From the Department of Anatomy and Cell Biology, University of
Melbourne, Victoria, Australia 3052, the ¶ Walter and Eliza Hall
Institute of Medical Research, Royal Melbourne Hospital, Victoria,
Australia 3050, and the § CRC Medical Oncology Department,
University of Manchester, Christie CRC Research Centre,
Manchester, M20 4BX United Kingdom
Heparan sulfate (HS) glycosaminoglycans are
essential modulators of fibroblast growth factor (FGF) activity and
appear to act by coupling particular forms of FGF to appropriate FGF
receptors. During neural development, one particular HS proteoglycan is
able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal
differentiation occurs. Using various analytical techniques, including
chemical and enzymatic cleavage, low pressure chromatography, and
strong anion-exchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of
6-O-sulfation, total chain length, and the number of
sulfated domains of the HS from the more mature embryonic brain. These
changes correlate with a switch in the ability of the HS to potentiate
the actions of FGF-1 in triggering cell differentiation. It thus
appears that each HS pool is designed to function in the modulation of
an intricate interaction with a specific growth factor and its cognate
receptor, and suggests tightly regulated expression of specific,
bioactive disaccharide sequences. The data can be used to construct a
simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.
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INTRODUCTION |
Heparin-binding fibroblast growth factors
(FGFs)1 are essential
regulators of mitogenesis and differentiation for the precursor cells
of the mammalian central nervous system (1). As in many tissues, the
bioactivity of the FGFs is partially regulated by the glycosaminoglycan
heparan sulfate (HS). These carbohydrate chains are normally found
attached to core proteins and cells lacking these heparan sulfate
proteoglycans (HSPGs) are unable to transduce an FGF signal (2, 3). One
current hypothesis is that HSs serve to couple FGFs to specific
HS-binding regions on competent FGF receptors (FGFRs) to form
activating ternary complexes (4, 5). We have previously purified two
different species of HSPG from embryonic murine neuroepithelia: from
embryonic day 10 (E10) cells, when an HSPG with an affinity for FGF-2
was found and whose sugar chains are herein designated as HS2, and from
E12 neuroepithelial cells, when an HSPG with an affinity for FGF-1 is
expressed, and whose sugar chains are called HS1 (6, 7). This switch in
HSPG activating activity was coincident with a similar switch in FGF
expression. As the HSPG core protein carrying the two HS species
appeared to be the same (6) then presumably differences in HS structure
between E10 and E12 must account for the change in FGF specificity.
As specific HS species are essential to FGF activation, knowledge of
the structures is clearly crucial to understanding their potentiating
functions. Variation in HS species arises from the synthesis of
non-random, highly sulfated sequences of sugar residues which are
separated by unsulfated regions of disaccharides containing N-acetylated glucosamine. The initial conversion of
N-acetylglucosamine to N-sulfoglucosamine creates
a focus for other modifications, including epimerization of glucuronic
acid to iduronic acid and a complex pattern of O-sulfations
on glucosamine or iduronic acids (8, 9). In addition, within the
non-modified, low sulfated, N-acetylated sequences, the
hexuronate residues remain as glucuronate, whereas in the highly
sulfated N-sulfated regions, the C-5 epimer iduronate
predominates (10-12). This limits the number of potential disaccharide
variants possible in any given chain but not the abundance of each.
Most modifications occur in the N-sulfated domains, or
directly adjacent to them, so that in the mature chain there are
regions of high sulfation separated by domains of low sulfation
(12-15). This pattern distinguishes HS from heparin, which is
essentially highly sulfated along its entire length. Some of these
modifications have been shown to be essential in creating unique
binding sites for molecules such as antithrombin III, FGF-2, hepatocyte
growth factor, and interferon-
(16-21). The possibility therefore
exists that such modifications may create specific binding sites for
different members of the FGF family and many other extracellular matrix
molecules.
This study presents a detailed structural comparison of two HS
preparations: HS1 from E12 primary murine neuroepithelia and HS2 from
E10 primary murine neuroepithelia. (22). It has now been well
established that cells are capable of changing both the
glycosaminoglycan moiety attached to a specific core protein, and the
sulfation patterns of HSs in culture (23-27), just as they do over the
course of development, injury, and disease (23, 28, 29). The main aim
of this study was to elucidate the structures of the two HS species
which mediate either mitogenesis or differentiation in vivo,
and to determine the structural properties that may account for the
selectivity for the two FGFs. Although direct sequencing of whole HS
chains is not yet feasible, methods for identifying specific structural
elements within a pool of similar chains are available. The conjoint
use of heparin lyases and nitrous acid digestion, low pressure
chromatography, HPLC, and a unique tetrasaccharide analysis have
allowed us to elucidate subtle structural and compositional differences
between mixtures. The results of the structural analysis have led to
the construction of a model for the changing HS pools.
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EXPERIMENTAL PROCEDURES |
Materials--
Trypsin was supplied by Calbiochem and DNase from
Boehringer Mannheim. D-[6-3H]Glucosamine
(specific activity 21 Ci/mmol) was obtained from Amersham Life Science.
Heparitinases I (EC 4.2.2.8), II (no EC number assigned), and III (EC
4.2.2.7) and chondroitin ABC lyase (EC 4.2.2.4) were obtained from
Seikagaku Kogyo Co., Tokyo, Japan. Heparitinase IV was from Sigma
(Sydney, Australia). Cell culture media was supplied by Life
Technologies, Inc. Bio-Gel P-2 and P-10 were from Bio-Rad Laboratories.
CL-6B gel, DEAE-Sephacel, columns, peristaltic pumps, fraction
collectors, and tubing were from Pharmacia Biotech Inc. (Sydney,
Australia). ProPac PA1 analytical columns for the HPLC were from Dionex
(Surrey, United Kingdom). Centriflo CF25 Membrane Cones were supplied
by Amicon (Sydney, Australia). Scintillant (Ultima Gold) was from
Packard (Melbourne, Australia) as were the scintillation vials. All
chromatographic procedures were carried out a minimum of 3 times, and
the standard errors on all peaks reported in this study were never more
than 8%.
Cell Culture and Labeling--
Primary neuroepithelial cells
were grown in 10% FCS/Dulbecco's modified Eagle's medium and 2 ng/ml
FGF-2 in 24-well tissue culture plates at a density of 100,000 cells/well for E10, and 200,000 cells/well for E12. Post-plating
(30-60 min), 20 µCi/ml [3H]glucosamine was added.
Cultures were further incubated for 3-4 days, with the E12 cells being
at confluence. HS from these preparations was designated HS2 and HS1
for E10- and E12-derived HS, respectively. The conditioned medium was
removed and centrifuged (1000 rpm for 5 min) to remove any cell debris
and stored at
20 °C until required.
Preparation of Intact Heparan Sulfate Chains--
The
conditioned media was subjected to ion-exchange chromatography on a
DEAE-Sephacel column (3 ml) equilibrated in 150 mM NaCl
with phosphate-buffered saline, pH 7.2. The media was manually loaded
onto the column and eluted under gravity. The column was washed with 10 column volumes of 250 mM NaCl in 50 mM
phosphate-buffered saline, pH 7.2. The bound material was eluted with 1 M NaCl in 50 mM phosphate-buffered saline and
2-ml fractions collected. Fractions containing the
[3H]glucosamine-labeled glycosaminoglycans were pooled,
concentrated, and desalted on Centriflo Cones (as per manufacturer's
instructions), freeze dried, and resuspended in a minimal volume
(100-500 µl) of neuraminidase buffer (25 mM sodium
acetate, pH 5.0). Samples were treated with neuraminidase (0.25 unit/sample) for 4 h. Five volumes of 100 mM Tris
acetate, pH 8.0, were then added to the sample which was then digested
with chondroitin ABC lyase (0.25 unit/sample) for 4 h at 37 °C
and further digested overnight with an equal amount of fresh enzyme.
Finally, the core protein and the lyases were digested away with
Pronase (1/5 total volume of 10 mg/ml Pronase in 500 mM
Tris acetate, 50 mM calcium acetate, pH 8.0) at 37 °C
for 24 h. The entire mixture was then diluted 1:10 with deionized
water, passed through a 2-ml DEAE-Sephacel column, eluted as described
previously, and 1-ml fractions collected. The sample was finally
desalted on a 1 × 35-cm Bio-Gel P-2 column, the
Vo fraction collected and freeze dried.
Heparan Sulfate Characterization--
To remove HS chains from
trace amounts of core protein, samples were incubated in 500 mM NaOH, 1 M NaBH4 for 16 h at
4 °C and neutralized to pH 7 with glacial acetic acid. Concentrated ammonium bicarbonate was added and after the bubbling stopped, samples
were run on a CL-6B column (1 × 120 cm) for sizing of the
released HS chains (30). Binding experiments were performed on these HS
preparations as described under "Results."
Nitrous Acid Treatment of HS Chains--
HS was chemically
depolymerized by low pH HNO2 (pH <1.5) as described
by Shively and Conrad (31) and modified by Bienkowski and Conrad (32).
Linkages susceptible to low pH HNO2 are those containing
N-sulfates of the type
GlcNSO3(±6S)
1-4UA(±2S) (11), whereas the resistant
linkages are UA
1-4
(GlcNAc(±6S)
1-4GlcA)n-GlcNSO3. A small portion
of the mixture was run on a Bio-Gel P-10 column (1 × 200 cm) to
obtain a profile of the fragments released by this treatment and the
rest of the mixture was separated on a Bio-Gel P-2 column (1 × 120 cm) to isolate disaccharides and tetrasaccharides for strong anion
exchange-high pressure liquid chromatography (SAX-HPLC).
Lyase Depolymerization of HSPGs--
Heparitinase (heparitinase
I), heparitinase II, and heparitinase IV were used at a concentration
of 25 milliunits/ml in 100 mM sodium acetate, 0.2 mM calcium acetate, pH 7.0. Heparinase was used at a
concentration of 50 milliunits/ml in the same buffer. Heparitinase I
(also known as heparinase III) cleaves mainly at GlcNR(±6S)
1-4
GlcA linkages (R = N-acetyl or
N-sulfate moiety) (11, 33) creating resistant products with
the structural motif GlcA
1-4(GlcNSO3(±6S)
1-4IdoA(+2S))n
1-4
GlcNR. Heparitinase II (also known as heparinase II) has a wide
spectrum of activity cleaving linkages of the type
GlcNSO3(±6S)
1-4IdoA/Glc leading primarily to the
generation of disaccharides from HS (33). Heparinase (also known as
heparitinase III) cleaves at linkages of the type GlcNSO3(±6)
1-4IdoA(2S) (11, 33) creating resistant
sequences of structure
IdoA(2S)
1-4(GlcNR(±6S)
1-4UA)
1-4GlcNSO3(±6S)
(R = N-acetyl or N-sulfate
moiety) (11, 33). Samples were digested in the presence of 100 µg of
non-labeled carrier HS. For single enzyme digests, samples were
separately incubated at 37 °C for 16 h and then a second
aliquot of enzyme added and incubated for a further 4 h.
Sequential digests for recovery of disaccharides for SAX-HPLC analysis
were performed in the presence of 100 µg of nonlabeled HS at 37 °C
as follows: heparinase for 2 h, heparitinase for 1 h,
heparitinase II for 18 h, and finally an aliquot of each lyase and
heparitinase IV for 6 h. Sample volumes were decreased to less
than 100 µl by desiccation and run on a Bio-Gel P-2 column to isolate
disaccharides.
Gel Chromatography--
Gel chromatography of intact chains or
scission products was performed on Sepharose CL-6B (1 × 120 cm)
or Bio-Gel P-2 (1 × 120 cm) or Bio-Gel P-10 (1 × 200 cm)
columns. The running buffer for the CL-6B and Bio-Gel P-10 columns was
0.5 M NH4HCO3 and for the Bio-Gel
P-2 column was 0.25 M NH4HCO3.
Samples were routinely eluted at 4 ml/h with 1-ml fractions collected.
For preparative runs, the radioactivity of a small aliquot of each
fraction (1-10 µl) was monitored by liquid scintillation counting to
ensure good separation and accurate isolation of fragments for further
analysis. Estimates of the size of fragments resolved on Sepharose
CL-6B were based on published calibrations (30).
SAX-HPLC Analysis of Disaccharides and
Tetrasaccharides--
Disaccharide composition of the HS was analyzed
on SAX-HPLC after either complete depolymerization with a mixture of
lyases as described above or HNO2 treatment. Disaccharides
and/or tetrasaccharides were recovered by Bio-Gel P-2 chromatography
and fractions corresponding to disaccharides or tetrasaccharides were
pooled, freeze dried, and stored at
20 °C. The lyase-derived
disaccharides were subjected to SAX-HPLC on a ProPac PA1 analytical
column (4 × 250-mm Dionex Ltd.) as follows. After equilibration
in the mobile phase (double-distilled water adjusted to pH 3.5 with
HCl) at 1 ml/min, samples were injected and disaccharides eluted with a
linear gradient of NaCl from 0 to 1 M over 45 min in the
same mobile phase. The eluant was collected in 0.5-ml fractions and
monitored for tritium-labeled disaccharide content for comparison with
lyase-derived disaccharides standards. Nitrous acid-derived
tetrasaccharides were subjected to the same conditions (with 0.25- or
0.5-ml fractions collected) and compared with double labeled standard
results which were supplied by Dr. Gordon Jayson (Christie Hospital,
Manchester, UK). Alternatively, HNO2-derived disaccharides
were separated using two ProPac PA1 columns in series in the same
mobile phase. A shallow, non-continuous gradient was used over the
course of 97 min. After a 1-min injection phase, a 50-min gradient from
0 to 150 mM NaCl was employed, followed by a 70-min
gradient of 150-500 mM NaCl. The eluant was collected as
described above and compared with standards. Major peaks are labeled in
Fig. 3A and three minor disaccharide peaks eluted as follows: GlcA(2S)-AManR between 43.75 and 44 min,
GlcA-AManR(3S) between 45.75 and 46.5 min, and
GlcA-AManR(3,6S) between 104 and 106 min.
Nitrocellulose Filter Binding Assays--
Nitrocellulose filter
binding assays were performed as described previously (34) with
modifications. Briefly, 3H-labeled HS samples were
incubated with 5 µg of the appropriate FGF for 10 min at 37 °C in
0.5 ml of 10 mM Tris-HCl buffer, pH 7.0, and the complex
immobilized by vacuum filtration onto a 25-mm diameter nitrocellulose
filter. The HS was then step eluted by consecutively vacuum filtering a
series of 10 mM Tris-HCl, pH 7.0, solutions (5 ml each)
containing increasing concentrations of NaCl (250, 500, 750, 1000, 1250, 1500, and 2000 mM) through the nitrocellulose
membrane. Radioactivity in the filtrate at each NaCl concentration was
measured by scintillation counting.
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RESULTS |
The structural features of the HS chains were investigated using
established protocols (12, 35-37). HS chains (derived by Pronase and
mild alkali treatment) were chromatographed on Sepharose CL-6B columns
either intact, or after heparinase treatment (Fig. 1 and Table
I). Both pools of HSPGs showed a partial
resistance to proteolysis by Pronase, characteristic of proteoglycans,
which are densely substituted with polysaccharide chains. This data indicates that there are at least two HS chains per core protein in
each preparation, and that their attachment sites are located close
together. HS2 and HS1 eluted
at Kav of 0.46 and 0.36, respectively, which
corresponds to average molecular masses of 25 and 40 kDa when compared
with published calibrations (30). Assuming an average molecular mass of
400 Da per disaccharide, HS2 and HS1 are 60 and 100 disaccharides in
length, respectively. The HS expressed early in development is thus
smaller than the more mature chains, indicating significant structural
changes underlying the functional differences exhibited in previous
studies.

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Fig. 1.
Gel filtration on Sepharose CL-6B of HS
chains and fragments. Heparan sulfate from primary neuroepithelial
cells (A and B) was isolated and the size of the
full-length chains (A) and heparinase-resistant fragments
(B) determined using a Sepharose CL-6B gel filtration column
(HS2, ; HS1, - - -). The results are summarized in Table I.
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Table I
A summary of the estimated Mr of extracellular-HS from the two
samples examined in this study
All HS samples were subjected to separation on a 1 × 120-cm
Sepharose CL-6B column after a variety of treatments. The size of
purified full-length HS was determined both before and after mild
alkali treatment to determine the presence of more than one chain per
protein core. In addition, the approximate distance between
heparinase-sensitive disaccharides was determined by isolating the
non-resolved, large oligosaccharides from a Bio-Gel P-10 column (Fig.
2B, Vo peak) and re-running them on a
Sepharose CL-6B column.
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Fig. 2.
Gel filtration on Bio-Gel P-10 of
oligosaccharides produced by various depolymerizing agents. HS
from primary neuroepithelial cells (A-C) was isolated as
described in the text and was fractionated on a Bio-Gel P-10 column
(1 × 200 cm) after the following treatments. A, low pH
HNO2 depolymerization. A small aliquot was fractionated on
a Bio-Gel P-10 column and the rest of this digested sample was run on a
Bio-Gel P-2 column (1 × 120 cm) to isolate the tetrasaccharides and disaccharides for SAX-HPLC analysis. This profile was used to
calculate the percentage of HNO2 susceptible linkages
(Table II). A large fraction of this digest sample was run on a Bio-Gel P-2 column (1 × 120 cm) to isolate the tetrasaccharides and
disaccharides for SAX-HPLC analysis. C, depolymerization by
heparitinase: the susceptibility of each species to heparitinase was
calculated from this profile and tabulated in Table II. The degree of
polymerization of each peak is represented by the number
above that peak and was subsequently used in the
calculations. B, depolymerization by heparinase:
inset, fractions 64-115 of the heparinase scission profile
with an expanded scale to reveal the proportions of low Mr products. The nonresolved
Vo peak was pooled, freeze dried, and resolved on a
Sepharose CL-6B (Fig. 1).
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Low pH HNO2 Treatment--
To determine the number and
general organization of N-sulfated disaccharides the HS
samples were subjected to low pH HNO2 treatment. Low pH
scission releases the N-sulfate groups from heparan sulfate
with subsequent cleavage of the adjacent hexosaminidic bond. This gave
elution profiles (Fig. 2A) with a typical distribution of
N-sulfated disaccharides characteristic of HS (38). From these profiles it is possible to calculate the percentage of linkages susceptible to this treatment and thus the percentage of
N-sulfated glucosamine residues in the HS chains. HS2 was
significantly more susceptible to nitrous acid cleavage than HS1 (Table
II). As the similarities between the
profiles confirm, the results also show the strong tendency of the
N-sulfated disaccharides to occur in contiguous or mixed
sequences. There is a small decrease in the proportion of repeating
N-acetylated disaccharide sequences in HS1 that might be
due to a reduction in the spacing between the sulfated domains.
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Table II
Proportion of the linkages susceptible to low pH HNO2,
heparitinase and heparinase in HS2 and HS1
Radiolabeled heparan sulfate from the two different sources was treated
with low pH HNO2, heparinase, or heparitinase and fractioned on
a Bio-Gel P-10 column as described in the text. The percentage of the
total treatment-sensitive linkages was determined in two separate
experiments by An/n, where An is the percentage of total radioactivity in that peak, and n
is the number of disaccharide repeat units in the oligosaccharides as
determined by the elution position.
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Chromatography of Lyase-treated HS--
Heparinase-treated
oligosaccharides were separated on a Bio-Gel P-10 column (Fig.
2B). The molecular mass of the heparinase-resistant domains
in HS corresponds to the average distance between the centers of highly
sulfated regions which contain heparinase-susceptible linkages.
Analysis of the percentage of small oligosaccharides (degree of
polymerization 2-6) (inset Fig. 2B) released by
this treatment revealed that HS2 released approximately 50% fewer
small oligosaccharides then HS1. Estimation of the molecular size of the material excluded from the P-10 column by CL-6B chromatography (Fig. 1B) showed that the major peaks correspond to
molecular weights of 7,000 and 5,000 (Table I), respectively, for HS2
and HS1, although there is considerable overlap of the two peaks, indicating a broad similarity in size distribution. The results do
suggest a tendency for closer average positioning of the sulfated domains in the more mature E12 chains, although more work will have to
be done to confirm this point. Heparinase therefore cuts the HS2 chains
into 3-4 segments but in contrast cuts the HS1 chains into 7-8
segments. HS2 is therefore less complex in domain structure than HS1.
Heparitinase treatment of the samples yielded elution profiles (Fig.
2C) which were also characteristic of HS (12). The
heparitinase cleavage also tends to confirm that the sulfated domains
are broadly similar in size from E10 to E12, the only significant
difference being a decrease in the tetrasaccharide peak in HS1.
Quantitative analysis of the profiles demonstrated that 61 and 65% of
the linkages in HS2 and HS1 were susceptible to this treatment,
respectively (i.e. GlcA-containing disaccharides), a result
which indicates that the sizes of the (heparitinase-resistant) sulfated
domains in both E10 and E12 material are broadly similar. The remaining
disaccharides must contain IdoA or IdoA(2S), indicating that there is a
slightly higher content of IdoA(±2S) residues in HS2 chains. Since HS2
is less susceptible to heparinase (Table II), which cleaves at IdoA(2S)
residues, and contains less 2-O-sulfated disaccharides (see
Tables III and IV) this indicates that it contains a higher level of
unsulfated iduronate than HS1.
SAX-HPLC of Nitrous Acid-generated
Disaccharides--
Oligosaccharides derived by HNO2
treatment of HS were reduced with sodium borohydride, separated on a
Bio-Gel P-2 column, pooled, and freeze dried. SAX-HPLC separation of
disaccharides resulted in the elution profiles in Fig.
3. Nitrous acid cleaves hexosaminidic
bonds in HS leading to the production of saccharides with an authentic
nonreducing end uronic acid, but the loss of N2 from the
reducing end glucosamine and its conversion to a 2,5-anhydromannose residue. For SAX-HPLC, this moiety was reduced to 2,5-anhydromannitol. Each disaccharide peak was identified according to its elution time in
comparison with standard HNO2-derived disaccharides. These results are summarized in Table III where
the area under each peak has been integrated and shown as a percentage
of total disaccharides released by HNO2 digestion. The
major differences are a decrease in IdoA(2S)-AManR and
increases in both IdoA(2S)-AManR(6S) and GlcA(2S)-AManR accompanying the transition from E10 to E12.
Overall, there is a slight increase in total sulfation in the
contiguous N-sulfated domains of HS1 when compared with HS2.
HS2 is therefore less sulfated than its more mature counterpart
HS1.

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Fig. 3.
Strong anion exchange-high pressure liquid
chromatography of HNO2-generated disaccharides.
Disaccharides produced by low pH HNO2 were isolated by
Bio-Gel P-2 low pressure chromatography, freeze dried, and separated by
SAX-HPLC. Disaccharides were eluted as described under "Experimental
Procedures." The elution times of the peaks were compared with those
of authentic standards and labeled accordingly. A representative
elution profile from HS2 disaccharides is shown in A and HS1
disaccharides in B. The relative amounts of each of the
peaks has been calculated and summarized for both HS pools in Table
III.
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Table III
Nitrous acid-derived disaccharide composition of heparan sulfate from
HS preparations from two developmental stages of neuroepithelia
Radiolabeled HS was depolymerized by deaminative cleavage with low pH
HNO2. Disaccharides were isolated on a 1 × 120-cm Bio-Gel P-2 column. The resulting disaccharides were fractionated by SAX-HPLC as described in the text. The area under each peak in Fig. 3 was integrated to give the percentage composition in each sample. The
numbers in the table are the average of three experiments which did not
vary >5%.
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Analysis of the Total Disaccharide Composition of the HS
Pools--
To fully characterize the composition of the samples of HS,
the chains were subjected to complete lyase depolymerization using heparitinases I, II, III, and IV. The products of this digestion were
separated on a Bio-Gel P-2 column with over 95% of the radioactivity accounted for in the disaccharide peaks (data not shown), indicating sufficiently complete digestion to provide a representative
compositional analysis. For analysis on SAX-HPLC, the disaccharides
were pooled and freeze dried. The peaks in Fig.
4 were identified by reference to
authentic standards (38, 39). Overall, the disaccharide compositions of
the HS samples were broadly similar (Table
IV). However, there were significant
increases in the trisulfated disaccharide
HexUA(2S)-GlcNSO3(6S) and in the disaccharide
HexUA(2S)-GlcNAc in the E12-derived HS1. Based on this data,
comparisons of the sulfation characteristics can be made (Table
V). N-Sulfation was identical,
whereas O-sulfation was slightly higher in HS1 relative to
HS2, due mainly to a higher level of 2-O-sulfation, with
only a small increase in 6-O-sulfation apparent.

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Fig. 4.
Strong anion exchange-high pressure liquid
chromatography of HS disaccharides. Disaccharides produced by
lyase depolymerization were separated by SAX-HPLC. The elution times of
the peaks were compared with whose of authentic standards and
numerically labeled accordingly. The numbers correspond with
numbers in Table IV which summarizes the relative abundance of each of
the disaccharides. Representative elution profiles from HS2
disaccharides (A) and HS1 disaccharides
(B).
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Table IV
Lyase-derived disaccharide composition of heparan sulfate from the two
sources of HS
Heparan sulfate was isolated and completely depolymerized with a
mixture of heparan lyases. The resulting unsaturated disaccharides were
isolated on a P-2 column and fractionated by strong anion exchange-HPLC. The area under each peak in Fig. 4 was integrated to
calculate the percentage of each disaccharide in each sample. The
numbers represent the average of three experiments which did not vary
>5%. Over 97% disaccharides were recovered from each sample.
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Table V
Sulfation characteristics of disaccharides from both HS pools
The calculations on sulfation characteristics shown are based on the
overall disaccharide composition data (Table IV). The data indicate
different levels of O-sulfation and sulfation ratios in
these two HS samples.
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SAX-HPLC of Nitrous Acid-generated Tetrasaccharides--
The
tetrasaccharide products isolated after an HNO2
depolymerization were separated by SAX-HPLC (Fig.
5). The general structure of
tetrasaccharides is HexA-GlcNac-Glc-AHM, and O-sulfation can occur at C-6 of the amino sugars, C-2 of the nonreducing terminal uronic acid, and at C-3 of GlcNSO3. Current knowledge of HS
biosynthesis and structure indicates that HNO2-resistant
tetrasaccharides are likely to flank the contiguous
N-sulfated domains (12, 20). The positions of non-, mono-,
and disulfated peaks were established by comparison with dual
35S/3H-labeled samples run under identical
conditions. The percentage of each of the designated peaks as compared
with the total population is summarized in Table
VI. The level of the nonsulfated peak
(UA
1-4GlcNAc
1-4GlcA
1-4AManR tetrasaccharides)
was similar in the HS from E10 and E12. However, the proportions of
nearly all of the monosulfated peaks changed significantly, especially
peaks 1 and 7, which decreased by 60 and 29%, respectively, and peak 5 which increased by 125%. The disulfated and trisulfated peaks also
show small but significant changes in levels, and overall there is a
slight trend toward increased sulfation at E12 in these
tetrasaccharides. Thus, the tetrasaccharide analysis approach revealed
considerable structural differences between the two HS pools,
confirming the increased complexity in sulfation patterns during
development.

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Fig. 5.
SAX-HPLC of the tetrasaccharides produced by
HNO2. Tritiated tetrasaccharides produced by low pH
HNO2 were isolated from Bio-Gel P-2 low pressure
chromatography, freeze dried, and separated by high pressure liquid
chromatography and compared with dual
35S/3H-labeled standard results. The
numbers correspond to the order of the left
column of Table IV. HS2 and HS1 profiles are presented in
A with a complete profile, and B, an expanded
axis highlighting the fractions which are low in abundance (HS2, ;
HS1, ···).
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Table VI
SAX-HPLC separation of the HS-derived tetrasaccharides produced by
HNO2
Tritiated tetrasaccharides derived by low pH HNO2-treated
heparan sulfates were originally separated by low pressure
chromatography on a Bio-Gel P-2 column and were then further resolved
by high pressure liquid chromatography. The percentage of each
tetrasaccharide was determined by calculating the radioactivity in each
peak and comparing it to the total radioactivity in all peaks combined. Tetrasaccharide peak numbers in the left column correspond to the peaks
in Fig. 5. Minor peaks comprise less than 12% of total tetrasaccharides. The degree of sulfation was determined by comparison of these tritiated samples with peaks generated by dual
35S/3H-radiolabeled samples (from Dr. Gordon Jayson,
University of Manchester) run on the same column under identical
conditions. The numbers represent the average of three separate
experiments.
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Binding Assays--
To confirm that the isolated heparan sulfate
chains retain their binding specificities for particular FGFs, filter
binding assays were performed. FGF-2 or FGF-1 were pre-mixed with
either HS2 or HS1, and the complexes formed captured on a
nitrocellulose filter and step eluted with increasing concentrations of
NaCl (Fig. 6). The results showed that
the E10-derived HS pool, HS2, binds FGF-2 strongly and with a markedly
greater affinity than for FGF-1. The E12-derived HS pool, HS1, also
binds FGF-2, albeit a little more weakly than HS2 and would also appear
to have, relative to HS2, an enhanced affinity for FGF-1.

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Fig. 6.
Filter binding assays for HS2 and HS1.
Filter binding assays were carried out as described under
"Experimental Procedures" to assess the ability of HS1 and HS2 to
interact with FGF-1 and FGF-2. 3H-Labeled HS was incubated
with the appropriate FGF and the complex immobilized on a
nitrocellulose filter. The HS was then step eluted with increasing
concentrations of NaCl shown. For the HS1 and HS2 assays the results
represent the mean and standard errors for two independent
experiments.
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DISCUSSION |
A mixture of structurally complex chain sequences are generated
during the biosynthesis of HS and the data generated here represent the
average for each pool of HS isolated. The analysis reported here makes
clear that chain organization and fine structure differs systematically
between key developmental stages, although the core protein carrying
these different chains remains constant (6). By integrating all the
structural data we have produced a simplified model to summarize the
most significant changes in structure of the neuroepithelial HSs (Fig.
7). Previous studies characterizing skin
fibroblast HS led to a model which posited the now widely accepted
domain structure model of HS (11, 12). It was concluded that
heparinase-sensitive disaccharides are located in short domains
consisting of GlcNSO3(±6S)-IdoA(±2S) repeats. These are
separated by regions of polysaccharide that are heparinase resistant,
enriched with N-acetylated disaccharides, and low in both
iduronic acid and sulfate moieties. Following this discovery, many
other HS pools have been characterized which are similar in their
overall organization (36, 37, 39, 40); this study demonstrates that HS
derived from primary neuroepithelial cells conform to this structural
theme, and to our knowledge represents the most detailed
characterization to date of HS derived from primary cells. Apart from
the increase in chain size and number of sulfated domains per chain in
HS1, the data indicate basic similarities in domain structure, but with
distinct O-sulfation patterns imposed on these domains.
These alterations must be the basis for the changes in binding and
activation of FGF2 and FGF1 previously reported (6). The main
difference in fine structure between the two HSs is an obvious increase
in sulfation at E12, which indicates that 2-O- and
6-O-sulfotransferases are significantly more active at the
E12 as opposed to the E10 stage of development. However, it is when the
disaccharides released by HNO2 are studied that the
distinctions become most clear, with HS1 showing a reproducibly higher
amount of 6-O-S specifically in the disulfated species IdoA(2S)-AManR(6S); this shows that a highly regulated
sulfotransferase activity is targeted to specific GlcNSO3
residues within contiguous sequences of N-sulfated
disaccharides. Data on the tetrasaccharides released by
HNO2 also support the view that regulation of
O-sulfotransferases creates subtle differences in patterns
of O-sulfation in the HS at different developmental
stages.

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Fig. 7.
Hypothetical models of the heparan sulfate
structures derived from the analysis of growing and differentiating
neuroepithelial cells. The model, based on the data presented
here, proposes that HSs with altered structures are secreted by
neuroepithelium to deal with changing expression patterns of
extracellular FGFs. The most significant changes observed are increases
in size, number of sulfated domains, and sulfation complexity (in
particular the patterns of O-sulfation) as the cells mature.
The circles denote the amino acids of the core protein, the
rectangular blocks denote the sulfated domains, the
solid circles denote 6-O-sulfate groups, and the
lines between the blocks denote regions of
N-acetylation.
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Analysis of the disaccharides from HNO2-degraded HS from
the neuroepithelium corresponds with the relative amounts of HS-derived disaccharides found in the conditioned medium of a hepatocyte cell
line, in that there is a high proportion of IdoA(2S)-AManR, followed by IdoA(2S)-AManR(6S) and
IdoA/GlcA-AManR (26). There is a significant decrease in
IdoA(2S)-AManR and an increase in GlcA-AManR(6S) in the transition from E10, the
proliferative influence, to E12, the differentiative influence. In
addition, HS1 is significantly different from HS2 in that it contains
approximately 50% more of the trisulfated disaccharide
HexA(2S)-GlcNSO3(6S). Both of these results may reflect the
crucial role suggested for 6-O-sulfates in binding FGF-1 and
potentiating its activity in vivo (41, 42). Similar to
hepatocytes, there is an increase in total sulfation in the E12 cells
over less differentiated, E10 cells (26). Fedarko and Conrad (26)
hypothesized that this reflects HS chains with longer sulfated domains
and shorter non-sulfated domains in the confluent as opposed to
dividing cells. In contrast, our data indicate that the two
neuroepithelial HS species are very similar in domain sizes, with the
main overall structural change in the E12-derived HS being the extra
chain length, presence of a smaller proportion of
N-acetylated sequences (which may contribute to the closer
spacing of the heparinase cleavage sites) and a larger number of
sulfated domains. Differences in the fine structure of HS recovered
from syndecan-1 proteoglycan have been observed in terms of
disaccharide composition, the arrangement of lyase cleavage sites, and
in the length and number of highly sulfated domains. These changes
appeared to underlie differences in the ability of HS to bind collagen
but not FGF-2 (27, 40). This supports the idea proposed here that cells
preferentially modify HS chains attached to a specific core protein to
enable the HSPG to perform specific functions (43).
The filter binding assays suggest that HS2 has an enhanced affinity for
FGF2 relative to HS1 and that this situation is reversed with respect
to their binding of FGF1. While the differences may appear slight, HS1
and HS2 are, in fact pools of HS made in the developing tissue at the
different ages. Consequently the subtle differences found here must
reflect significant structural changes taking place in these pools
during development which are necessary to appropriate function in an
increasingly complex environment. These changes complement the
expression of these two growth factors in the developing
neuroepithelium (6, 7). It is also important to remember that this
binding assay can assess one event only, the HS-FGF interaction, of the
ternary interaction (HS-FGF-FGFR) thought to be required to initiate
specific cell signaling. Thus, it is likely that distinct sequences
required for biological activation of specific FGFs could be masked in
simple binding assays by the presence of other inactive sequences which
are nevertheless capable of binding FGFs. It is interesting to note
that the intact HSPG binds growth factors with higher affinities than
the heparan sulfate chains alone
(6).2
One of the most interesting observations in this study is that the HS
from undifferentiated, dividing cells is both smaller and simpler in
structure than the HS from more differentiated, contact-inhibited
cells. We have also undertaken studies on HS in an in vitro
model with the 2.3D neuroepithelial cell line derived from E10 cells,
in which the transition from growth to post-confluence parallels the
E10 to E12 transition (6); overall, the differences between the samples
of HS from less differentiated, growing cells and the HS from more
mature, confluent cells follow similar trends to those observed in the
primary cells (47). In view of the fact that the HS from the two less
differentiated sources (E10 primary cells and growing 2.3D cells) bear
the same FGF-2 potentiating activity (6), it follows that a particular
HS sequence motif embedded within the sulfated domains may be common to
both, and thus responsible for the formation of an FGF-2·FGFR ternary
complex. A similar argument would hold for an FGF-1 activating
subdomain within HS from E12 and confluent 2.3D cells. We know that HS
from the growing 2.3D cell line contains a subdomain which promotes the
interaction of FGF-2 with an FGFR (4). The exact composition of the
sulfated domain which interacts with any specific FGF and its cognate
FGF receptor remains to be determined; almost certainly the differences
between chains that generate such significant differential specificity
between ligands are quite subtle (18, 42, 43). However, the data
presented here strongly suggest that regulation of patterns of
6-O-sulfation are a critical element. In addition, the
spacing between the growth factor-binding and any receptor-binding
regions of an HS molecule is likely to be significant to its
recruitment into a ternary complex and thus its bioactivity (4,
44-46). Our results also carry the important implication that the use
of heparin or heparin fragments in tissue culture experiments as
analogues of HS to augment the activity of such agents as FGFs and
other HS-binding proteins is likely to mask crucial and complex
in vivo control mechanisms. Unlike HS, heparin does not
consist of discrete sulfated domains; heparin is a more extensively
epimerized and sulfated form of HS, with much larger proportions of
disaccharides that are trisulfated.
As HS must serve a variety of different functions in vivo,
the structural differences between HS chains must be designed to selectively produce specific functional properties. It is highly likely
that these differences are the key to the individual and specific
functions of HS in the extracellular environment, although methods for
direct sequencing of HS saccharides will be required to substantiate
this view. The fundamental hypothesis underlying the present study was
that the functional changes in the FGF activating activities of
secreted HS from either dividing or differentiating cells were due to
systematic changes in the saccharide sequences of the HS chains. The
differences in the structure of the two kinds of HS species found in
this study are compatible with the idea that distinct HS sequences are
expressed to selectively activate different FGFs, and that during
embryonic development a non-committed cell type has the potential to
vary the HS structure in response to epigenetic or environmental cues.
Taken together with similar results from the structural analyses of
other HS-binding molecules such as antithrombin III, hepatocyte growth
factor, and interferon-
, we predict that every molecule that
requires HS for activation will have a distinct and specific
disaccharide sequence motif dedicated to regulation of its biological
activity. Thus, HS chains with different repertoires of distinct
sequences will be generated by cells dependent on their need to
orchestrate the activity of specific HS-binding proteins. Currently
such molecules encompass members of the transforming growth factor-
family, the platelet-derived growth factor family, all of the FGFs, the
pleiotropin-like family, and structural molecules such as the laminins,
the fibronectins, the amyloid precursor protein family and the
collagens, among many others. We further suggest that such HS-binding
molecules will have configurations of basic amino acids which are
spatially distinctive for the binding of such HS sequences.
Another important question raised by our data is how a single cell type
regulates the switching of the production of one class of HS chain for
another, closely related chain. We have as yet no data which informs us
whether the sudden production of FGF-1 in E12 cells induces a change in
HS synthesis, or whether the new HS is made in readiness for the growth
factor switch. In vivo, this is an interesting question, as
differentiation of hundreds of different subclasses of neurons and glia
commences at these stages of mouse brain development. Recent evidence
from our laboratory has tended to confirm our original hypothesis that
the sudden synthesis of FGF-1 is a key event in the subsequent
emergence of the neuronal lineage; if this is true, the change in HS
specificity toward promotion of FGF-1 bioactivity becomes a seminal
event in the appearance of neurons. This hypothesis can now be tested by specifically interfering with the enzymatic processes of those 6-O-sulfotransferases which construct the subtle differences
in HS structure at each developmental stage, and monitoring for changes in subsequent rates of neuronal differentiation.