From the Department of Medical Biochemistry and
Microbiology, Uppsala University, The Biomedical Center, Box 575, S-751
23 Uppsala, Sweden and the ¶ Department of Oncology, Hadassah
University Hospital, P.O. Box 12000, Jerusalem 91120, Israel
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ABSTRACT |
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Heparan sulfate proteoglycans, attached to cell surfaces or in the extracellular matrix, interact with a multitude of proteins via their heparan sulfate side chains. Degradation of these chains by limited (endoglycosidic) heparanase cleavage is believed to affect a variety of biological processes. Although the occurrence of heparanase activity in mammalian tissues has been recognized for many years, the molecular characteristics and substrate recognition properties of the enzyme(s) have remained elusive.
In the present study, the substrate specificity and cleavage site of
heparanase from human hepatoma and platelets were investigated. Both
enzyme preparations were found to cleave the single
-D-glucuronidic linkage of a heparin
octasaccharide. A capsular polysaccharide from Escherichia
coli K5, with the same (-GlcUA
1,4-GlcNAc
1,4-)n structure as the unmodified backbone of heparan sulfate, resisted heparanase degradation in its native state as well as after chemical N-deacetylation/N-sulfation or partial
enzymatic C-5 epimerization of
-D-GlcUA to
-L-IdceA. By contrast, a chemically
O-sulfated (but still N-acetylated) K5
derivative was susceptible to heparanase cleavage.
O-Sulfate groups, but not N-sulfate or IdceA
residues, thus are essential for substrate recognition by the
heparanase(s). In particular, selective O-desulfation of
the heparin octasaccharide implicated a 2-O-sulfate group
on a hexuronic acid residue located two monosaccharide units from the
cleavage site, toward the reducing end.
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INTRODUCTION |
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Heparin is synthesized by connective tissue type mast cells, as part of serglycin proteoglycans, and is subsequently stored intracellularly in cytoplasmic granules. Heparan sulfate (HS),1 structurally related to heparin, is also elaborated in proteoglycan form; contrary to heparin, heparan sulfate chains may be associated with a variety of core proteins (1, 2). Heparan sulfate proteoglycans typically occur at the surface of most types of cells as well as in the extracellular matrix. They have been attributed a variety of biological effects that generally appear to be mediated through interactions between the constituent polysaccharide chains and various proteins (2-5).
The highly extended polysaccharide chains, Mr
60,000-100,000, of heparin proteoglycans may be cleaved shortly after
completed biosynthesis, in the mast cell, into fragments similar in
size to commercially available heparin (Mr
5000-25,000). The enzyme responsible for this conversion was
identified as an endo--D-glucuronidase (6).
Endoglycosidases capable of partially depolymerizing HS chains have
been demonstrated in a variety of cells and tissues, such as placenta
(7), skin fibroblasts (8), platelets (9), melanoma (10), lymphoma (11),
hepatocytes (12), CHO cells (13), and endothelial cells (14). Such
enzymes, commonly referred to as "heparanases," contribute to the
intracellular degradation of HS proteoglycans (15, 16) but are also
released into the extracellular matrix, where they appear to modulate
several processes of pathophysiological importance. Heparanases thus
contribute to the remodeling of extracellular matrix and basement
membranes prerequisite to angiogenesis (17) and to the egress of
metastatic tumor cells (18) and other types of blood-borne cells (19) from the vasculature. They may regulate growth factor action by releasing heparan sulfate-bound growth factors such as basic fibroblast growth factor from cell surfaces or from the extracellular matrix (20,
21). Heparanases have also been implicated in the release of
subendothelial bound lipoprotein lipase (22). Moreover, cleavage of HS
chains on the vascular endothelium may affect the local concentration
of HS with blood anticoagulant properties, thereby modulating the
coagulation process (9).
Studies by Oldberg et al. (9) suggested that the heparanase
obtained from human platelets cleaves
endo--D-glucuronidic linkages in HS, thus mimicking the
action of the heparin-degrading enzyme in mast cells. Similar
endoglucuronidase activity was reported in subsequent similar studies
on heparanases from different sources, such as human placenta (7),
human platelets (23), mouse melanoma (24), and CHO cells (25). In
contrast, Hoogewerf et al. (26) attributed
endo-
-D-glucosaminidase properties to a heparanase purified from human platelets and further proposed, based on amino acid
sequencing, that this enzyme was identical to connective tissue-activating peptide-III/neutrophil-activating peptide-2. These
components and
-thromboglobulin are differently truncated forms of
platelet basic protein, a member of the CXC-chemokine family.
Several attempts have been made to define the substrate recognition properties of heparanases from various sources (6, 9, 16, 25, 27-31). The approaches taken have involved structural comparison between polysaccharides susceptible or resistant to an enzyme, sequence analysis of fragments generated by enzymatic cleavage, and studies of inhibitory effects of chemically modified heparin derivatives. While most of these studies pointed to the importance of sulfate groups, no unified pattern emerged, and the minimal structural requirements for substrate recognition have remained to be defined.
The aim of the present study was to pinpoint the substrate recognition
properties of two heparanases from different sources, human hepatoma
and platelets. Both enzymes were identified as endo--D-glucuronidases, based on their action on a well
defined oligosaccharide substrate. Further model substrates were
generated by systematic modification of the capsular polysaccharide
generated by Escherichia coli K5, which has the same
structure as the precursor polymer in heparin/HS biosynthesis. Testing
various K5 polysaccharide derivatives as substrates for the heparanases
revealed that whereas neither N-sulfate groups nor IdceA
units are required for substrate recognition, the presence of
O-sulfate groups, particularly on the hexuronic acid units,
appears to be essential for enzyme action.
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EXPERIMENTAL PROCEDURES |
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Enzymes--
Platelet heparanase was partially purified from 500 ml of human platelet-rich plasma (kindly given by Dr. C.-H. Heldin, The Ludwig Institute, Uppsala, Sweden) according to a modification of a
previously described procedure (23). Briefly, the enzyme was recovered
following fractionation on heparin-Sepharose, phenyl-Sepharose CL-4B
and (passage through) DEAE-Sephacel. The partially purified enzyme
contained <3 µg of protein/ml and was stored in portions at
20 °C. Hepatoma heparanase was partially purified from a human hepatoma cell line (Sk-hep-1), essentially as described for the placental heparanase (32), omitting the ammonium sulfate precipitation and adding a second cation exchange (CM-Sepharose) chromatography at pH
7.2. Briefly, the cells were grown in suspension in Dulbecco's modified Eagle's medium (4.5 g of glucose/liter) containing 10% calf
serum to a concentration of 106 cells/ml in batches of 50 liters. Cell lysate was subjected to 1) cation exchange (CM-Sepharose)
chromatography performed at pH 6.0; 2) cation exchange (CM-Sepharose)
chromatography at pH 7.2; 3) heparin-Sepharose chromatography at pH
7.2; and 4) ConA-Sepharose chromatography at pH 6.0. Active fractions
were eluted by NaCl gradients in 10 mM phosphate citrate
buffer (steps 1-3) and by phosphate citrate buffer containing 1 M NaCl and 0.25 M
-methyl mannoside (step
4). The partially purified (~240,000-fold purification) preparation
eluted from ConA-Sepharose contained 10-20 µg of protein/ml and
consisted primarily of a ~46-kDa protein. Commercially available human heparanase was purchased as
-thromboglobulin from
Calbiochem.
K5-derived Saccharides-- Capsular polysaccharide from E. coli K5 was kindly given by Dr. V. Cavazzoni (Department of Industrial Microbiology, University of Milan, Italy).
A 35S-labeled heparin-like (or HS-like) polysaccharide was generated by incubating 25 µg of K5 polysaccharide with 4.8 mg of solubilized mouse mastocytoma microsomal enzymes and 3.5 MBq of 3'-phosphoadenosine-5'-phospho[35S]sulfate (PAPS) (specific activity, 6.6 TBq/mol), essentially as described by Kusche et al. (33). Anion exchange chromatography of the product on DEAE-Sephacel showed an elution position similar to that of standard chondroitin 4-sulfate from cartilage (data not shown). Metabolically 3H-labeled K5 polysaccharide was prepared by growing E. coli K5 in the presence of D-[1-3H]glucose (34). The products were partially or exhaustively chemically N-deacetylated (by hydrazinolysis) (35) and were then N-sulfated (36). A portion of ~50% N-sulfated [3H]K5 polysaccharide was incubated with D-glucuronyl C-5 epimerase, purified from bovine liver, yielding a product in which ~30% of the D-glucuronyl residues located between adjacent N-sulfated GlcN units had been converted into IdceA (34). Chemically O-sulfated K5 polysaccharide, obtained by treating the native product with pyridine-sulfur trioxide (according to route A as described by Casu et al. (37), except that no N-deacetylation/N-sulfation was performed) was kindly given by Dr. A. Naggi (Ronzoni Institute, Milan, Italy). NMR analysis of this material indicated a fully 6-O-sulfated polysaccharide with some sulfate groups also on GlcUA.2 The absence of N-sulfate groups was shown by the resistance of the polysaccharide to depolymerization by HNO2 at pH 1.5 (38) (data not shown). To radiolabel this material, 0.5 mg of the polysaccharide was first partially N-deacetylated by hydrazinolysis (in 0.55 ml of hydrazine hydrate (Fluka) containing 1% hydrazine sulfate) at 100 °C for 5 min (35). The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) in 10% aqueous ethanol and lyophilized. The polysaccharide was cleaved at the generated N-unsubstituted GlcN residues by nitrous acid treatment at pH 3.9 (38) and labeled by reduction of the resultant terminal anhydromannose units with 0.5 mCi of NaB3H4 (39). The 3H-labeled product (specific activity, ~100 × 103 cpm/µg of hexuronic acid) was recovered by gel filtration on Sephadex G-15 (1 × 150 cm) in 0.2 M NH4HCO3 and further separated on a Superose 12 gel filtration column in 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4. Fractions containing material of Mr >5000 (specific activity, ~10 × 103 cpm/µg of hexuronic acid) were pooled, dialyzed against H2O, and lyophilized. Chemically N- and O-sulfated K5 polysaccharide was as described (40) (preparation Bb-2, fraction 4, recovered after chromatography on antithrombin-Sepharose). This material was subjected to partial depolymerization by limited deamination at pH 1.5 followed by reduction of the products with NaB3H4 (40). Labeled polysaccharide (Mr >5000; specific activity, ~15 × 103 cpm/µg of hexuronic acid) was isolated by gel chromatography on Bio-Gel P-10 (1 × 150-cm column; fine grade) in 0.5 M NH4HCO3.Heparin/Heparan Sulfate-derived Saccharides-- N-[3H]Acetyl-labeled heparin (specific activity, 88 × 103 cpm/µg of hexuronic acid) was prepared as described by Höök et al. (41) and separated into fractions with high and low affinity for antithrombin by affinity chromatography (42).
An octasaccharide with high affinity for antithrombin was recovered following partial deaminative cleavage of heparin. Samples were labeled at the reducing end by reduction with NaB[3H]4 (specific activity of product, ~100 × 103 cpm/µg of hexuronic acid) or by treatment with [14C]acetic anhydride following N-deacetylation by hydrazinolysis (specific activity of product, ~130 × 103 cpm/µg of hexuronic acid) (28). A sample of 3H-labeled octasaccharide was degraded to labeled hexasaccharide by N-deacetylation followed by deamination (pH 3.9), and the hexasaccharide product was further converted to pentasaccharide by digestion with exo-De-O-Sulfation of Saccharides--
Selective
2-O-desulfation was performed according to Jaseja et
al. (Ref. 44; see also Ref. 45). An
N-[14C]acetyl-labeled heparin octasaccharide
(670 × 103 cpm, 5.3 µg of hexuronic acid) with high
affinity for antithrombin was mixed with 4 mg of heparin
tetrasaccharide (derived from bovine lung heparin by partial
deamination at pH 1.5) in a total volume of 1 ml of 0.13 M
NaOH, pH 12.5. The mixture was frozen at 70 °C and lyophilized.
The radiolabeled material was separated from the tetrasaccharide
carrier on a column (1 × 200 cm) of Sephadex G-25 (superfine
grade) in 0.2 M NH4HCO3 and
lyophilized.
Heparanase Incubation--
Samples of radiolabeled saccharide
substrate, typically ~10,000 dpm, were mixed with heparanase
(generally ~1 µg of hepatoma enzyme, ~0.5 µg of platelet
enzyme, 5 µg of -thromboglobulin) in 10 mM citrate
phosphate buffer, pH 5.8, 0.5 mM CaCl2, 100 mM NaCl, 0.5 mM dithiothreitol in a total
volume of 200 µl. Occasionally, an internal standard of
35S-labeled heparan sulfate (10,000 dpm) was included as a
positive control of enzyme activity. After incubation at 37 °C for
16 h, the samples were heated at 95 °C for 5 min. The digests
were mixed with 200 µl of 2 M NaCl, 100 mM
Tris-HCl, pH 7.4, 0.2% Triton X-100 and were then centrifuged for 10 min at 13,000 rpm before gel chromatography. Control samples were
incubated in parallel without the addition of enzyme.
Analytical Procedures-- For compositional analysis of chemically O-sulfated K5 polysaccharide, the sample was N-deacetylated by hydrazinolysis for 5 h and was then subjected to deaminative cleavage by treatment with HNO2 at pH 3.9 (38). The resultant disaccharides were reduced with NaB3H4, isolated by gel chromatography, and finally analyzed by anion exchange HPLC on a Partisil-10 SAX column. For additional experimental details, see Ref. 47.
Structural analysis of N-sulfated saccharide was performed by deaminative cleavage (pH 1.5) at N-sulfated GlcN residues (38), followed by reduction with NaB3H4. The resultant 3H-labeled disaccharide was isolated by gel chromatography and analyzed by anion exchange HPLC. Digestion of oligosaccharides with exo- ![]() |
RESULTS |
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Incubations of radiolabeled heparan sulfate (Fig. 1A) or heparin (Fig. 1B) with heparanase derived from hepatoma cells resulted in appreciable degradation of both polysaccharides, as shown by gel chromatography. Similar results were obtained with the platelet enzyme (data not shown). Heparin preparations with high or low affinity for antithrombin appeared equally susceptible to cleavage (data not shown). Experiments were undertaken to define the type of glycosidic bond cleaved by the enzymes and to elucidate their substrate recognition properties.
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Heparanases Cleave -D-Glucuronidic
Linkages--
Previous experiments demonstrated that platelet
heparanase cleaves a single glycosidic bond in a heparin octasaccharide
having high affinity for antithrombin (28). Gel chromatography of the cleavage products along with oligosaccharide standards, obtained by
partial deaminative cleavage of heparin, suggested that cleavage had
occurred between monosaccharide units 3 and 4 from the nonreducing terminus (indicated by the open arrow
in Fig. 2), in accord with the postulated
-D-glucuronidase properties of the enzyme. By contrast,
a platelet heparanase more recently isolated was claimed to be an
-D-glucosaminidase (26). The putative glucosaminidase activity was associated with purified connective tissue-activating peptide-III, which occurs in commercially available
"
-thromboglobulin" (a protein 4 amino acid residues shorter than
connective tissue-activating peptide-III at the N terminus).
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O-Sulfate Groups Are Essential for Heparanase Activity--
The
capsular polysaccharide generated by E. coli K5 has the same
(-GlcUA1,4-GlcNAc
1,4-)n structure as the precursor polymer in heparin/HS biosynthesis. Completed HS chains contain major
regions of such sequence that have escaped biosynthetic polymer
modification (47). Incubation of a 3H-labeled K5
polysaccharide preparation, obtained by growing the E. coli
K5 strain in the presence of [1-3H]glucose, with hepatoma
heparanase did not lead to any appreciable degradation of the polymer
(Fig. 4, A and B).
By contrast, a sample of 35S-labeled heparan sulfate that
was included as an internal control showed a drastic reduction in size
(indicated by the arrows in Fig. 4, A and
B). The glucuronidic linkages in the native K5
polysaccharide thus were resistant to enzymatic cleavage.
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Specification of O-Sulfate Requirement--
The
O-sulfated K5 polysaccharide was subjected to compositional
analysis, involving complete N-deacetylation, deaminative cleavage, reduction/3H-labeling of the resultant
disaccharides, and separation of the products by anion exchange HPLC
(Fig. 7). A major peak (67% of the total
labeled disaccharides) appeared at the elution position (~45 min) of
GlcUA-aManR(6-OSO3) that would correspond to a
-GlcUA-GlcNAc(6-OSO3)- sequence in the intact polymer. In
addition, a smaller (25% of total disaccharides), more retarded peak
was observed after 90 min, presumably representing a
di-O-sulfated disaccharide species. Treatment of the
disaccharide mixture with exo--D-glucuronidase eliminated the mono-O-sulfated species, as expected, but did
not affect the di-O-sulfated component (data not shown).
This result indicates that the latter compound contains a sulfate group
on the GlcUA residue that confers resistance toward exoenzymatic cleavage of the corresponding glucuronidic linkage.
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DISCUSSION |
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The existence of heparanase type endoglycosidases has been recognized for over 2 decades, yet several basic features of the enzymes remain poorly understood. Although early reports pointed to the occurrence of distinct enzyme species with different substrate specificities (28, 48), neither the number of enzymes nor their substrate recognition properties have been defined. A recent comparison of the enzyme characteristics such as molecular size, pI, and pH optimum of heparanases from mouse melanoma cells, mouse macrophages, and human platelets suggested that the enzymes were highly similar, although the human platelet enzyme was found to be smaller than the murine homologs (49). On the other hand, Bame et al. (25) concluded, in a study of heparanases from CHO cells, that a single type of cells may contain several heparanases with different substrate specificities. While the present investigation does not provide any information as to the number of different enzymes, it is noted that the heparanase preparations recovered from human platelets and from hepatoma cells could not be distinguished on the basis of substrate specificity.
Identifying the target glycosidic bond for heparanase action has been a
matter of controversy. Early studies of endoglycosidases from mouse
mastocytoma (6) and from human platelets (9, 23) implicated
-D-glucuronidic linkages. More recently, however, it was
proposed that a heparanase from platelets has
endo-
-D-glucosaminidase activity (26). The results of
the present study, based on identification of fragments generated by
heparanase cleavage of a well defined heparin octasaccharide, clearly
point to glucuronidic linkages as the site of cleavage. These results
applied also to the commercially available
-thromboglobulin
preparations that were claimed to express endoglucosaminidase activity
and thus contradict the conclusion of Hoogewerf et al.
(26).
The simplest polysaccharide available that is basically related to
heparin/HS is the (-GlcUA1,4-GlcNAc
1,4-)n species, which
has a structure identical to that of the initial polymer formed in
heparin/HS biosynthesis (50). This polysaccharide may be isolated in
large quantities from cultures of E. coli K5 and thus
provides a well defined starting point for attempts to elucidate the
substrate specificity of heparanase. The resistance of this polymer to
heparanase digestion indicates that substrate recognition depends on
one or more of the structural features introduced during biosynthetic
polymer modification. Indeed, exposing the K5 polysaccharide to the
action of solubilized mastocytoma microsomal enzymes led to the
generation of a product that was susceptible to heparanase-induced
cleavage (Fig. 5). Notably, the relatively sparse distribution of
susceptible bonds pointed to a composite recognition site involving a
combination of different structural features. More specific information
was obtained by screening a series of variously modified K5
polysaccharide samples for heparanase susceptibility (Table I). These
experiments revealed that neither N-sulfate groups (see also
Ref. 51) nor IdceA units were required, whereas O-sulfate
groups were found to be essential for enzyme action.
Selective O-desulfation of the antithrombin-binding heparin octasaccharide, previously found to be cleaved by the enzyme, provided more detailed insight into the substrate recognition properties of the heparanase(s) studied. While 6-O-desulfation of this molecule did not prevent the predicted degradation upon incubation with heparanase, the 2-O-desulfated analog not only resisted cleavage but also appeared to inhibit the enzyme (Fig. 8). The single 2-O-sulfated hexuronic acid residue in position 5 of the octasaccharide substrate (Fig. 2) would therefore seem to be essential for substrate recognition and subsequent cleavage of the linkage between units 3 and 4. While we cannot exclude the possibility of a promoting effect on heparanase action, an obligatory requirement for the 3-O-sulfate group on unit 4 appears unlikely for several reasons. First, structural analysis of the chemically O-sulfated K5 polysaccharide, found to be a substrate for the heparanase preparations, revealed no evidence of 3-O-sulfated GlcN units. Moreover, heparin with low affinity for antithrombin was degraded by heparanase to about the same extent as high affinity heparin, despite an ~10-fold lower content of GlcN 3-O-sulfate groups (39). Finally, Bai et al. (16) recently found aberrant turnover of HS in CHO cells defective in a sulfotransferase required for 2-O-sulfation of hexuronic acid residues and concluded that 2-O-sulfated IdceA units are important for cleavage of HS by intracellular heparanase.3 The results of the present study suggest that the single 2-O-sulfated hexuronic acid unit required for substrate recognition by the heparanase may be either GlcUA or IdceA. Furthermore, the N-sulfate groups that are located close to the target glucuronidic linkage for the heparanase in the antithrombin-binding region (Fig. 2) and in HS (16) are prerequisite to the incorporation of the essential 2-O-sulfate groups during polysaccharide biosynthesis (3, 4) but apparently are not per se required for heparanase action. The structural characteristics of a minimally O-sulfated HS sequence required for substrate recognition by the heparanase(s) (cleavage at the arrow) thus could be summarized as follows (see also Bai et al. (16)).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Annamaria Naggi and Dr. Benito Casu (Ronzoni Institute, Milan, Italy) for the gift of O-sulfated K5 polysaccharide and for constructive criticism of the manuscript.
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Addendum |
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The predicted selective 2-O-desulfation was ascertained by structural analysis of alkali-treated octasaccharide. Deaminative cleavage of (reduced) octasaccharide (treatment with nitrous acid at pH 1.5), followed by reduction of the products with NaB3H4, yielded a labeled disaccharide that corresponded to units 5 and 6 of the structure shown in Fig. 2. While this disaccharide was identified (by anion exchange HPLC; see "Experimental Procedures" and the legend to Fig. 7) exclusively as the disulfated species, IdceA(2-OSO3)-aManR(6-OSO3), the corresponding component from the alkali-treated octasaccharide was IdceA-aManR(6-OSO3) (data not shown). The 2-O-sulfate group thus had been removed, whereas the 6-O-sulfate group remained bound to the GlcN residue.
The interpretation of the results of heparanase incubations are potentially complicated by alkali-induced modifications other than 2-O-desulfation of the octasaccharide. These include N-deacetylation of GlcNAc residues as well as conversion of 3-O-sulfated GlcNSO3 units to an aziridine derivative that resists cleavage by nitrous acid (Ref. 45; A. Naggi and B. Casu, personal communication). In accord with these observations ~30% of the initial N-[14C]acetyl label was released from the octasaccharide due to the alkali treatment (data not shown). However, the yield of 3H-labeled disaccharide derived from units 5 and 6, as compared with that of the hexasaccharide 1-6 (Fig. 2), following HNO2/NaB3H4 treatment (see above) indicated that the GlcNSO3 unit 4 remained largely susceptible to deamination (data not shown). We therefore conclude that more than half of the total 2-O-desulfated octasaccharide molecules would have escaped the side reactions, hence that the observed resistance of the modified octasaccharide to heparanase cleavage should be ascribed to the lack of the 2-O-sulfate group.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grant 2309, European Commission Grant BIO4-CT95-0026, and Polysackaridforskning AB (Uppsala, Sweden).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. Tel.: 46-18-4714242; Fax: 46-18-4714209; E-mail: Dagmar.Pikas{at}medkem.uu.se.
1 The abbreviations used are: HS, heparan sulfate; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; aManR, 2,5-anhydro-D-mannitol; CHO, Chinese hamster ovary; HPLC, high pressure liquid chromatography.
2 B. Casu, personal communication.
3 While this conclusion is probably correct, it is recalled that a 2-O-sulfated IdceA unit was identified as part of the substrate recognition site for the 3-O-sulfotransferase involved in the biosynthesis of the antithrombin-binding region (52); a polysaccharide devoid of HexA 2-O-sulfate groups thus will probably lack GlcN 3-O-sulfate substituents.
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REFERENCES |
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