Heparan sulfate interacts with growth factors,
matrix components, effectors and modulators of enzymatic catalysis as
well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is
known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced
binding to isoforms of platelet-derived growth factor A and B chains
containing polybasic cell retention sequences. By contrast, the binding
to fibroblast growth factor 2 is affected to a much lesser extent. The
enhanced binding of aorta heparan sulfate to platelet-derived growth
factor is suggested to be due to an age-dependent increase
of GlcN 6-O-sulfation, resulting in increased abundance of
the trisulfated L-iduronic acid
(2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.
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INTRODUCTION |
Interactions of the sulfated glycosaminoglycan
(GAG)1 heparan sulfate (HS)
with various proteins affect the biological activity, tissue
localization, and turnover of the protein ligands (1-3). Such
interactions, generally electrostatic in nature, regularly involve
specific oligosaccharide domains generated by an elaborate biosynthetic
machinery in the Golgi apparatus. HS formation starts by assembly of an
initial (GlcA-GlcNAc)n polymer. Parts of the nascent polymer
are subsequently modified by
N-deacetylation/N-sulfation of GlcNAc residues,
and further modifications, including C-5 epimerization of GlcA residues
into IdceA residues as well as O-sulfation at various
positions, occur mainly in the vicinity of the previously incorporated
N-sulfate groups (3, 4). The O-sulfate groups are
predominantly found at the C-2 position of IdceA residues and the C-6
position of GlcN residues. The protein-binding HS domains typically
reside within the N-sulfated regions, their functional
specificity being determined by the pattern of modification, particularly the positioning of sulfate groups. Although information regarding the structures of the recognition sites for individual proteins (5-11) and the general features of HS biosynthesis (3) is
accumulating, the biological control of HS structure and function remains poorly understood. Nevertheless, HS species from various cells
and tissues clearly differ in their structure and in some studies such
differences have been correlated to differential protein-binding
properties (12-15). These and other findings (discussed in Refs. 1 and
3) suggest that the biosynthesis of HS is subject to regulation during
development or aging. Control of the appropriate expression of
functional HS domains in given organs or at particular developmental
stages would appear essential, whereas, conversely, perturbed
regulation could contribute to various pathologies. In the present
study we have explored the aging aortic wall as a model to gain insight
into the control of HS structure and function in humans. Aging is a
strong predisposing factor to atherosclerosis, characterized by
endothelial damage, lipid accumulation, and cell proliferation in
arterial wall plaques (16). HS has been attributed multiple roles in
these processes by interacting with lipoprotein lipase (17) and with
growth factors such as basic fibroblast growth factor (FGF-2) and
platelet-derived growth factors (PDGFs) (18-21). HS binds to and
enhances the mitogenic activity of FGF-2 (22) and regulates the tissue
localization of PDGFs (20). PDGFs are homo- or heterodimers of two
closely related, A and B, polypeptide chains. The PDGF-A chain exists as two variants due to alternative mRNA splicing. The longer
variant (PDGF-AL) contains a C-terminal polybasic sequence
that serves as a cell retention signal and is associated with the
localization of PDGF to the surface of the producer cell or to the
extracellular matrix (21), presumably due to interactions with HS (20). A similar but not identical retention signal is found at the C terminus
of the PDGF-B propeptide chain (PDGF-BL) (21). This propeptide may undergo various N- and C-terminal proteolytic processing events that give rise to multiple forms of cell-associated PDGF-B species (23).
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MATERIALS AND METHODS |
Isolation and Radiolabeling of Heparan Sulfate--
Tissue
samples from human abdominal aorta were obtained at autopsy and stored
at
70 °C until processed for HS isolation. The surrounding
connective tissue was removed, and the sample, encompassing the entire
thickness of the vessel wall, was cut into fine pieces with a scalpel.
The samples were defatted essentially as described (15) with the
exception that ethyl ether was replaced by ethanol. Defatted samples
(dry weight, 0.15-2.0 g) were subjected to protease digestion with
papain (Sigma; 5 mg/g of defatted tissue) in 25 ml of 0.05 M Tris, pH 5.5, 0.01 M EDTA, 2 M
NaCl, 0.01 M cysteine/HCl at 60 °C for 18 h and
centrifuged (10 min at 2000 × g), and the supernatants
were applied to columns of DEAE-Sephacel (1.5 × 5 cm; Amersham
Pharmacia Biotech) equilibrated with 0.05 M Tris, pH 7.2. The column was washed with 0.05 M sodium acetate, pH 4.0, followed by elution of the DEAE-bound material (containing sulfated
GAGs) by a linear gradient of LiCl (0.15-2.0 M) in the acetate buffer (15). 2-ml fractions were collected and analyzed for
uronic acid content by the carbazole reaction (24). Fractions containing GAGs were pooled, dialyzed against water, lyophilized, and
digested with 1 unit of chondroitinase ABC (Seikagaku Corporation) and
125 units of endonuclease (Benzonase, Benzon Pharma A/S) in 0.05 M Tris-HCl, pH 8.0, 1 mM MgCl2,
0.05 M sodium acetate at 37 °C overnight (25). The
digest was heated for 2 min and applied to a DEAE-Sephacel column
(1.5 × 5 cm) equilibrated with 0.2 M NH4HCO3 and eluted by a linear gradient of
NH4HCO3 (0.2-2.0 M). Fractions
containing HS were identified by the carbazole reaction, pooled, and
freeze-dried, and the material was stored at
20 °C until further
use. Treatment of the purified HS preparations with HNO2 at
pH 1.5 resulted in quantitative degradation of the material into lower
molecular weight species as demonstrated by chromatography of intact
and HNO2-treated samples on a column of Superose 12 (data
not shown), indicating that the purification procedure yielded pure
HS.
For radiolabeling, HS samples (80-150 µg) were
N-deacetylated in hydrazine hydrate (Fluka Chemie AG)
containing 30% water and 1% hydrazine sulfate (Merck) for 2-4 h at
100 °C (26), desalted, and reacetylated using
[3H]acetic anhydride (Amersham Pharmacia Biotech) as
described (15). The specific activity was calculated after measurement
of the uronic acid content of the [3H]HS samples by the
carbazole reaction.
Assay of Heparan Sulfate-Protein Interaction--
The binding of
[3H]HS preparations to recombinant PDGF-AAL
(27), PDGF-BBL (33 pmol/incubation) (prepared as described
for PDGF-AAL
(27)),2 and FGF-2 (29 pmol/incubation) (Pepro Tech EC) was studied by a nitrocelloluse filter
trapping assay as described previously (8, 11).
PDGF Affinity Chromatography--
5 mg of recombinant
PDGF-AAL was mixed with an equimolar amount of heparin and
immobilized to 3 ml of CH-Sepharose CL4B (Amersham Pharmacia Biotech)
according to the manufacturer's instructions (8). The column was
equilibrated with Tris-buffered saline (50 mM Tris-HCl pH
7.4, 150 mM NaCl) prior to the application of
[3H]HS. Subsequently, the column was washed with 10 ml of
Tris-buffered saline, and the bound material was eluted using a linear
NaCl gradient (0.15-2.0 M in 50 mM Tris-HCl,
pH 7.4). Fractions of 1 ml were collected and analyzed for
radioactivity in a liquid scintillation counter.
Structural Analysis of Heparan Sulfate--
Samples of HS (~30
µg) were subjected to cleavage with nitrous acid at pH 1.5 (28). The
cleavage products were end-labeled by reduction with 250 µCi of
NaB3H4 (Amersham Pharmacia Biotech) overnight
as described previously (4) and desalted on a Sephadex G-15 column
(1 × 190 cm, Amersham Pharmacia Biotech) in 0.2 M
NH4HCO3. Fractions containing disaccharides were analyzed by anion exchange HPLC on a Partisil-10 SAX column (4.6 × 250 mm, Whatman Inc.) (29).
To calculate the degree of N-sulfation of GlcN residues the
[3H]aManR end group-labeled HS
oligosaccharides resulting from the HNO2 pH
1.5/NaB3H4 treatment were separated by
chromatography on a column of Bio-Gel P-10 (1 × 160 cm, Bio-Rad)
in 0.5 M NH4HCO3. Fractions of 1 ml were collected and analyzed for radioactivity. The degree of
N-sulfation was calculated from the elution profiles using
the following formula.
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(Eq. 1)
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where n is the number of monosaccharide units and
a is the total radioactivity in a given oligosaccharide
species.
In order to isolate 3H-labeled tetrasaccharides, aliquots
of the HS oligosaccharides resulting from cleavage with
HNO2 at pH 1.5 were separated by gel chromatography on a
Superdex 30 (Amersham Pharmacia Biotech) column in 0.5 M
NH4HCO3. Fractions corresponding to
tetrasaccharides were pooled and desalted. Tetrasaccharide species were
separated by high voltage paper electrophoresis on Whatman number 3MM
paper in 6.5% HCOOH, pH 1.7.
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RESULTS |
We first examined the binding of radiolabeled human aorta HS from
a young and an old individual to FGF-2 and to dimers of the long
isoforms of PDGF A or B chains (designated PDGF-AAL and PDGF-BBL below) (30). A filter trapping procedure was
employed, involving interaction of labeled HS and the protein ligand in solution followed by rapid passage of the mixture through a
nitrocellulose filter. Protein and protein-bound HS chains (but not
free HS chains) were retained on the filter (8). We found that the
binding capacity for PDGF-AAL and PDGF-BBL of
HS from a 76-year-old individual was 4-5 times higher than that of HS
from a 21-year-old individual (Fig.
1A), whereas the binding to
FGF-2 differed only marginally between the two HS samples. To assess
the inter-individual variation in these interactions, we next compared
the binding of six HS preparations, from three young and three old
subjects, to PDGF and FGF-2. The results of this experiment (Fig.
1B) indicated a virtually invariable level of binding of HS
to a given protein ligand within each of the two age groups but marked
differences in binding characteristics between these groups. The effect
of age thus was selectively expressed for different proteins, in accord
with the data shown in Fig. 1A. These findings clearly point
to the occurrence of age-dependent differences in HS
structure that affect the binding of HS to PDGF and FGF-2 in distinct
ways. The effect of this structural transition with regard to PDGF
binding was further examined by affinity chromatography of HS from
young and old subjects on a column of immobilized PDGF-AAL
(Fig. 1C). Both types of HS contained material that remained
bound to the growth factor at physiological pH and ionic strength, as
well as a fraction of unbound material. The PDGF-binding chains
amounted to 50.0 ± 2.4% versus 81.8 ± 1.5%
(mean ± S.E. from three and two samples, respectively) of the
total HS from young and old subjects, respectively. These results thus
confirm not only the correlation between individual age and PDGF
binding ability of aortic HS but also the striking inter-individual
similarity within each age group.

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Fig. 1.
Effect of age on binding of HS from human
aorta to growth factors. A, PDGF-AAL,
PDGF-BBL, and FGF-2 were tested for binding to
[3H]HS isolated from a young (21 years) and an old (76 years) individual. Increasing amounts of [3H]HS (up to 19 pmol) were incubated with recombinant PDGF-AAL,
PDGF-BBL (33 pmol/incubation), and FGF-2 (29 pmol/incubation) as described under "Materials and Methods." The
data shown represent means of duplicate incubations. B,
samples of [3H]HS (7.5 pmol/incubation) from three young
(20, 21, and 22 years) and three old (70, 76, and 81 years) subjects
were tested for binding to PDGF-AAL, PDGF-BBL,
and FGF-2 (same amounts as in A). The binding of HS from old
subjects is set as 100%. The means ± S.D. are shown.
C, samples of [3H]HS from three subjects aged
20 (solid line), 21(dashed line), and 22 (dotted line) years (Young subjects) and two
subjects aged 76 (dashed line) and 80 years (solid
line) (Old subjects) were subjected to chromatography
on a column of PDGF-AAL- Sepharose as described under
"Materials and Methods." The arrow indicates the start
of the gradient.
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Binding of HS to PDGF-AAL and FGF-2 involves
structurally distinct oligosaccharide domains. The former domain is
comprised by N-sulfated ~8-mer sequences with at least one
trisulfated
IdceA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit (8), whereas the minimal FGF-2-binding site contains
an essential IdceA(2-OSO3) residue but no
6-O-sulfate groups (5, 7, 31). Given these data, we wanted
to examine whether the differential protein binding of aorta HS from
young and old subjects would correlate with the O-sulfate
substitution pattern of the N-sulfated regions of HS. We
therefore determined the disaccharide composition of these regions in a
total of 15 HS preparations from subjects aged 20-84 years (including
the six preparations used in the binding
experiments). The results of this
analysis (Table I) demonstrated an
age-dependent increase in the proportion of the
IdceA(2-OSO3)-GlcNSO3(6-OSO3) unit
(Fig. 2). Calculation of the overall
extent of 2-O- and 6-O-sulfation indicated that
this increase was due to an approximate doubling of the level of
6-O-sulfate substitution of GlcNSO3 residues in the old subjects (Fig. 2), whereas the IdceA 2-O-sulfation
remained high and essentially unchanged (Fig. 2). The
6-O-sulfation of GlcNSO3 residues showed an
almost linear increase between the age of 20 and 40 years; in the still
older subjects the levels of 6-O-sulfation were somewhat
scattered but consistently higher than in the young individuals (Fig.
2).
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Table I
Analysis of O-sulfated disaccharide species derived from the contiguous
N-sulfated regions of human aorta heparan sulfate
Samples of HS were treated with HNO2 at pH 1.5, resulting in
deaminative cleavage at GlcNSO3 residues. The cleavage products
were radiolabeled by reduction with NaB3H4.
Disaccharides were recovered by gel chromatography and analyzed by
anion exchange HPLC as described.
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Fig. 2.
Age-dependent changes in
O-sulfate substitution of human aorta HS. The
composition of O-sulfated disaccharide units within the
contiguous N-sulfated HS domains was determined in 15 HS
preparations (Table I) by anion exchange HPLC as shown in Fig. 2. The
panels show the proportion of the trisulfated disaccharide units
(IdceA(2-OSO3)-GlcNSO3(6-OSO3), as
present in the corresponding intact polysaccharides), and the
contribution of GlcN 6-O- and HexA 2-O-sulfate
groups to total O-sulfation.
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We further determined the degree of N-sulfate
substitution of GlcN residues from the pattern of HS depolymerization
following cleavage with HNO2 (at pH 1.5; see "Materials
and Methods"). Five of the HS samples (subject aged 20, 21, 74, 76, and 80 years) were analyzed (Fig. 3) and
found to contain essentially similar proportions of GlcNSO3
units (means ± S.D., 39 ± 2% of total GlcN units).

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Fig. 3.
Analysis of the extent of
N-sulfation of GlcN residues. Samples of aortic HS
from a young (20 years) and an old (76 years) subject were subjected to
cleavage with HNO2 at pH 1.5. The cleavage products were
radiolabeled by reduction with NaB3H4 and
separated by chromatography on Bio-Gel P-10 as described under
"Materials and Methods."
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Finally, we assessed the degree of sulfation of the sequences comprised
of alternating N-sulfated and N-acetylated
disaccharide units. These domains typically harbor GlcN
6-O-sulfate groups but few or no IdceA
2-O-sulfate groups (4) and are recovered as tetrasaccharides
after cleavage of HS with HNO2 at pH 1.5. Analysis of
tetrasaccharides from four HS samples (subject aged 20, 30, 50, and 80 years) indicated essentially similar proportions of nonsulfated,
monosulfated, and disulfated species (76 ± 3, 20 ± 3, and
4 ± 1% of all tetrasaccharides, respectively, expressed as
means ± S.D.). The increase in 6-O-sulfate
substitution of GlcN units in the old individuals thus is essentially
confined to the contiguous N-sulfated domains of HS.
The major alteration in human aorta HS in association with aging is
increased 6-O-sulfation of GlcNSO3 residues. The
6-O-sulfate groups were largely incorporated into
GlcNSO3 units linked at C-4 to IdceA(2-OSO3)
residues, leading to increased formation of the trisulfated
IdceA(2-OSO3)-GlcNSO3(6-OSO3)
disaccharide units. These results are in good agreement with the
protein binding properties of the polysaccharide because
IdceA(2-OSO3)-GlcNSO3(6-OSO3) units
have been implicated in the binding of HS to PDGF-AAL. By contrast, the expression of FGF-2-binding HS domains was not affected by aging, as might be expected because regardless of the subject age
the HS species were rich in IdceA(2-OSO3) residues and had proportions similar to those of N-sulfate groups.
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DISCUSSION |
The present study demonstrates that human aging is accompanied by
specific alterations in the fine structure of HS in the aortic wall.
The increased degree of 6-O-sulfate substitution of
GlcNSO3 residues in old subjects is reflected by an
elevated proportion of trisulfated, "heparin-type"
IdceA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide units and enhanced binding of the HS to PDGFs. Such progressive, age-dependent structural alteration represents
a previously unrecognized type of functional modulation of a mammalian macromolecule. Unlike rapid and reversible modifications such as
phosphorylation, the alterations in sulfation described here occur
during an extended time span of several decades. The remarkable structural and functional similarities of HS specimens from different age-matched subjects suggest a strictly controlled process of HS
assembly.
Our findings point to 6-O-sulfation of GlcNSO3
units as a key regulator of the biological properties of HS in the
human aortic wall. The 2-O- and 6-O-sulfation
reactions appear to be separately regulated during HS biosynthesis,
because the former sulfation type is largely confined to the contiguous
N-sulfated sequences, whereas the latter modification occurs
within as well as outside these domains (4). Intriguingly, the enhanced
6-O-sulfation appeared to selectively involve the contiguous
N-sulfated domains, because the sulfation of alternating
domains (recovered as tetrasaccharides after cleavage of HS with
HNO2 at pH 1.5) was essentially similar in HS from young
and old subjects. This finding suggests the involvement of two (or
more) HS GlcN 6-O-sulfotransferase species that differ in
substrate specificity and mode of regulation. Unfortunately, our
knowledge regarding the regulatory mechanisms in HS biosynthesis is
still fragmentary (3). Could the abundance of a given sulfate group
simply reflect the expression level for a corresponding sulfotransferase?
Atherosclerotic lesions are frequently encountered in human aorta, and
HS-binding growth factors such as FGFs and PDGFs are thought to
contribute to the pathological smooth muscle cell migration and
proliferation characterizing the disease (16). The expression level of
the PDGF-AL chain is high in human arterial smooth muscle cells and increases markedly during the conversion of monocytes into
macrophages (32). It has been shown that PDGF isoforms containing the
AL or B chains are retained at cell surfaces or in the
extracellular matrix by HS (20, 21). In the arterial wall, increased
binding of PDGF to HS could thus result in extracellular accumulation
of PDGF. Such early changes might facilitate later pathophysiological
processes such as aberrant smooth muscle cell migration and growth in
individuals prone to develop atherosclerotic disease. Moreover, we note
that the trisulfated
IdceA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide units found to promote binding of PDGF to HS has also been
implicated in binding of lipoprotein lipase (6), a cell surface-bound
enzyme that catalyzes the breakdown of triglycerides and affects the
cellular uptake of lipids (17). The observed change in HS structure
thus is likely to have more widespread functional implications than
those emphasized in this study.
We thank Drs. F. Lustig and G. Fager (Wallenberg Laboratory for Cardiovascular Research,
Gothenburg, Sweden) for generous gifts of recombinant PDGFs
and T. Lehtipalo, U. Elinder, and T. Östberg for help with
isolation and analysis of HS samples.