water at a flow rate at 1 ml/min. 0-5-min
fractions were collected and counted
(6) .
H. pomatia Lectin Affinity
Chromatography
H. pomatia agglutinin linked to
agarose beads was obtained from EY Laboratories and packed into a 0.5
7-cm column and equilibrated with PBS, pH 7.2. Samples of 1000
cpm in 0.1 ml of PBS were mixed with internal marker of
[
S]SO4, applied to the column, and washed
immediately with PBS. Approximately 30-40 5-drop fractions were
collected. This was followed by elution with PBS containing 50
m
M GalNAc which did not elute any additional material.
Recovery was always >90%. Protein estimations were by the method of
Lowry
(8) . Enzyme Digestions For all enzyme digestions the samples were dried and dissolved in 50
µl of buffer, incubated at 37 °C overnight, and analyzed
directly on QAE-Sephadex or C-18 cartridges. Samples for HPLC analysis
were diluted with an equal volume of water, heated at 100 °C for
2-3 min, and the insoluble material removed by centrifugation in
a microcentrifuge for 1 min at top speed. Digestions with individual or
combinations of enzymes were as follows:
-glucuronidase (0.3 unit)
in 100 m
M sodium acetate, pH 5.5; sialidase (8 milliunits) in
100 m
M sodium acetate, pH 6.0, containing 4 m
M
calcium acetate; jack bean
-hexosaminidase (50 milliunits) in 50
m
M sodium formate, pH 5.0; human placental
-hexosaminidase A (2 units) in 25 m
M sodium formate
buffer, pH 4.5;
- N-acetylgalactosaminidase (4 milliunits)
in 100 m
M sodium citrate-phosphate buffer, pH 4.0 (supplied by
Oxford Glycosystems); chicken liver
-galactosidase in 100
m
M sodium citrate-phosphate, pH 4.0, containing 1 mg/ml bovine
serum albumin and 0.15
M NaCl (buffer supplied by Oxford
Glycosystems for
-galactosidase); sialidase and
-glucuronidase, 100 m
M sodium acetate, pH 6.0, containing
4 m
M calcium acetate;
- N-acetylgalactosaminidase
and
-glucuronidase, 100 m
M sodium citrate-phosphate
buffer, pH 4.0;
- N-acetylgalactosaminidase,
-glucuronidase, and
-galactosidase, 100 m
M sodium
citrate-phosphate buffer, pH 4.0, containing 1 mg/ml bovine serum
albumin. Measurement of [
H]Galactos in GAG
Chains-[
H]Gal-labeled GAG chains made
on Xyl
MU after 6 h was measured by precipitation with
cetylpyridinium chloride
(9) .
Digestion with
Since the amount
of the purified material described in the previous article was small,
we again turned to the analysis of the same
[6--GalNAcase
H]Gal-labeled material that was used as a
tracer in the accompanying article
(1) . Based on the previous
results, this labeled material consisted of approximately 65%
-GalNAc-terminated core structure and about 35% of several other
partially characterized products. Using the labeled material focuses on
galactose-containing xylosides and excludes the others. The results in
Table I show that digestion with
-glucuronidase alone or in
combination with
-hexosaminidase did not convert any of the
labeled anionic product into a neutral species. This confirms that
[6-
H]galactose preferentially labels the
galactose-containing species. Digestion with
-GalNAcase followed
by
-glucuronidase is the only treatment that neutralized
65-70% of the material. Longer digestion times or increased
amounts of enzyme did not change this amount. The neutralized material
was recovered and analyzed by amine adsorption HPLC; it co-eluted with
a standard of Gal
3Gal
4Xyl
MU showing that it had the
predicted core structure (data not shown). These results show that the
majority of the radiolabeled material prepared from these cells had the
same structure as the major component analyzed in the previous paper
(1) . The structure of the remaining anionic material is under
investigation.
Binding to H. pomatia Agglutinin
Column
H. pomatia agglutinin is a lectin that
binds terminal -GalNAc residues and also recognizes the Tn antigen
(GalNAc-
-Ser/Thr) that initiates O-GalNAc linked
oligosaccharides
(10) . To determine whether the
-GalNAc-terminated molecule would bind to this lectin, an aliquot
of the [
H]galactose-labeled material was applied
to a column of immobilized H. pomatia agglutinin. To find the
elution position of unbound molecules, an internal marker of
SO
was added to each sample before applying it
to the column. Fig. 1 shows that about 40% of
[
H]galactose-labeled material eluted exactly with
SO
, while the remainder bound to the column
and eluted by continued buffer wash in the absence of any competing
saccharide (GalNAc). Recovery was 90-95%. The broad elution
profile is typical of molecules that interact weakly with commercial
immobilized lectins. To be certain that the samples ran true, they were
pooled separately and rerun over the same column again. The bound
material completely rebound and the unbound material ran through the
column (data not shown). Digestion of the total material with chicken
liver
-GalNAcase or inclusion of 50 m
M GalNAc at the
beginning of the run eliminates binding, and all of the label runs in
the void volume, exactly co-incident with
SO
.
In contrast, inclusion of 50 m
M Glc does not prevent binding
of the labeled material to the column (see Fig. 1).
Figure 1:H.
pomatia agglutinin affinity chromatography of sialidase and
glucuronidase-resistant anionic [H]Gal-labeled
Xyl
MU products. Anionic [
H]Gal-labeled
xylosides which were resistant to sialidase and glucuronidase
digestions were passed over a column of immobilized H.
pomatia agglutinin which binds oligosaccharides with terminal
GalNAc residues as described under ``Experimental
Procedures.'' Panel A, sample applied directly to the
column and eluted with buffer (
), or following digestion with
GalNAcase (
). Panel B, a similar aliquot is added
to a column pre-equilibrated in 50 m
M Glc (
), or 50
m
M GalNAc (
). In each case the arrowhead ()
marks the elution position of an internal standard of
SO
which marks the void volume of the column.
Occurrence of
We previously showed that melanoma, Chinese hamster
ovary cells (CHO), and U937 cells make an unusual sialylated xyloside,
Sia-GalNAc Capped
-Xylosides in
Other Cells
3Gal
4Xyl
MU
(6) . These cells and several other
cell types also made anionic xyloside products that were resistant to
sialidase and
-glucuronidase digestions. Considering the results
shown in the companion study
(1) , we re-examined the resistant
products secreted in the presence of 1 m
M Xyl
MU. In each
case, an aliquot was digested with an individual glycosidase or a
combination of glycosidases and then analyzed by QAE-Sephadex ion
exchange chromatography to determine the amount converted into a
neutral product. Their size was then determined by HPLC. Table II shows
the results of these digestions and the likely structure based on that
of the core saccharide. It is clear that the relative proportions of
the products varies with the cell type. Human neuroblastoma (IMR-32)
and human leukemia cells (HL-60) are rich sources of
-GalNAc-capped material, while CHO and the human monocyte cell
line (U937) appear to make relatively little. It was surprising that
none of the cell lines made any labeled material that was neutralized
by sequential
-hexosaminidase and
-glucuronidase digestions,
even though a structure with a terminal
-GalNAc residue is
expected to be one of the intermediates in chondroitin sulfate chain
synthesis. The reason for this is not known, but the most likely
explanation is that the addition of
-GalNAc residues leads to
rapid formation of highly charged chondroitin sulfate chains that do
not bind to C-18 cartridges and are discarded during preparation (data
not shown).
Effects of the Aglycone Structure on the Synthesis of
Most studies with -GalNAc-capped Xyloside
-xylosides
use a variety of aglycone ( e.g. MU and pNP) derivatives
interchangeably, since they generally have the same effects on GAG
chain biosynthesis
(11, 12, 13, 14) .
However, recent evidence clearly shows that the structure of the
aglycone and its concentration substantially influence the proportion
of chondroitin sulfate or heparan sulfate chains made on these
acceptors
(14, 15) . To determine whether the aglycone
effects the relative amount of
-GalNAc-capped xylosides, both
melanoma and neuroblastoma cells were incubated with 1 m
M MU
or pNP-xyloside and labeled with [
H]Gal. The low
molecular weight secreted products were purified on C-18 cartridges and
the results are shown in Table III. Xyl
MU is a better acceptor
than Xyl
pNP and both cell lines show a characteristic distribution
of neutral and anionic products. Table IV shows the results of
digesting the xylosides having one negative charge with various
exoglycosidases. Both cells make a greater proportion of
-GalNAc-capped material using Xyl
pNP as an acceptor compared
to Xyl
MU. In keeping with this finding, the proportion of total
anionic labeled pNP product from neuroblastoma cells which binds to the
H. pomatia agglutinin column is correspondingly
increased, and
-GalNAcase treatment eliminates this binding (shown
in Fig. 2).
Xyloside Concentration Determines the Proportion of
In previous studies we found that
the proportion of Sia-GalNAc-capped Material
3Gal
4Xyl
MU made by melanoma and CHO
cells varied with the concentration of xyloside
(6) . At low
concentration (<0.1 m
M), the amount was very small
(
10% of total) while at 1-2 m
M it became the
predominant product (60-65%). Free GAG chains usually accounted
for 10% of the label found in the low molecular weight xyloside
products. Other cell lines, such as the neuroblastoma cell line
(IMR-32) made almost no sialylated material using 1 m
M
xyloside. At the time of this study, the other anionic products could
not be analyzed; however, the present results prompted a
reinvestigation. As shown in Fig. 3, GAG chains account for 5-10%
of the total incorporation. The total amount of
-xyloside product
is nearly maximal by 0.3 m
M in melanoma cells, but continues
to increase up to 1 m
M in CHO cells. However, in both cell
lines, the proportions of the various products change. The
-GalNAc-capped molecule is the major product (45-65%) made
by both melanoma and CHO cells, but only at low (0.03 m
M)
xyloside concentration. At higher concentrations, the proportion
decreases to 5-10%, and is replaced by the sialylated molecule
which can account for 70% of the products. The absolute amount of
-GalNAc capped material decreases also. The basis for this change
is not clear, but in rat liver Golgi the first galactosyltransferase
(GalT I) is co-localized with an
2,3-sialyltransferase but not
with the second galactosyltransferase (GalT II)
(16) . Perhaps
at high ``effective concentration'' of Xyl
MU the
co-localized GalT I and sialyltransferase consume the Xyl
MU before
it ever reaches the compartment containing the next enzyme(s) in the
GAG-core biosynthesis. Whatever the reason may be for the changes in
amounts of different products, the predominant synthesis of the
GalNAc-capped pentasaccharide at low acceptor concentration
strongly suggests that it is probably a naturally occurring structure,
rather than an artifact of incubation with xylosides.
-xylosides by human melanoma, leukemia, monoblast, neuroblastoma,
and Chinese hamster ovary cells. The structure is identical to the
typical carbohydrate core linkage glycan except for the presence of a
terminal
GalNAc residue. Thus, this modification is probably
common to many types of cells. In addition, a search of the CarbBank
data base showed there are no other known carbohydrate structures that
contain a GalNAc
-GlcA
- terminal disaccharide. In preliminary
experiments, we found that microsomes from melanoma cells could
catalyze the addition of
-linked [
H]GalNAc
from UDP-[
H]GalNAc to GlcA
MU, and this
material was sensitive to
- N-acetylgalactosaminidase
digestion.
(
)
Further work is needed to define
this enzymatic activity.
-GalNAc-terminated structure has
not been found in naturally occurring GAG chains. All studies to date
show the presence of either
-GalNAc or
-GlcNAc at this
position. Perhaps the core resembles another naturally occurring
molecule and becomes an alternate, but inappropriate acceptor of the
-GalNAc residue. Sia
3Gal
4Xyl
-MU
(6) and
Glc-Glc-Xyl
-MU
(5) , and Xyl
4Xyl
MU and
GlcA
Xyl
MU
(7) and those reported in the companion
article
(1) seem to be unrelated to the known biosynthetic
pathway of GAG core synthesis. However, preferential synthesis of
GalNAc-terminated product at low xyloside concentration
(0.03-0.1 m
M) argues that its occurrence is not an
artifact. These concentrations of
-xylosides typically give nearly
maximum inhibition of proteoglycan synthesis
(11, 13, 14, 17, 18, 19, 20) .
2,3-sialyltransferase in rat liver Golgi
(16) .
-GalNAc residue occurs at a
critical location in the assembly of GAG chains. Even though
chondroitin sulfate and heparan sulfate have the same core saccharide
structure terminating in GlcA, the next sugar to be added is
-GalNAc in chondroitin/dermatan sulfate chains or
-GlcNAc in
heparan sulfate chains. All of the factors that control the type of
chains added to the common core are not known; however, it is clear
that the amino acid sequence of the core protein (or structure of the
aglycone in
-xyloside acceptors) influences the decision
(25) . Also, the core saccharide region of some chondroitin
sulfate molecules are modified by phosphorylation of xylose residues
and sulfation of one or both galactose residues or the first
-GalNAc residue
(26, 27, 28) . Heparan
sulfate core structures do not appear to have these modifications
(29) , suggesting that they may serve as a signal and influence
the type of chain added. In at least one instance, the enzymes that
polymerize and modify chondroitin sulfate are segregated from those
used in heparan sulfate biosynthesis
(30) , so the reactions may
not be strictly competitive.
-GalNAc
residue at this crucial location is unknown. It could be a stable
intermediate in more complex pathway or a transient intermediate that
is later replaced by the typical
-GalNAc or
-GlcNAc residues
in chondroitin sulfate or heparan sulfate, respectively. If
-GalNAc halts further elongation of the GAG chains, this could be
involved in the synthesis of part-time proteoglycans where only a
portion of the core proteins contain GAG chains
(31, 32) . This will require identification of such an
oligosaccharide on a protein.
Table: QAE-Sephadex chromatography of
anionic [H]Gal xyloside
H]Gal-labeled xyloside products to QAE-Sephadex
were done using aliquots (1000 cpm) of sialidase and
-glucuronidase-resistant material was digested with the indicated
glycosidase(s) and then analyzed on QAE-Sephadex to determine the
percentage neutralized.
Table: Composition of anionic -xyloside products secreted by various cells
MU and 10
µCi/ml of [6-
H]galactose and the anionic
xyloside products were purified and then digested with either
sialidase,
-glucuronidase alone,
-glucuronidase plus
-hexosaminidase, or
- N-acetylgalactosaminidase plus
-glucuronidase as described under ``Experimental
Procedures.'' The digest was fractionated on QAE-Sephadex to
measure the percentage neutralized by the digestion. The size of the
neutralized product was determined as described under
``Experimental Procedures.''
Table: Distribution of C-18 cartridge bound
xyloside products from melanoma and neuroblastoma cells
MU or
Xyl
pNP as described under ``Experimental Procedures.''
The secreted xyloside products were fractionated on QAE-Sephadex into
neutral molecules, those with 1 negative charge, and those with at
least 2 negative charges. The results are expressed as
H
cpm x 10
/100 µg of protein. Numbers in
parentheses are the % of cpm in each fraction.
Table: Analysis of xyloside
products with 1charge from human
melanoma and neuroblastoma cells
MU,
4-methylumbelliferyl-
-xyloside; GAG, glycosaminoglycan; MU,
4-methylumbelliferyl; Xyl
pNP,
p-nitrophenyl-
-xyloside; CHO, Chinese hamster ovary; PBS,
phosphate-buffered saline; HPLC, high performance liquid
chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.