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INTRODUCTION |
Heparan sulfate proteoglycans
(HSPGs)1 are critically
involved in a wide variety of biological phenomena including
organogenesis, angiogenesis, regulation of blood coagulation, growth
factor/cytokine action, cell adhesion, lipid metabolism, and wound
healing (1-4). All these activities are elicited primarily through the
heparan sulfate (HS) chains at the cell surface. The high degree of
structural diversity observed in these HS chains is believed to impart
the specificity of function of these macromolecules. Such diversity of
structure is imposed by tightly regulated patterns of sulfation and
epimerization upon the basic polysaccharide backbone during synthesis
within the Golgi (2, 5). Highly regulated HS synthesis permits HSPGs to
accomplish their distinct biological roles, such as basic FGF binding
and activation, which are mediated through specific sequences within
the chains (6) and which can be markedly modulated by the physiological
degradation of HS in vivo (7). Recently, HSPGs have been
shown to regulate signaling by many diverse growth factors, including
FGFs, members of the TGF-
superfamily, Wg/Wnt,
heparin-binding-EGF, and hepatocyte growth factor through HS
(reviewed in Refs. 4, 8, and 9). Thus, the HS chains are believed to be
the principal determinants of the binding and regulatory activity of
HSPGs on the external surface of the plasma membrane.
Syndecans are a major family of four kinds of transmembrane HSPGs (4,
10-13). The mammalian syndecans are similar in primary sequence in the
cytoplasmic and membrane-spanning regions, but the extracellular
domains (ectodomains) are largely non-homologous. According to the
chromosomal localization, exon organization, and sequence relationships
with Drosophila syndecan, the syndecan gene is thought to
have arisen by gene duplication and divergent evolution from a single
ancestral gene and that syndecan-1 and -3 and syndecan-2 and -4 comprise separate subfamilies (14). These members of the syndecan
family are expressed in distinct cell-, tissue-, and developmental
stage-specific patterns (15, 16). It suggests that each syndecan family
member may have distinct functions (17), although some shared
activities of, for example syndecan-1 and -4, have been observed (4,
11, 18, 19). Thus far, evidence from knock-out mice indicates that no
critical step in development depends on a specific syndecan gene.
Syndecan-1 and -4 promote intercellular adhesion following their
transfection into human B lymphoid cells, suggesting that both of them
are important mediators of this event (20). Both syndecan-1 and -4 are
induced during wound repair, but their cellular localization is
different (21), suggesting that although both are involved in the wound
healing process their functions may be dissimilar. Kinnunen et
al. describe a unique role for syndecan-3 in the binding and
activity of heparin-binding growth-associated molecule at E11 in the
rat rhomboencephalon during neurite outgrowth (22), while syndecan-4
has a distinctive role in the generation and maintenance of focal
adhesion complexes (23, 24) and in signal transmission during dendritic
process formation (25). The ectodomains of all syndecans may be shed
intact by proteolytic cleavage of the core protein in a site adjacent
to the plasma membrane. Proteolytic activity causes the release of
syndecan-1 and -4 ectodomains in acute human dermal wound fluids (26)
where they modify the protease/antiprotease balance (27). Recently, it
was shown that the shedding of syndecan-1 and -4 ectodomains is
regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive cell surface metalloproteinase (28). Although it is
well documented that these molecules may display sometimes similar and
sometimes very different biological activities, it is unclear whether
it is the protein core component or the GAG chains of these
proteoglycans that are the major determinants of differences in
biological activity between syndecan family members. Comparative
structural and functional analyses of the HS chains on syndecan-1 and
-4 have not been undertaken to date. In the present study we describe
such analyses on HS chains that were prepared simultaneously from the
same cell type, namely NMuMG (normal murine mammary gland epithelium),
and show that HS of syndecan-1 and -4 have highly similar, fine
structural profiles and ligand-binding activities. These data provide
further insights into the regulation of HS biosynthesis within a
particular cell population. The findings indicate that heparan sulfate
structure, although highly dependent upon cell type, appears to display
very little variability within the syndecan population of an individual cell type (in this instance NMuMG), an observation that may have notable implications in the understanding of the biological roles of
glycosaminoglycan chains.
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EXPERIMENTAL PROCEDURES |
Materials--
Na235SO4
(carrier-free, -specific activity, 1200-1400 Ci/mmol) was obtained
from PerkinElmer Life Sciences. Heparinase I (Flavobacterium heparinum, heparinase, heparin lyase, EC 4.2.2.7), heparinase II
(F. heparinum; no EC number assigned), and heparinase
III/heparitinase (F. heparinum; heparitin-sulfate
lyase, EC 4.2.2.8) were purchased from Grampian Enzymes (Aberdeen, UK).
Chondroitin ABC lyase (Proteus vulgaris; EC 4.2.2.4) was obtained from
Seikagaku Kogyo Co. (Tokyo, Japan). Sodium chloride (HiPerSolv grade)
for HPLC and all other analytical grade reagents were supplied from
BDH-Merck Ltd. (Lutterworth, Leics, UK). Ultra-pure water (resistance
18 megohms) for all HPLC analysis was dispensed from a Milli-Q
water system (Millipore, UK). Bio-Gel P-6 (fine grade) was from Bio-Rad (Hemel Hempstead, Hertfordshire, UK). ProPac PA-1 analytical columns were purchased from Dionex (Camberley, Surrey, UK). Sepharose CL-6B was
obtained from Pharmacia Biotech (Uppsala, Sweden). Low-melting point
agarose (SeaPlaque) and Gelbond were from FMC Corp. Bioproducts (Rockland, ME). Type I collagen from rat tail was from BD Biosciences. FGF-2 was from Intergen Co. (Purchase, NY). Antibodies specific to
syndecan ectodomains included monoclonal antibodies 281-2 against the
mouse syndecan-1 ectodomain (29), KY/8.2 against the mouse syndecan-4
ectodomain (25), and polyclonal antisera MSE-4 against the recombinant
syndecan-4 ectodomain (17). Rat monoclonal antibody 281-2 directed
against the mouse syndecan-1 core protein was conjugated to
CNBr-activated Sepharose 4B and used as described previously (15). Rat
monoclonal antibody KY/8.2 against the mouse syndecan-4 conjugated
Sepharose 4B was prepared and used. The 281-2 antibody was from
PharMingen (San Diego, CA), and the KY/8.2 monoclonal was kindly
supplied by Dr. P. W. Kincade (Oklahoma Medical Research Foundation, Oklahoma City, OK).
Cell Culture--
NMuMG cells were cultured as described
previously (30). Early passage (20) of NMuMG cells was used for all the
experiments. To purify labeled syndecan-1 and -4, the NMuMG cells were
labeled by 35S in conditions as described previously
(15).
Immunohistochemical Staining--
Cultured NMuMG cells were
stained by 281-2 for syndecan-1 and by MSE-4 (affinity purified rabbit
polyclonal antibody against mouse syndecan-4) or KY/8.2 for syndecan-4.
We used Cy3-conjugated donkey anti-rat antibody (minimal cross-react to
rabbit; Jackson ImmunoResearch, West Grove, PA) or fluorescein
isothiocyanate-conjugated donkey anti-rabbit antibody (minimal
cross-react to rat; Jackson ImmunoResearch, West Grove, PA) as a second
antibody on commercially recommended conditions. Immunolabeled cells
were observed using a fluorescence microscope (Olympus, Tokyo, Japan).
Photographs were taken using Kodak Tri-X pan film (Eastman Kodak Co.,
Rochester, NY). Immunohistochemical staining was performed on 10 differently prepared cells and gave reproducible results. A control for
nonspecific staining omitted the primary antibody. No staining was
observed in control samples.
Preparation of Cell Surface Syndecan-1 and -4--
To prepare
the extracellular domains of syndecan-1 and -4, the conditioned media
of NMuMG was used (15). After anion exchange chromatography, the
samples were applied to density gradient centrifugation, and the
fractions whose specific gravity were more than 1.35 g/ml were
collected. Syndecan-1 and -4 were purified by using 281-2 and KY/8.2
affinity columns, respectively. To purify the labeled syndecan-1 and
-4, mildly treated trypsinate was also used (15). For the
following HS structural and affinity co-electrophoresis analyses, the
labeled samples were digested by chondroitin ABC lyase before the
affinity chromatography.
Size Determination of the HS Chains of Syndecan-1 and
-4--
Proteoglycans were dissolved in 500 µl of distilled water
containing 0.1% (w/v) CHAPS (Sigma) and then subjected to eliminative cleavage and reduction by adjusting to 100 mM NaOH/1
M NaBH4 for 24 h at 37 °C. After
neutralizing samples by dropwise addition of 1 M HCl, free
HS chains were recovered by separation on a Sepharose CL-6B column
(1 × 97 cm) eluting in phosphate-buffered saline at a flow-rate
of 10 ml/h and collecting 1-ml fractions. Radioactivity in each
fraction was determined by liquid scintillation counting. The void
volume (Vo, fraction 25) and total volume
(Vt, fraction 63) of the column were established
using Dextran Blue and sodium dichromate, respectively.
Disaccharide Compositional Analysis of HS Chains--
Aliquots
of 7000 cpm (35S) each of syndecan-1 and -4 HS chains were
adjusted to 50 mM sodium acetate/0.5 mM calcium
acetate, pH 7.0, before addition of 20 mIU/ml each of heparinase
I, II and III in a total volume of 100 µl or less. Samples were
incubated for 4 h at 25 °C followed by a second addition of the
same amount of enzymes and incubation for a further 24 h at
25 °C. Each digest was then diluted to 1 ml with distilled water
adjusted to pH 3.5 by addition of HCl before application to a ProPac
PA-1 strong-anion exchange (SAX) column (4.6 mm × 25 cm, Dionex
UK) attached to a gradient HPLC pump (Dionex, Surrey, UK).
Disaccharides were eluted in a gradient of 0-1.0 M NaCl,
pH 3.5, over 45 min at a flow rate of 1 ml/min, collecting 0.5-ml
fractions (31). Radioactivity in each fraction was determined by liquid
scintillation counting, and disaccharides were identified by comparison
with unlabeled disaccharide standards of known composition that were
monitored by on-line UV absorption at A232 nm.
Recoveries of radio-labeled sample were typically between 85-95%.
Very little radioactivity was detected after elution of the most
sulfated disaccharide, indicating almost complete depolymerization of
GAG chains.
Analysis of Heparinase I, Heparinase III, and Nitrous
Acid-derived Oligosaccharides--
Aliquots of 7000cpm
(35S) each of syndecan-1 or -4 in distilled water
containing 0.1% CHAPS were adjusted to 50 mM sodium
acetate/0.5 mM calcium acetate, pH 7.0 before addition of
40 mIU/ml of either heparinase I or heparinase III
(heparitinase) and incubation at 25 °C for 24 h. Additionally,
similar aliquots were subjected to low pH nitrous acid cleavage (32)
for 20 min, followed by neutralization by dropwise addition of 2 M sodium carbonate. The resulting oligosaccharide digestion
products from the above treatments were then resolved by size exclusion
chromatography on either a Bio-Gel P-6 column (1 × 106 cm, eluted
at 3 ml/h in 0.1 M ammonium hydrogen carbonate) or on a
Sepharose CL-6B column (as described above), collecting 1 ml fractions.
Peaks of radioactivity were determined by liquid scintillation
counting. Additional digests, containing 13,000 cpm each of syndecan-1
and syndecan-4, were undertaken in which the resulting oligosaccharides
were then analyzed on a ProPac PA-1 SAX-HPLC column eluted with a
gradient of 0-1.2 M NaCl, pH 3.5, over 90 min at a
flow-rate of 1 ml/min and collecting 0.5-ml fractions for liquid
scintillation counting. Recoveries for the above were typically above
85% of added material.
Affinity Co-electrophoresis (ACE) Analysis--
NMuMG cells were
labeled with radio-sulfate, and the [35S]sulfate-labeled
syndecan-1 and -4 ectodomain from conditioned medium was purified as
described above. HS chains from syndecan-1 and -4 were released by
incubation with 0.5 M NaOH and 1 M
NaBH4 for 16 h at 4 °C. Binding of each ectodomain
or HS chains to type I collagen and FGF-2 was assessed by ACE as
described elsewhere (15, 27, 33). Briefly, 1% (w/v) low-melt agarose
gels were cast containing distinct lanes with various concentrations of type I collagen (0, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, and 5000 nM, assuming that the molecular mass of type I collagen is
300 kDa) or FGF-2 (0, 1, 2, 4, 8, 16, 32, 64, and 128 nM). [35S]sulfate-labeled syndecan-1 and -4 (12,500 cpm) was
electrophoresed through these lanes. The migration on syndecan-1 and -4 was detected on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
The pixel intensities were integrated and used to determine the
migration distance of the major peak of 35S-labeled
syndecan-1 and -4 in each lane. These mobilities were plotted as a
function of ligand concentration and used to estimate the apparent
Kd values as described earlier (33).
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RESULTS |
Simultaneously Expressed Syndecan-1 and -4 on the NMuMG
Cells--
Histochemical analysis was performed on the cultured NMuMG
cells to investigate the simultaneous expressions of syndecan-1 and -4. For this purpose, we used 281-2 monoclonal and MSE-4 polyclonal antibodies to detect syndecan-1 and -4, respectively (Fig.
1). Syndecan-1 was expressed at the cell
surfaces of NMuMG cells uniformly as shown previously (34, 35), while
the focal expression of syndecan-4 was seen at the extracellular matrix
regions as previously shown by Woods and Couchman (23). Syndecan-1
expressed rather strongly along the cell-cell interaction regions,
whereas the syndecan-4 was expressed faintly. No signals were detected
on the cells stained by second antibodies alone (data not shown). We
also performed the immunohistochemical staining of syndecan-4 on the
NMuMG cells using KY/8.2 monoclonal antibody that we used for the
purification of syndecan-4 for the following analyses. KY/8.2
monoclonal antibody also showed the focal expression of syndecan-4.
These results indicate that syndecan-1 and -4 are co-expressed in
NMuMG, albeit with spatially different distributions. Therefore,
metabolically labeled syndecan-1 and -4 were purified from these cells
for HS structural analyses.

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Fig. 1.
Expressing pattern of syndecan-1 and -4 on
the identical NMuMG cells. The expressions of syndecan-1 and -4 on
the same NMuMG cells were examined comparatively by the double
immunofluorescent staining with 281-2 monoclonal and MSE-4 polyclonal
antibodies, respectively. Similarly prepared NMuMG cells were also used
to stain syndecan-4 by KY/8.2 monoclonal antibody. Syndecan-1 and -4 were simultaneously expressed on the same NMuMG cells at the same time.
Syndecan-1 is uniformly expressed at the cell surfaces of NMuMG cells,
while syndecan-4 shows focal expression by both MSE-4 and KY/8.2
monoclonal antibodies. No signals were detected on the cells stained by
the second antibody alone (data not shown).
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Alkali/Borohydride Eliminative Cleavage of HS on
Syndecan-1 and -4--
NMuMG cells that were taken at 50% confluence
were metabolically radiolabeled for 48 h with
35SO4. Radiolabeled syndecan-1 and -4 were
purified from the radiolabeled conditioned medium and cells as
described under "Experimental Procedures." HS chains from
syndecan-1 and -4 were produced by alkali borohydride eliminative
cleavage from the respective proteoglycans and then applied to
Sepharose CL-6B column to compare the relative sizes of the HS chains
(Fig. 2). The resulting HS chains for
both syndecan-1 and -4 HS displayed a unimodal distribution with a Kav of 0.35, which corresponds to a molecular
mass of ~40 kilodaltons (according to the calibration of Ref.
36). No detectable difference in average chain length or distribution,
therefore, could be established between the HS derived from syndecan-1
compared with syndecan-4 by this method. The void volume
(Vo, fraction 25) and total volume
(Vt, fraction 63) of the column were established using Dextran Blue and sodium dichromate, respectively.

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Fig. 2.
Sepharose CL-6B gel filtration chromatograph
of syndecan-1 and syndecan-4 after alkali/borohydride treatment.
The partially purified cell surface proteoglycan fraction from
35S-radiolabeled NMuMG cells was subjected to chondroitin
ABC lyase to remove chondroitin sulfate chains, and the syndecan-1 and
-4 were isolated by 281-2 and KY/8.2 immunoaffinity column
chromatographies, respectively. HS chains from syndecan-1 and -4 were
produced by alkali/borohydride eliminative cleavage of the respective
proteoglycans and then applied to a Sepharose CL-6B column to compare
the relative sizes of the HS chains. There is no major size difference
on the HS chain sizes of syndecan-1 and -4.
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Low pH Nitrous Acid Scission of HS on Syndecan-1 and
-4--
The HS chains from both syndecans were individually
subjected to low pH nitrous acid degradation, which results in specific scission at N-sulfated disaccharides with the concomitant
loss of the N-sulfate groups (32). In these
35S-labeled HS preparations, wherever N-sulfated
disaccharides are situated consecutively within the chain, nitrous acid
digestion will result in the liberation of disaccharides and free
[35S]sulfate. When N-sulfated disaccharides
are interspersed by disaccharides containing
N-acetylated glucosamine residues (which are resistant to
attack by nitrous acid) fragments larger than disaccharides will
be generated, but with a 35S-label these will be visible
only if they contain additional O-sulfates. The size of
resistant oligosaccharides will be a reflection of the number of
consecutive N-acetylated disaccharides possessed by that
oligosaccharide. Fig. 3 indicates that
there is a close association between N- and
O-sulfation, which is a characteristic feature of
HS. Approximately 89 and 90% of the radiolabel from syndecan-1 and -4, respectively, is represented in the above peak. The
remainder of the sulfates, i.e. O-sulfates are
located in alternating sequences, represented by peaks of radioactivity
corresponding to tetrasaccharides, and containing 11 and 10% of the
total incorporated [35S]sulfate for syndecans-1 and -4, respectively. Larger, sulfated oligosaccharides are not observed,
demonstrating that in both HS species, there is a strong linkage
between N- and O-sulfates, such that
O-sulfates do not appear to occur more than one disaccharide away from an N-sulfate.

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Fig. 3.
Bio-Gel P-6 column chromatography of
syndecan-1 and -4 after scission with nitrous acid.
35S-radiolabeled HS chains were treated with low pH nitrous
acid and the resulting oligosaccharide product mixture separated on a
Bio-Gel P-6 column as described under "Experimental Procedures."
The column void volume (Vo) and total volume
(Vt) were determined using hemoglobin and sodium
dichromate, respectively. (dp, degree of
polymerization.)
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Heparinase III Scission--
Heparinase III (heparitinase) can
cleave only at unsulfated hexuronic acid (HexUA) (glucuronic acid
(GlcUA) or iduronic acid (IdoUA)) residues, especially if there are
relatively few SO4 groups on the adjacent residues.
Heparinase III largely cleaves HS at hexosaminidic linkages to
glucuronic acid residues (GlcN(NS or NAc, ±6S)-GlcUA) (37-39),
although the enzyme also shows activity against some glucosaminidic
linkages to iduronic acid (40) but with reduced efficiency. The samples
of digested syndecan-1 and -4 were subjected to heparinase III
digestion and then applied to a Bio-Gel P-6 chromatography to assess
the distribution and ratios of the resulting oligosaccharides. Fig.
4 illustrates the profiles of heparinase
III-resistant fragments derived from both syndecan-1 and -4 heparan
sulfates. This approach revealed no significant structural differences
between the two HS species under investigation, with both
displaying a similar pattern of resistant fragments ranging in size
from disaccharide (dp2) to around tetradecasaccharide (dp14) in very
similar proportions (Table I). Minor
variations in the proportional spread of radiolabel among the resistant
fragments of this digest may reflect incomplete enzymic scission at a
small proportion of its potential cleavage sites. The small peals of
radioactivity between fractions 84 and 85 arose due to partial
resolution of the disaccharides into lower and higher sulfated
constituents. The contents of 2-O-sulfation, 6-O-sulfation and N-sulfation in the labeled
syndecan-1 and -4 samples are ~54, 46-51, and 89% of the total
sulfated disaccharide population, respectively (see Table
II). Regions of the chains resistant to
heparitinase scission represent the higher sulfated domains (S-domains)
of HS.

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Fig. 4.
Bio-Gel P-6 column chromatography of
syndecan-1 and -4 after depolymerization with heparinase III.
Aliquots of each of the syndecan HS chains were subjected to heparinase
III digestion and then applied to a Bio-Gel P-6 column as described
under "Experimental Procedures" to assess the distribution and
ratios of the resulting oligosaccharides. Vo and
Vt were determined using hemoglobin and sodium
dichromate, respectively. The number associated with each peak
corresponds to the degree of polymerization of oligosaccharides
(e.g. dp 2, disaccharides).
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Table I
Distribution of 35S label within oligosaccharides produced by
Heparinase III digestion of syndecan-1 and -4 HS chains
Conditions for digestion are described under "Experimental
Procedures." Values represent the proportion of the total
radioactivity associated with each individual oligosaccharide size.
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Table II
Comparative disaccharide compositions of 35S-radiolabeled HS
samples on syndecan-1 and -4
35S-radiolabeled HS samples were degraded by combined
heparinase I, II, and III digestion, and the resulting disaccharides
were analyzed by HPLC anion-exchange chromatography as described under
"Experimental Procedures." Values represent the proportion of the
total disaccharides produced by combined enzymic digestion, and
depolymerization was greater than 95% complete. ND, not detected.
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The oligosaccharides generated by this enzyme were also subjected to
SAX-HPLC (Fig. 5) and similarly displayed
only minor differences between the two species under investigation.
Both the pattern and proportion of resulting oligosaccharides were similar for both syndecans, indicating a comparable distribution of
glucuronate residues within the two HS species. The small differences between profiles may be due to incomplete cleavage of a minority of the
potential susceptible linkages within the chains.

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Fig. 5.
Strong anion-exchange HPLC analysis of the
oligosaccharides of syndecan-1 and -4 after depolymerization with
heparinase III. Aliquots of syndecan-1 and -4 HS chains were
subjected to heparinase III (heparitinase) digestion, and the resulting
total oligosaccharide mix for each digest was separated by HPLC on a
ProPac PA-1 column as described under "Experimental Procedures."
The products of each digest are represented by peaks of radioactivity,
and the profiles compared with establish whether the same or different
oligosaccharides were the major products from the different HS species.
No standards are available for this procedure. The dotted
line represents the NaCl gradient conditions.
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Heparinase I Scission--
To gain further information on the
structure of HS chains, the enzyme heparinase I was used. This enzyme
cleaves HS essentially where GlcNS(±6S)-IdoUA(2S) residues occur (37,
38), although the enzyme is active against
glucuronate-2-O-sulfate (41), a rare constituent of HS (42,
43). This enzyme generated very similar amounts of di-, tetra-, hexa-,
and octasaccharide in both syndecan-1 and -4 HS (Fig.
6). Furthermore, the resistant sulfated fragments resulting from heparinase I digestion were the same average
size (Kav of 0.73 (7 kDa), which corresponds to
an average size of around 14-16 disaccharides), and had the same size
distribution when the total digest was analyzed by Sepharose CL-6B size
exclusion chromatography (Fig. 7).

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Fig. 6.
Bio-Gel P-6 column chromatography of HS
chains on syndecan-1 and -4 after depolymerization with heparinase
I. Aliquots of each of the syndecan chains were subjected to
heparinase I digestion and then applied to a Bio-Gel P-6 column to
assess the distribution and ratios of the resulting oligosaccharides.
The syndecan-1 and -4 HS gave very similar yields of low molecular
weight oligosaccharides. The number associated with each peak
corresponds to the degree of polymerization of oligosaccharides
(e.g. dp 2, disaccharides). Vo and
Vt were determined using hemoglobin and sodium
dichromate, respectively.
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Fig. 7.
Sepharose CL-6B gel filtration chromatography
of HS chains on syndecan-1 and -4 after depolymerization with
heparinase I. 35S-radiolabeled HS chains of syndecan-1
and -4 were treated with heparinase I, and the products subjected to
Sepharose CL-6B column chromatography as described under
"Experimental Procedures." Vo and
Vt were determined using Dextran Blue and sodium
dichromate, respectively. The major resistant fragments are represented
by the major peaks of radioactivity having a maximum at around fraction
53.
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Total Disaccharide Composition--
35S-radiolabeled
HS chains from both syndecan-1 and -4 were completely degraded to
disaccharides by the combined actions of heparinases I, II, and III.
The disaccharides were separated by gradient SAX-HPLC, and the results
are shown graphically (Fig. 8) and
numerically (Table II). Disaccharides were identified by comparison of
their elution positions relative to those of known, unlabelled
disaccharide standards. It was not possible to determine the levels of
the non-sulfated disaccharide,
UA-GlcNAc as the material was
metabolically radiolabeled with 35S alone. The major
35S-radiolabeled disaccharides in both labeled syndecan-1
and -4 were
UA(2S)-GlcNS(6S) and
UA-GlcNS, and
UA(2S)-GlcNS
and
UA-GlcNAc(6S). The
UA(2S)-GlcNAc(6S) was not detected in
syndecan-1 and -4. The data indicate no major differences between the
two types of HS chain at the gross level of analysis. The overall
levels of both N- and 2-O-sulfation (89.3/53.9%
and 88.9/54.0% for syndecan-1 and -4, respectively) of the two HS
types are in very good agreement with each other as are the total
sulfation levels (189.4% and 193.6% for syndecan-1 and -4, respectively). Similarly, the estimated levels of
N-sulfation for both syndecans by this enzymatic technique are in very close agreement with those established by the chemical method employing low pH nitrous acid (see above). A relatively small
difference in 6-O-sulfation of 4.5% was noted, though this is possibly within experimental variation for this system using this
quantity of radiolabeled material. These findings are compatible with
the same elution profiles of syndecan-1 and -4 treated with nitrous
acid, heparinase I, II, and III (Figs. 3-8).

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Fig. 8.
Strong anion-exchange HPLC analysis of
disaccharides derived from 35S-radiolabeled HS samples on
syndecan-1 and -4. HS chains from syndecan-1 and -4 were subjected
to complete depolymerization by heparinases I, II, and III as described
under "Experimental Procedures," and then disaccharides were
separated by HPLC on a ProPac PA1 column using a gradient of 0-1
M NaCl, pH 3.5, from 2.1 to 47.1 min, collecting 0.5-ml
fractions. Individual disaccharides were identified by reference to the
elution positions of known, unlabelled disaccharide standards.
UA-GlcNAc (1), UA-GlcNS (2),
UA-GlcNAc(6S) (3), UA(2S)-GlcNAc (4),
UA-GlcNS(6S) (5), UA(2S)- GlcNS (6),
UA(2S)-GlcNAc(6S) (7), and UA(2S)-GlcNS(6S)
(8). 2S, 2-O-sulfate;
6S, 6-O-sulfate; NS,
N-sulfate; UA, unsaturated hexuronic
acid; GlcNAc, N-acetyl-glucosamine;
GlcNAc(6S),
N-acetyl-6-O- sulfated glucosamine;
GlcNS, N-sulfated glucosamine;
GlcNS(6S),
N- sulfated-6-O-sulfated glucosamine.
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Summarizing the above, the following can be concluded about the two HS
species investigated in this study: 1) the chain lengths, the size
distribution of chains, and overall disaccharide compositions are
almost indistinguishable; 2) the distribution of N-sulfated residues within the chains are apparently the same; 3) the proportion and distribution of IdoUA(2S) and GlcUA residues within the chains are
almost identical. It can only be concluded that the two different syndecans (syndecan-1 and -4) produced by the same cell, although having very different core proteins, are decorated by substantially similar HS chains.
Syndecan-1 and -4 Show Same Specificity in Binding to
Type I Collagen and FGF-2--
To examine whether these two
proteoglycans, bearing essentially the same HS, bind type I collagen
and FGF-2 in a similar manner, metabolically labeled ectodomains or
alkali-released HS chains were purified from the culture medium of
NMuMG cells and subjected to ACE (33). Type I collagen and FGF-2 are
both known to bind syndecan-1 (15).
The ACE profiles with type I collagen and both syndecan-1 and -4 ectodomains showed similar binding activities (Figs.
9 and 10). Based on the median mobilities,
the apparent Kd values for the interaction of
syndecan-1 and -4 with type I collagen were 38 nM in both
instances, confirming previously determined values (15). Intact
syndecans bind type I collagen with a slightly higher affinity than the
isolated HS chains, presumably because of the multivalent nature of the
proteoglycan or possibly due to a small but significant contribution of
the protein core to the binding. The apparent Kd
values for the binding of isolated HS chains of both syndecans to type
I collagen were 175 nM.

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Fig. 9.
ACE of type I collagen with syndecan-1 and -4 from the same NMuMG cells. 35S-radiolabeled syndecans
or their isolated HS chains were electrophoresed through zone
containing various concentrations of type I collagen ligands embedded
in agarose gels as described under "Experimental Procedures." The
distribution of the radioactivity was visualized autoradiographically
by PhosphorImager analysis. The concentration of ligand is indicated in
each lane. PG, cell surface syndecan (proteoglycan form);
HS, isolated HS chains.
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Fig. 10.
Affinities for type I collagen or FGF-2
ligands of syndecan and its isolated HS chains. The binding of
35S-radiolabeled syndecan-1 and -4, or their isolated HS
chains to type I collagen (0-5000 nM) or FGF-2 (0-128
nM) was analyzed by ACE. Data are presented as the
distribution of pixel intensity from the PhosphorImager analysis ligand
concentrations near the apparent Kd. PG,
cell surface syndecan (proteoglycan form); HS, isolated HS
chains.
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The ACE profiles utilizing FGF-2 again revealed apparently identical
affinities for both syndecan-1 and -4 (Fig. 10). The calculated Kd values for intact syndecan-1 and -4 were both 28 nM, similar to values shown previously (15). Similar ACE
profiles were obtained with the isolated HS chains, and once more these Kd values were almost indistinguishable (56 and 52 nM for syndecan-1 and -4, respectively).
The agreement in the binding values for type I collagen and FGF-2 with
syndecan-1 and -4 support the hypothesis that, when prepared
simultaneously from NMuMG cells, these proteoglycans possess HS chains
that have almost identical structures. These data also indicate that
the binding of both of these proteoglycans to these protein ligands is
mediated primarily through their GAG chains with only a minor
contribution being made from their protein core components.
 |
DISCUSSION |
This paper describes detailed analyses of the structures and
ligand-binding properties of two related but functionally different members of the syndecan family, syndecan-1 and -4, derived from the
surfaces of identical cell types. Though related, these two proteoglycans and the other two members of the syndecan family display
dissimilar topological distribution and histotypic organization, and
although some of their biological activities overlap (for example
they are capable of activating bFGF in experimental systems), there are nonetheless some important functional differences between them (see the Introduction).
Studies to date have indicated that the fine structures of the HS
chains borne on syndecan-1 can vary considerably in their molecular
fine structures in an apparently cell type-specific manner (15, 44),
but prior to this study it was unclear whether different members of
this proteoglycan family, synthesized in the same cells, are modified
with structurally similar HS chains or with uniquely different ones.
The present study demonstrates that the HS moieties of syndecan-1 and
-4, synthesized simultaneously in NMuMG cells are, indeed, almost
indistinguishable at the gross and fine structural levels and with
respect to protein ligand binding.
The molecular mass of syndecan-1 may differ according to cell type
(NMuMG cell, NIH/3T3, BALB/3T3) due primarily to differences in the
length of its constituent HS chains as apposed to alterations in the
gross domain organization of the HS (15). We now reveal that syndecan-1
and -4 derived from the same NMuMG cells bear HS chains of very similar
molecular size (both ~40 kilodaltons as determined by CL-6B size
exclusion chromatography). Furthermore, the overall molecular
organization of the polysaccharides and their sulfation patterns are
remarkably similar as determined by comparison of the frequency and
disposition of N-sulfated glucosamine residues (Fig. 3
enzymic depolymerization profiles and Table I), disaccharide
compositions and O-sulfate ratios (Figs. 4-7 and Table I).
The syndecan core proteins share highly conserved transmembrane and
cytoplasmic domains, with the exception of the V-regions (10) and, as
illustrated by this study, essentially identical HS chains; therefore
the question arises as to how these proteoglycans exhibit their
biologically different functions. The V-regions of syndecans vary in
both length and amino acid sequence, such that, for instance,
syndecan-4 through binding and activation of protein
kinase-C
and phosphatidylinositol-4,5-diphosphate participates in the formation of focal adhesions, whereas that of
syndecan-2 being a substrate for a different array of kinases does not
(reviewed in Ref. 4). Another possibility derives from their
structurally divergent extracellular domains (ectodomains). Sequence
variability is greatest in these domains, with the exception of the
glycosaminoglycan attachment sites proximal to the N terminus, which
are relatively well conserved. The calculated Mr
values for the core proteins of syndecan-1 and -4, for example, are
quite different at 30.6 and 19.5 kDa, respectively. The syndecan-4
ectodomain core protein has a high affinity binding site for an unknown
ligand on the surfaces of several human and mouse cell types, whereas the syndecan-1 core protein ectodomain shows only weak binding to the
surface of Swiss 3T3 cells (45). The syndecan-4 interaction with cell
surfaces shows specificity in that its ectodomain, but not that of any
other proteoglycan, can block this binding (46). There is also a
potential contribution of chondroitin sulfate (CS) chains to consider;
however, there is limited information about the role of CS chains on
syndecans-1 and -4, but by analogy with other proteoglycans they could
modify the protein binding characteristics of these macromolecules (47,
48).
Core protein synthesis is cell type-specific and developmentally
regulated (reviewed in Ref. 9). Contained within the sequences of these
proteins is the information needed to instruct cells to initiate GAG
chain synthesis upon them (either HS or CS/DS), but increasing evidence
suggests that their role stops there and that subsequent postpolymeric
modification is dictated by the Golgi apparatus, utilizing a particular
repertoire of biosynthetic enzymes possessed by that cell. This was
illustrated by studies on the matrix proteoglycan perlecan, in which
three proteoglycans from three different cellular sources were
investigated. Essentially the same protein core was shown to bear HS
chains that were not only structurally dissimilar but also varied in
their ability to modulate the biological activities of FGF-1 and FGF-2
(49). Similarly, during glial cell progenitor differentiation down to astrocytic or oligodenditic lineages, it was observed that a marked change from the expression of heparin to heparan sulfate occurred upon
the surface of these cells, although no concomitant alteration in the
core protein expression pattern was detectable (50). Additionally, two
unrelated HSPGs extracted from fibroblasts, namely syndecan-4 (membrane
intercalated) and glypican-1 (GPI anchored), were demonstrated to
possess HS chains with no major domain or fine structural differences
between them and almost inseparable affinities for the Hep-II domain of
the matrix component fibronectin (51). Thus, it would appear that HS
structure, per se does not appear to be dictated by the core
protein. Instead the available evidence supports the hypothesis that
the synthesis of HS chains and core proteins of individual HSPGs are
independently regulated in any particular cell type and possibly
tailored for the desired role(s) of that cell. Interestingly, various
cloned and non-cloned vascular endothelial cells, which form part of the non-thrombogenic surfaces of blood vessels, were all shown to
synthesize similar, albeit low, levels of anticoagulantly active heparan sulfate, whereas in the same study smooth muscle cells did not
(52).
The reproducibly high levels of structural similarity observed between
the HS chains of both syndecan-1 and -4 may suggest that not only the
cell type but also the cell status could affect the HS structures of
the cell surface of HSPGs. The composition of HS derived from the
culture medium, the cell surface, the intracellular, and nuclear pools
of a rat hepatoma cell line are clearly different (43, 53). Furthermore
the amount of heparan sulfate as well as its composition in these pools
changes when growing cells reach confluence (43). At the time of these
publications we had no information on the structure of HSPG core
proteins. A similar scenario was illustrated convincingly by Brickman
et al. (54), who isolated two separate pools of HS from
neuroepithelial cells derived from embryonic day 10 mice; one from
cells in log-phase growth, which greatly potentiated the activity of
FGF-2, and a second from contact-inhibited cells, which preferentially
activated FGF-1.
In the present study, we used two different in vitro assay
systems to evaluate the ligand-binding activities of syndecan-1 and -4. Although these limited assays may not reflect all biological activities
of HS chains on syndecans, it does suggest that these two HSPGs may
display the same activities in vivo. These results, however,
do mirror those outlined above in which HS derived simultaneously from
fibroblast syndecan-4 or glypican-1 displayed very similar affinities
for the Hep-II domain of fibronectin. Similarly, in a study into the
production of proteoglycans in L cells by Shworak et al.
(55), the investigators were unable to identify any major structural
differences between HS chains (in terms of sulfation/epimerization) produced in cultures overexpressing transfected syndecan-4 (Ryudocan) and those of control cultures. Additionally, the proportion of antithrombin III binding sites (which are uniquely specific for high
affinity binding to antithrombin III) in both of these HS species were
remarkably constant. The above article supports findings that the
production of anticoagulant HS does not require a unique protein core
(56).
One can only speculate as to the benefits of expressing cell surface
proteoglycans that all possess the same potential to bind a vast array
of ligands. In normal, undamaged tissues, these proteoglycans may not
necessarily encounter all of these ligands, but after perturbation of
the tissue, for instance during wounding, previously unseen soluble
effectors may need to be sequestered via HS chains to initiate the
correct repair process. Many of these soluble effectors are, indeed,
heparin-binding molecules (reviewed in Ref. 4).
In conclusion, the findings from the present study indicate that the HS
chains on syndecan-1 and -4 derived from the same NMuMG cells display
minimal detectable fine structural or domain structural differences.
The implication is that the HS chains on HSPGs synthesized
simultaneously by other cell types may also show this characteristic.
This apparent identity in structure leads one to speculate that protein
core-independent sulfation and epimerization of HS chains during
biosynthesis in a particular cell type may be determined not only by
the repertoire of HS synthetic enzymes possessed by that cell but also
by other factors, such as its growth status or developmental role.
Precisely how these factors dictate HS structure merits further investigation.