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J Biol Chem, Vol. 274, Issue 26, 18455-18462, June 25, 1999
From the Cancer Research Campaign and University of Manchester
Department of Medical Oncology, Christie Hospital NHS Trust,
Manchester, M20 4BX, United Kingdom and The heparan sulfates (HS) are hypervariable
linear polysaccharides that act as membrane co-receptors for growth
factors, chemokines, and extracellular matrix proteins. In most
instances, the molecular basis of protein recognition by HS is poorly
understood. We have sequenced 75% of the sulfated domains (S-domains)
of fibroblast HS, including all of the major ones. This analysis
revealed tight coupling of N- and 2-O-sulfation
and a low frequency but precise positioning of
6-O-sulfates, which are required functional groups for
HS-mediated activation of the fibroblast growth factors. S-domain sequencing was conducted using a novel and highly sensitive method based on a new way of reading the sequence from high performance liquid
chromatography separation profiles of metabolically labeled HS-saccharides following specific chemical and enzymatic scission. The
implications of the patterns seen in the sulfated domains for better
understanding of the synthesis and function of HS are discussed.
Many biological macromolecules have information encoded in their
primary structure. In proteins, the amino acid sequence determines the
secondary and tertiary structural characteristics of the folded protein, whereas in DNA and RNA the nucleotide sequence is the means of
storing coded information that can be read by the transcriptional and
translational machinery of the cell. Information for ligand binding and
activation is also present within the structure of complex saccharides
(1, 2). Heparan sulfate (HS)1
is one of a class of polysaccharides known as glycosamino glycans, and
recent research indicates that it expresses important protein recognition domains within its primary sequence (3, 4). However, unlike
nucleic acids and protein, there is no established method available to
read this primary information.
The near ubiquitous occurrence and strategic positioning of heparan
sulfate proteoglycans, both at the surface of most mammalian cells and
in the extracellular matrix, is a good indicator of their role in
cell-cell recognition and cell-matrix adhesion (5, 6). Heparan sulfate
proteoglycans are key sites of interaction and signaling when cells
form focal adhesions on extracellular matrix substrates such as
fibronectin (7). A very extensive range of growth factors and
cytokines, including key angiogenic factors such as basic fibroblast
growth factor (bFGF) (8, 9), hepatocyte growth factor (10), and
vascular endothelial growth factor (11), have been shown to not only
bind HS in vitro but to require its presence as a membrane
co-receptor for optimal activation of their cognate, high affinity
signaling receptors. The similarity between the co-receptor role of HS
and the effect of heparin in facilitating the inactivation of thrombin
by antithrombin is indicative of many biological processes being
driven by HS catalysis. It may therefore be possible to exploit an
understanding of HS-growth factor interactions to modulate the effects
of these ligands (12) in a similar way to the current clinical use of heparin. Although there is a substantial body of information concerning the general similarities (and differences) in structure between HS from
different sources (13-17), regrettably little is still known about its
detailed primary structure. Furthermore, although common molecular
designs in the form of spatially discrete sulfated domains (S-domains)
can be recognized within the HS family, cells impose their own imprint
on this design through variations in sugar sequence and patterns of
sulfation, which are known to be of physiological significance.
The HS chain is initially formed in the Golgi as a polymer of
alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) residues. A number of modification steps then follow. First,
selected GlcNAc residues undergo linked N-deacetylation and
N-sulfation. This N-sulfation is a prerequisite
for the epimerization of adjacent GlcUA to iduronic acid (IdoUA).
Following epimerization, O-sulfation takes place at various
positions around these saccharides, i.e. at C-2 of IdoUA,
C-6 (and rarely C-3) of N-sulfated glucosamine (GlcNS), and
C-6 of GlcNAc. These modifications, which are clearly nonrandom, are both interdependent and quantitatively incomplete and
generate regions of high sulfation that alternate with areas with
little or no modification. The controlled variability of S-domain
structure and spacing may be a method used by cells to direct which
signaling molecules they respond to or to regulate their adhesion
and migration in different extracellular matrix environments
(14).
What is clear is that HS binds to effector proteins via S-domains with
specific sugar sequences and sulfation patterns, and the importance of
these interactions has been revealed in studies of development. Studies
in mice indicate the key role of sulfation of HS for normal kidney
growth (18, 19). Also, HS and its associated core proteins are
mediators in the decapentaplegic and wingless pathways in
Drosophila (20-23).
The best known example of the involvement of HS and heparin in
molecular recognition is the antithrombin III binding sequence, the
elucidation of which was a landmark discovery that required the
concerted use of a variety of specialized techniques and many years of
work (24). More recently, high affinity binding sites have been
identified for bFGF (25, 26). These sites are of relatively simple
structure, being mainly composed of 4-5 repeat units of
IdoUA(2S)-GlcNS, but intriguingly, these sequences fail to
elicit a biological response to bFGF unless substituted by one or more
6-O-sulfate groups (27, 28). This has led to the view that
HS may act as a template for cell activation, with the 6-O-sulfate groups being needed to dock an HS-bFGF complex
onto the signaling receptor (29). Other members of the FGF family (27)
and also unrelated growth factors, e.g. hepatocyte growth factor (10), are responsive to different HS structures than those that
affect bFGF activity, but their active-site sequences are unknown.
Likewise, the Hep II domain of fibronectin accommodates an extended
S-domain of 14-16 sugar residues, but the sulfation pattern of the
binding sequence is unknown (30). The development of a rapid sequencing
method for HS is vital if we are to understand its detailed
structure-function relationships and exploit this knowledge for
therapeutic use.
The method we describe here allows the progressive sequencing of
regions of metabolically radiolabeled HS purified from cells grown in
culture. We have been able to sequence trace quantities of HS
oligosaccharides rapidly and accurately. All the major S-domains falling within the size range of hexa- to octasaccharides, together with a proportion of the decasaccharides have been sequenced without selection by ligand binding. This approach has yielded new and intriguing information on the molecular structure of HS.
Reagents
Cell culture media and donor calf serum were from Life
Technologies, Inc. Heparinase I (Flavobacterium heparinum;
heparin lyase EC 4.2.2.7), heparinase II (F. heparinum; no
EC number assigned), and heparinase III (F. heparinum;
heparitin-sulfate lyase EC 4.2.2.8) were all purchased from Grampian
Enzymes (Orkney, UK). Bio-Gel P-10 (fine grade) was from Bio-Rad.
ProPac PA-1 analytical columns were obtained from Dionex (Camberley,
Surrey, UK). Cell Culture and HS-Oligosaccharide Preparation
NIH-3T3 fibroblasts were maintained at 37 °C (5%
CO2 in air) in Dulbecco's modified essential medium
supplemented with 5-10% donor calf serum, 2 mM glutamine,
penicillin (100 units ml Disaccharide Analysis
Aliquots containing 5000 cpm of 3H were taken from
each of the isolated oligosaccharides and lyophilized. These were then
digested with a combination of heparinase I, II, and III enzymes and
assayed for their specific disaccharide composition as detailed
previously (26).
S-domain Sequencing
Partial Nitrous Acid Scission--
Nitrous acid affects
deaminative scission of glycosidic linkages of GlcNS-HexA, releasing
inorganic 35SO4, and converting GlcNS to
anhydromannose (aMan). Each oligosaccharide was subjected to partial
depolymerization using dilute nitrous acid as described (34), with
aliquots of the reaction stopped at a number of time points to generate
a range of intermediates of the depolymerization process. Briefly, each
oligosaccharide was lyophilized, then resuspended in 80 µl of
H20 to which was added 10 µl each of 10 mM
NaNO2 and 190 mM HCl. At each stop point (usually 30 min, 1 h, 2 h, 3 h, and 4 h), 20 µl
of the reaction mixture was removed and added to a common vial
containing 25 µl of 0.2 M sodium acetate, pH 5.0. All
procedures and reagents were at 4 °C. As can be seen in Fig. 2,
cleavage of a hexasaccharide, with two internal GlcNS residues, will
produce two distinct tetrasaccharides, one of which will contain a
Lysosomal Exoenzyme Digestions--
Following nitrous acid
depolymerization, samples to be digested with enzymes were desalted
by passage over PD-10 size exclusion columns eluted with
H20, then lyophilized. Digests were set up in a total
volume of 25 µl of 40 mM sodium acetate, pH 4.5. 2-Sulfatase and iduronidase were used either singly or combined
sequentially; 6-sulfatase was only used following use of the former two
enzymes. Each individual enzyme digest was incubated for 12 h at
37 °C. Iduronidase was used at 0.29 milliunits/digest, 2-sulfatase
was used at 0.54 milliunits/digest, and 6-sulfatase was used at 0.096 µunits/digest. Occasionally, glucuronidase (1,000 units/digest) was
also used for sequence analysis. After treatment with the enzymes,
samples were adjusted to 1 ml by addition of H2O/HCl, pH
3.5.
HPLC Separation Conditions for Sequence Analysis--
A single
ProPac PA-1 SAX column (4.6 × 250 mm) was used with a linear
gradient running from 0-0.75 M NaCl over 110 min. Samples were loaded using a 1-ml sample injection loop. Following sample loading, the loop was washed onto the column with 1 ml of
H2O before application of the gradient. The column was
eluted at a flow rate of 1 ml min S-domains generated by heparinase III digestion were separated by
Bio-Gel P10 gel filtration and SAX-HPLC to yield a range of
oligosaccharide species differing in length, sugar sequence, and
sulfation pattern. The HPLC separations for dp6-10 are shown in Fig.
1.
Individually purified oligosaccharides from Fig. 1 were partially
depolymerized using dilute nitrous acid (Fig.
2), and the scission products were
separated by SAX-HPLC. A typical profile is shown for the major
hexasaccharide species designated 6a (Fig. 3A). In this profile, we can
recognize peaks that correspond to the nonsulfated disaccharides
(fraction 18, labeled non-S; IdoUA-aMan, GlcUA-aMan, and
Highly Sensitive Sequencing of the Sulfated Domains of
Heparan Sulfate*
, and The Lysosomal
Diseases Research Unit, Department of Chemical Pathology, Women's and
Children's Hospital, North
Adelaide, South Australia 5006, Australia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-Glucuronidase from bovine liver
(glucuronidase, EC 3.2.1.31) was from Sigma. 
-Glycuronidase was
kindly supplied by Dr. K. Yoshida, Seikagaku Co. (Kobe, Japan). Human
-L-iduronate-2-sulfatase (2-sulfatase (31)), human
-L-iduronidase (iduronidase (32)), and caprine -D-N-acetylglucosamine-6-sulfatase
(6-sulfatase (33)) were all purified recombinant enzymes and are now
available from Oxford Glycosciences (Abingdon, UK).
D-[6-3H]glucosamine hydrochloride (20-45 Ci
mmol
1) and Na235SO4
(1050-1600 Ci mmol1) were from NEN Life Science
Products. PD-10 prepacked, disposable Sephadex G-25 M
columns were from Amersham Pharmacia Biotech. 3H-Labeled
anhydromannose-containing disaccharide standards derived from heparin
were purchased from Chirazyme (Urbana, IL). All HPLC solutions were
prepared using MilliQ (Millipore; Watford, UK) ultra pure water.
1), and streptomycin (100 µg
ml1l). Antibiotics were removed from the culture medium
for one week before radiolabeling. 3H-Glucosamine (up to 40 µCi ml1) and
Na235SO4 (up to 80 µCi
ml1) were added to the culture medium of cells at ~80%
confluency for 24 h. Radiolabeled HS chains were extracted and
digested with heparinase III, and the enzyme-resistant fragments were
further purified as described previously (10). Briefly, following
heparinase III digestion, enzyme-resistant fragments were separated by
size using a Bio-Gel P-10 gel filtration column (170 cm × 1 cm).
Samples were eluted at 4 ml h1 in 0.1 M
ammonium hydrogen carbonate, and 0.5-ml fractions were collected.
Aliquots were monitored for radioactivity by scintillation counting.
Peaks of radioactivity were pooled separately and freeze-dried several
times to remove residual salt. Individual size-fractionated samples
corresponding to degree of polymerization dp6-dp10 (dp = degree of
polymerization, or number of saccharide units, e.g. dp6 = hexasaccharide) were then further resolved by strong anion-exchange (SAX) HPLC chromatography as described previously (28). Samples were
applied to a ProPac PA-1 (4.6 × 250 mm) column preequilibrated in
distilled water adjusted to pH 3.5 with HCl and eluted with increasing
NaCl (maximum of 1.5 M). Oligosaccharides were fractionated using a range of linear gradients optimized for the separation of each
size group. Fractions of 0.5 ml were collected, and the aliquots were
subjected to scintillation counting (see Fig. 1). By collection of only
the central fractions from each peak, single species were obtained that
were then desalted by passage over a PD-10 column eluted in
H2O and lyophilized. Each peak was checked for purity by
reapplication onto a ProPac PA-1 column and elution using the extended
gradient conditions described below. Only those oligosaccharides
observed as single peaks under these conditions were further analyzed
by sequencing.
4,5-unsaturated uronate at the nonreducing end (from the action of
heparinase III) and one with a natural uronate at this position. Also
present in the reaction mixture will be three disaccharides and some
remaining intact dp6. If a hexasaccharide has one internal GlcNAc
residue, then only one tetrasaccharide can be formed. If two internal
GlcNAc residues are present, the entire hexasaccharide will be
resistant to nitrous acid cleavage.
1, collecting 0.5-ml
fractions. Radioactivity in each fraction was measured by scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Preparative SAX chromatography of sized HS
oligosaccharides. Sized dp6, dp8, or dp10 oligosaccharide
populations were applied to a single ProPac PA-1 column, eluted with a
linear gradient of NaCl in MilliQ water, pH 3.5, at a flow rate of 1 ml/min, and 0.5-ml fractions were collected. The gradients used were
dp6, 0-1 M NaCl over 80 min (A); dp8, 0-1.2
M NaCl over 90 min (B); dp10, 0-1.2
M NaCl over 180 min (C). Elution profiles were
monitored by scintillation counting (solid line,
3H; dashed line, 35S), and
individual peaks pooled sharply (one or two central fractions only).
For dplo only, no fractions were collected for the first 46 min of the
gradient.
UA-aMan all co-elute as a single peak), the sulfated disaccharide
IdoUA(2S)-aMan (fraction 45, labeled ISM), free
35SO4 (fraction 38), two tetrasaccharides
(fractions 78 and 95, designated R4 and U4 in
Fig. 2), and the original hexasaccharide at fraction 118. The
disaccharides were identified by comparison with known standards. R4
and U4 were confirmed as tetrasaccharides by their size elution
position on a Bio-Gel P-10 column (data not shown). The appearance of
two tetrasaccharides indicates that there are two internal GlcNS
residues in dp6a. One of these tetrasaccharide fragments, in common
with the original dp6 fragment, will contain a 4,5-unsaturated
uronate at its nonreducing end and will be resistant to lysosomal
enzymes; the other will be linked to the reducing end and will be
susceptible to the enzymes. It is possible to distinguish between
tetrasaccharides R4 and U4 by comparing the profiles in Fig. 3,
panels B-D. Digestion with 2-sulfatase causes the R4 peak
(at fraction 78) to shift to fraction 45 (R4'); further
digestion with iduronidase causes an additional shift to fraction 31 (R4''). This tetrasaccharide is therefore unequivocally identified as being derived from the reducing end of dp6a
(thus designated R4), and it contains a terminal IdoUA(2S). The other tetrasaccharide is not affected by the enzymes and is derived from the
unsaturated nonreducing end of dp6a (thus designated U4).
As expected, the original dp6a is also unchanged after exoenzyme digestion. The disaccharide identified as IdoUA(2S)-aMan moves as
expected after 2-sulfatase treatment but not with iduronidase alone,
providing a useful internal control for the action of the enzymes. The
last piece of information to be extracted from the profiles is the
identity of the residues in the reducing terminal disaccharide of dp6a.
UA-GlcNAc was seen in the disaccharide analysis of the
hexasaccharide, and iduronidase generates a peak in the position of
free GlcNAc from the nonsulfated disaccharides. Nitrous acid scission
showed that all the internal amino sugars were N-sulfated;
therefore the deduced IdoUA-GlcNAc disaccharide must be at the reducing
end. The entire sequence of dp6a is therefore UA-GlcNS-IdoUA(2S)-GlcNS-IdoUA-GlcNAc. The disaccharide analysis of
dp6a (Table I) is compatible with this
sequence.
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Fig. 2.
Representation of the various fragments
generated by partial nitrous acid scission of a hexasaccharide
containing two internal GlcNS residues. Partial depolymerization
generates two tetrasaccharides and three disaccharides in addition to
some uncleaved hexasaccharide. One tetrasaccharide (U4)
retains the nonreducing end unsaturated uronate, and the
other (R4) retains the Reducing end. An aMan
residue is formed at all cleaved GlcNS sites. Nitrous acid scission
requires a GlcNS residue; therefore, if a hexasaccharide contained a
GlcNAc residue at position b, only one tetrasaccharide
(sequence a-d) would be generated by nitrous acid, together
with one disaccharide, IdoUA-GlcNAc (IdoA-GlcNAc).
Alternatively, if the GlcNS residue at position d were
changed to GlcNAc, nitrous acid would still only generate one
tetrasaccharide (sequence c-f) together with one
disaccharide UA-aMan. The blue arrows represent exoenzyme
digest steps; 1, 2-sulfatase; 2, a combination of
2-sulfatase and iduronidase. Neither of these enzymes have any effect
on fragments with a UA residue at position a. The
exoenzyme digest steps are used to identify the nonreducing terminal
sugars. For example, the tetrasaccharide R4 is digested during both
enzyme steps (to generate R4' and R4''), indicating a terminal
IdoUA(2S) (IdoA(2S)) residue, whereas the disaccharide
IdoUA-GlcNAc (IdoA-GlcNAc) is unchanged by the first enzyme
step but digested by the second, indicating a terminal IdoUA
residue.
View larger version (15K):
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Fig. 3.
Sequence analysis of hexasaccharide 6a.
A, fragment profile generated by partial nitrous acid
scission resolved on a ProPac PA-1 column eluted with a linear 0-0.75
M NaCl gradient over 110 min. Aliquots of these fragments
( 
5,000 cpm of 3H/run) were subsequently digested with
2-sulfatase (B), iduronidase (C), or both enzymes
sequentially (D). Fractions (0.5 min) were collected and
counted for radioactivity (solid line, 3H;
dashed line, 35S). Identified peaks are
nonsulfated disaccharide pool (nonS, fraction 18), free
35SO4 (fraction 38), free GlcNAc
(NAc, fraction 5), disaccharide IdoUA(2S)-aMan
(IdoA(2S)-aMan (ISM), fraction 45),
tetrasaccharide R4 (green line), tetrasaccharide U4
(blue line), and intact hexasaccharide 6a (fraction
118).
Disaccharide compositions of HS oligosaccharides
UA-GlcNS, which occurs once at the nonreducing end of
each oligosaccharide.
The major octasaccharide 8a was subjected to a similar analysis,
generating the profiles seen in Fig. 4.
Again, by observing the ways in which each of the peaks seen in the
profile of the initial partial nitrous acid scission (Fig. 4,
panel A) are affected by the sequencing enzymes, it is
possible to assign fragments for sequencing. A number of peaks were
observed, but the key ones for sequencing are the hexa- and
tetrasaccharides R6 and R4. The enzyme digestions (panels
B-D) show that both R6 and R4 contain terminal IdoUA(2S) units.
Another tetrasaccharide derived from the middle of dp8a
(thus designated M4) was also present after partial nitrous acid
treatment, and this fragment also contained an IdoUA(2S) terminal unit,
whereas a third tetrasaccharide (U4) was enzyme-resistant and,
therefore, from the unsaturated end. The presence of three
tetrasaccharides (R4, M4, and U4) after partial nitrous acid shows that
the three internal GlcN residues of dp8a are N-sulfated. An
N-acetylated disaccharide is also present in dp8a, and this
must therefore be at the reducing end. This is compatible with the fact
that iduronidase yields free GlcNAc from the nonsulfated disaccharide
peak. Furthermore, the elution characteristics of R4 in dp8a were
identical to those of R4 in dp6a, indicating a reducing end sequence of
IdoUA(2S)-GlcNS-IdoUA-GlcNAc. The complete sequence of dp8a is
therefore UA-GlcNS-IdoUA(2S)-GlcNS-IdoUA(2S)-GlcNS-IdoUA-GlcNAc, and
this again is compatible with the disaccharide analysis (Table I). The
presence of U4 and U6 fragments after nitrous acid depolymerization is
further confirmation that the sequence has three internal
N-sulfated units. The elution position of U4 in dp8a was
identical to that of U4 in dp6a, and this would be predicted from the
sequences of dp6a/dp8a. Although not strictly necessary to sequence an
unknown oligosaccharide, the predictable nature of these common
fragments did prove useful, particularly in reducing the number of
peaks to be classified within a set of profiles.
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The profiles for the sequencing of the major decasaccharide 10a are
shown in Fig. 5. Once again, U4 and U6
are clearly identified by their resistance to the exoenzymes. Fragments
R4, R6, and M4 all move with 2-sulfatase but not with iduronidase
alone. It is not possible to accurately identify either R8 or U8.
Fragments U6 and R6, being derived from opposite ends of the dp10
sequence, overlap in the central disaccharide repeat, and this fact
essentially solves the sequence. Other identified fragments
(e.g. R4 and U4) are all compatible with the final sequence
shown in Fig. 5.
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More complex species are also easily solved using this technique.
When the products of partial nitrous acid scission of octasaccharide 8d
were separated by HPLC, the presence of 6-O-sulfation was
indicated by a peak corresponding to the disaccharide
IdoUA(2S)-aMan(6S) (Fig. 6). Therefore an
additional enzyme digestion step was included in this analysis,
combining 6-sulfatase, 2-sulfatase, and iduronidase. Once again, it is
easy to identify fragments that are not affected by any of the enzyme
treatments (i.e. U6 and the intact dp8) as well as one
tetrasaccharide (R4, the same as was seen in 6a and 8a), which shifts
with the 2-sulfatase alone and further with 2-sulfatase plus
iduronidase. An additional peak is also observed that elutes at
fraction 190 (R6) and that shifts with 2-sulfatase to fraction 150 (R'). This latter peak displays only a small shift when subsequently
treated with iduronidase (fraction 148, R6''), but the combination of
these two enzymes must be removing the hexuronate as the subsequent
addition of 6-sulfatase causes a further significant shift to fraction
98 (R6'''). Fragment R6 therefore contains a nonreducing terminal
sequence of IdoUA(2S)-GlcNS(6S) and is a reducing end fragment of dp8d.
The presence of R6 after nitrous acid scission shows that the
unsaturated disaccharide of dp8d is N-sulfated. Confirmation
that the unsaturated uronic acid lacks 2-sulfation comes from the
presence of the nonsulfated disaccharide peak in all of the profiles
(fraction 18); specifically this peak is still observed after the
combined 2-sulfatase and iduronidase digest. Combining the sequence
data of R6 with the presence of R4, the sequence of dp8d is
UA-GlcNSIdoUA(2S)-GlcNS(6S)-IdoUA(2S)-GlcNS-IdoUA-GlcNAc.
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This same principle of observing the results of digesting partial nitrous acid-generated fragments with exoenzymes was therefore used to deduce the sequences of all the oligosaccharides labeled in Fig. 1, panels A and B, as well as two decasaccharides from Fig. 1, panel C (see Table II). In each case, the ability to match common fragments between analyses (e.g. R4 in 6a, 8a, and 10a) and to independently ascertain the disaccharide composition by multiheparinase digestion helped to confirm the sequences.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The present study describes a method by which trace quantities of metabolically radiolabeled HS oligosaccharides can be directly sequenced without the need to chemically end-label samples. The idea of using the inherent specificities of the lysosomal exoenzymes to identify the nonreducing terminal residues within HS is nothing new in itself (35). The key to their specific use in the present method is the ability of the controlled partial cleavage with nitrous acid to open up the internal sequence of the oligosaccharide for analysis and the method of reading the sequence from the HPLC separation profiles. The positions of N-sulfation are read from the partial nitrous acid profiles, as illustrated in Fig. 3 for the hexasaccharide 6a. Other sulfation positions and sugar residues are identified by the use of the specific exoenzymes. Although this investigation has so far been limited to dp6-dp10 oligosaccharides, the method could in principle be extrapolated to the analysis of longer complex sequences within the range of resolution of the HPLC column.
Recently, there have been a limited number of advances in the development of methodologies for routinely sequencing small quantities of HS. The use of mass spectrometry, although sensitive (36), suffers from a significant drawback in that it cannot by itself discriminate between isomeric mass forms within the oligosaccharides, such as IdoUA/GlcUA stereoisomers, or 3S/6S positional isomers of GlcN. Another approach has been to specifically end-label oligosaccharides with a fluorescent tag. Partial depolymerization then generates a range of fragments similar to those used in this study but simplified in their analysis by the exclusion of all those unlabeled fragments that are not co-terminal with the end-label (37). Tagging methods require additional handling procedures and µg quantities of material for sequencing. The present method is applicable to the analysis of trace quantities of HS produced in cultured cells, the main requirement being the availability of approximately 30,000-40,000 cpm of each 3H-oligosaccharide to be sequenced (which includes a supportive disaccharide analysis). Also, this is realized without recourse to additional chemical modification, with the increased handling and losses this entails, and with access to only standard laboratory equipment (i.e. HPLC and a scintillation counter). Fluorescent tagging is valuable, however, where the oligosaccharide for sequencing is not available metabolically radiolabeled.
Nitrous acid is a specific scission agent that is reported to act with equal probability at all GlcNS residues within an oligosaccharide (38). It would therefore be theoretically possible to obtain an evenly proportioned mixture of fragments from a partial nitrous acid depolymerization. However, when used to cleave the substrates described above, we observed a degree of unexpected selectivity. With all the substrates, the reducing end tetrasaccharide proved to be more resistant than other regions, with fragment R4 always being more abundant than U4, irrespective of the nature of the oligosaccharide. The presence of the specific R4 peak (IdoUA(2S)-GlcNS-IdoUA-GlcNAc) was in fact a significant aid to sequencing, as can be seen in the profiles of Figs. 3-6. It is possible that the local chemical environment of the reducing end makes these tetrasaccharides relatively nitrous acid-resistant. It was also noted that the longer or more complex oligosaccharides were more susceptible to nitrous acid scission, with the reaction times needing to be adjusted accordingly.
A minority of the sequenced S-domains (i.e. 6b, 8b, and 8e)
contain a single glucuronate, whose position in the reducing end disaccharide was determined by the additional use of glucuronidase to
release free GlcNAc from the nonsulfated disaccharide peak (data not
shown). Although not strictly necessary, it is also possible to
positively confirm the unsaturated nature of a completely enzyme-resistant fragment by either specific chemical (mercuric acetate
treatment (39)) or enzymatic (-glycuronidase digestion) removal of
the terminal unsaturated glycuronate residue.
We have elucidated the structures of all the main hexa- and
octasaccharides as well as the two most abundant decasaccharides (Fig.
1), which are compiled in Fig. 7. In
total, these represent approximately 75% of all the S-domains (dp6)
derived from 3T3-fibroblast HS. Although it has been known for some
time that the biosynthetic mechanisms that generate HS impose certain
restraints upon the resulting structures (14, 40-42), it is only when
a number of S-domains of comparable size are actually sequenced that
the true pattern of regulation of individual modifications is fully
revealed. In the present examples, all sequences terminate in GlcNAc.
Internal IdoUA residues flanked by GlcNS are consistently modified by
2-O-sulfation. Variation is seen at the reducing end, where
the hexuronate positioned between GlcNS and the terminal GlcNAc is
either IdoUA, IdoUA(2S), or in a minority of cases, an un-epimerized
GlcUA. The relative proportions of each of these variants is similar
within the hexa- and octasaccharides, with the majority of S-domains
having an IdoUA-GlcNAc-reducing end sequence. An unexpected and
potentially important observation was that the positioning of
6-O-sulfation on a centrally disposed GlcNS residue was a
very tightly controlled modification, occurring only sparingly in the
hexasaccharides (12%) and slightly more often in the octasaccharides
(29%). The fact that this is not a random modification is most clearly
observed from the relative abundance of the various octasaccharides
(Fig. 1). Each of the three dp8 variants, with IdoUA, GlcUA, or
IdoUA(2S) at the reducing end (8a, b, and c, respectively) can be
modified by the addition of a single 6-O-sulfate (giving 8d,
e, and f). However, only a minor although variable fraction of each
variant was modified, although in each case the central positioning of the 6-O-sulfate was conserved. Tightly controlled, nonrandom
patterns such as these may well be highly significant, particularly in relation to basic and acidic FGFs, which require
6-O-sulfates in their activation sequences (27, 28), and
also hepatocyte growth factor, where 6-O-sulfate groups
appear to be the only sulfates required for binding (10). Because of
this regulation, small alterations at the HS biosynthetic level can
cause changes that stand out from a background pattern and may be more
easily and rapidly recognized by HS binding proteins.
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It is becoming apparent that the precise positioning of modifications
within the HS chain is important not only for directing which ligand
can bind (such as the differences between bFGF and hepatocyte growth
factor binding sites) but also for determining whether a bound ligand
is activated or held in an inactive state (28, 43, 44). Although we
have not as yet attempted to sequence the longer and less abundant
S-domains (dp12), which will also be involved in the activation of HS
binding growth factors, it would seem likely that, for at least the
3T3-fibroblast HS, the patterns observed within the smaller S-domains
will be mirrored in the larger ones but perhaps with more complex
patterns of 6-O-sulfation.
The use of specific exo-glycosidases has, for a number of years, formed the basis of a method for sequencing branched N-linked oligosaccharides. The reagent array method initiated a flood of interest in the field of glycobiology, as investigators began to realize its potential to open up the information held in these complex branched sugars (45, 46). As a result of the use of this technique, many biological problems have been addressed; for example the importance of the glycosylation of IgG in rheumatoid arthritis (47, 48). Glycosaminoglycan sequence analysis, on the other hand, still retains the reputation of being a somewhat arcane art, with detailed, accurate sequence information only available in very few cases. The technique described here can be used to routinely and reliably sequence HS from cultured cells, the most commonly studied source of HS. It is hoped that this technique will make HS sequencing a practical proposition for any reasonably equipped laboratory interested in the structure and function of these hypervariable regulatory polymers. With only a few modifications, the same method can be applied to saccharides released from HS by other scission methods such as heparinase I or partial nitrous acid. Depending upon the availability of a partial scission method and an appropriate array of specific exoenzymes, this basic protocol could in theory also be adapted to the sequencing of oligosaccharides derived from other complex glycosaminoglycans, e.g. dermatan sulfate.
The data presented in this paper reveals new and important
characteristics of S-domain sequence and diversity in 3T3 fibroblast HS. In addition to helping to elucidate specific protein recognition domains in HS, this approach should form the basis for the future elucidation of HS sequence patterns that define different cell types,
stages in embryonic development, and disease processes (e.g.
tumorigenesis) and may also contribute toward realizing a true whole
chain analysis of HS.
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ACKNOWLEDGEMENTS |
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We thank S. Stringer, D. Pye, and R. Vivés for their helpful advice and K. Beckman and C. Boulter for production of the recombinant enzymes.
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FOOTNOTES |
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* This work was supported by grants from the Cancer Research Campaign (to M. L., J. A. D., and J. T. G.), the National Health and Medical Research Council of Australia (to J. J. H.), the Biotechnology and Biological Sciences Research Council (to C. L. R. M.), and Glaxo Wellcome (to C. L. R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Cancer Research Campaign and University of Manchester Department of Medical Oncology, Christie Hospital NHS Trust, Manchester, M20 4BX, UK. Tel.: 44 161 446 3201; Fax: 44 161 446 3269; E-mail:jgallagher{at}picr.man.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are:
HS, heparan sulfate;
GlcNAc, N-acetylglucosamine;
GlcUA, glucuronic acid;
IdoUA, iduronic acid;
GlcNS, N-sulfated glucosamine;
bFGF, basic
fibroblast growth factor;
SAX-HPLC, strong anion exchange high pressure
liquid chromatography;
dp, degree of polymerization or number of
saccharide units, e.g. dp6 = hexasaccharide;
aMan, anhydromannose;
2-sulfatase, -L-iduronate-2-sulfatase;
iduronidase, -L-iduronidase;
6-sulfatase, -D-glucosamine-6-sulfatase;
S-domain, sulfated domain;
HexA, hexuronic acid.
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ABSTRACT
INTRODUCTION
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