 |
INTRODUCTION |
Interactions between adherent cells and the extracellular
environment influence maintenance of cellular functions such as proliferation, differentiation, and migration. Heparan sulfate proteoglycans (HS-PGs)1 are
covalently linked protein-HS glycosaminoglycan (GAG) conjugates found
in extracellular matrix (ECM) and on the cell surface of most cells and
have been demonstrated to be key components of the cell-cell and
cell-ECM interactions (for reviews, see Refs. 1-4). Most of the
biological properties of HS-PGs are conferred by the HS moiety, which
is a sulfated polydisperse copolymer of alternating GlcN and HexUA
(GlcUA or IdoUA) residues (for reviews, see Refs. 5 and 6).
HS-GAG binds to and co-localizes with structural proteins, such as
fibronectin and collagen in the ECM, providing a framework for matrix
organization (for reviews, see Refs. 7-9). Both cell surface and ECM
HS-PGs also tether various growth/differentiation factors and cytokines
as storage depots of bioactive signaling molecules. There is clear
evidence that an association of a ligand with HS-GAGs can activate or
stabilize the ligand and also facilitate signal transduction via its
high affinity receptor (for reviews, see Refs. 10-12).
The interaction of a ligand with ECM or cell surface HS-PGs is
regulated by biosynthesis of specific sulfation sequences, which bind
to the ligand (for reviews, see Refs. 5, 13, and 14). Indeed, fine
sulfation structures of HS-GAGs change during development, aging, or
tumor progression to malignancy, and the abilities of specific ligands
to bind HS-PGs are switched (15-18). Another way to alter the
functional state of HS-PGs is to degrade and release HS-GAGs from the
core proteins, which is achieved by the specific action of an
endoglycosidase, heparanase (for reviews, see Refs. 19 and 20).
Heparanase is the name of mammalian endoglucuronidase capable of
specifically cleaving HS-GAGs and differs from bacterial eliminases
such as heparinase and heparitinase (21). The extracellular heparanase
has been implicated in basement membrane remodeling after injury or at
inflammation sites by destroying HS-GAG chains and in the regulation of
cell growth and differentiation by releasing growth factors that are
bound to extracellular HS-PGs. There is also evidence that the
heparanase activity correlates with the metastatic potential of tumor
cells in animal models and increases in the sera of human patients with
metastatic cancers (22-24).
The heparanase research had been hampered by the limited abundance and
unstable enzyme activity as well as the lack of a simple assay method.
Recently, several laboratories have developed a variety of assay
methods, purified human heparanase, and isolated the cDNA (25-29).
It became clear that heparanases previously purified from various
sources are identical and that the heparanase is initially synthesized
as an inactive 65-kDa glycoprotein and then processed into the active
50-kDa enzyme by cleavage of the N terminus peptide (for reviews, see
Refs. 19 and 20). A direct role of the heparanase in tumor cell
invasion was confirmed by the transfection of the sense and antisense
heparanase cDNA into cells, which acquired highly and poorly
metastatic phenotypes, respectively (26, 30). High expression of the
heparanase mRNA was also observed in advanced stage tumors and
metastatic cell lines derived from various tissues (27, 28, 31,
32).
The aim of the present study was to explore the substrate recognition
property of the human recombinant heparanase. Previously, several
attempts were made to define the substrate specificity of the
heparanase partially purified from different animal sources (for
reviews, see Refs. 19 and 20). Most approaches have involved the
structural analysis of the fragments generated by enzymatic cleavage of
polymer HS from various tissues and heparin (Hep) polysaccharides
derived from the lung and intestine. Specificity studies on human
heparanase using structurally defined oligosaccharide substrates have
been limited to those performed using partially purified enzyme
preparations (33, 34). In the present study, we systematically
investigated the specificity of the purified recombinant human
heparanase using a number of structurally defined oligosaccharides as
substrates and revealed the hitherto unreported specificity of the
enzyme, which will aid establishing the quantitative assay methods and
designing inhibitors for new therapeutic strategies in highly
metastatic cancer and other heparanase-related diseases, notably
inflammatory or cardiovascular diseases.
| |
EXPERIMENTAL PROCEDURES |
Materials--
HS (sodium salt) from bovine kidney and
concanavalin A-agarose was purchased from Seikagaku Corp. (Tokyo,
Japan). Stage 14 Hep (sodium salt) from porcine intestinal mucosa was
obtained from American Diagnostic Inc. (New York). TSK-GEL
G3000SWXL was from TOSOH Corp. (Tokyo, Japan). Sephacryl
S-300HS and Hep-Sepharose CL-6B columns as well as a prepacked
disposable PD-10 column containing Sephadex G-25 medium were purchased
from Amersham Biosciences. Fluorescein isothiocyanate (FITC) was from
Sigma-Aldrich. Structurally defined oligosaccharides were isolated from
porcine intestinal Hep or bovine kidney HS, and their homogeneity was
judged by capillary electrophoresis as described previously
(35-41).
Preparation of FITC-HS and -Hep--
HS (sodium salt) from
bovine kidney and Hep (sodium salt) from porcine intestinal mucosa were
labeled with FITC as described previously (25).
Cell Culture--
Human melanoma A375M cells were cultured as
monolayers in RPM-I1640 (Nissui, Tokyo, Japan) supplemented with
heat-inactivated 10% fetal bovine serum, 2 mM
L-glutamine, penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 mg/ml) in humidified 95% air, 5%
CO2 at 37 °C.
Preparation of Human Heparanase--
The 1632-bp-long cDNA
coding for a human heparanase was inserted into an expression vector,
pcDNA3.1/Hygro (Invitrogen), and the vector was transfected into
human melanoma A375M cells using the LipofectAMINETM
reagent (Invitrogen). The transfectants were selected by hygromycin resistance and a stable transfectant cell line expressing a high level
of recombinant human heparanase was established. After the cells had
grown to confluence, they were harvested and homogenized in a lysis
buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.5% Triton X-100, 0.2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 µg/ml aprotinin). The cell lysate was then
loaded onto a Hep-Sepharose column (5.0 × 10 cm) preequilibrated
with 50 mM Tris-HCl, pH 7.5, containing 150 mM
NaCl at a flow rate of 3.0 ml/min, and heparanase was eluted with the
same buffer, containing 1.0 M NaCl as described previously (25). The 1.0 M NaCl-eluted fractions were diluted with an
equal volume of the dilution buffer (50 mM sodium acetate
buffer, pH 6.0, containing 0.4% CHAPS) and applied to a concanavalin
A-agarose column (2.5 × 10 cm) at a flow rate of 0.8 ml/min.
Heparanase was eluted with 50 mM sodium acetate buffer, pH
6.0, containing 0.5 M NaCl, 0.2% CHAPS, and 0.7 M 
-methylmannose as described previously (25). The
concanavalin A-agarose-eluted fractions were diluted 3.3-fold with 50 mM sodium acetate buffer, pH 6.0, containing 0.2% CHAPS,
and loaded onto an immunoaffinity column (1.0 × 5 cm) containing
the anti-heparanase polyclonal antibody (42) at a flow rate of 0.18 ml/min. Heparanase was eluted with 0.1 M glycine-HCl, pH
2.7, containing 0.05% CHAPS at a flow rate of 0.5 ml/min. Fractions of
1 ml were collected, and aliquots were used for heparanase assay and
SDS-PAGE, which was performed under reducing conditions employing
4-20% gradient polyacrylamide gels (84 × 91 mm) in the
Tris-glycine buffer system (43). After electrophoresis, gels were
subjected to silver staining. The human heparanase protein was
quantified by the BCA protein assay (Pierce).
Determination of Heparanase Activity--
Human heparanase
activity was determined from the gel permeation HPLC chromatogram of
the enzyme digest of FITC-HS by measuring a forward half area of the
peak of the intact FITC-HS as described previously (25). The decrease
in this peak area observed after heparanase treatment was measured
using an integrator, and the amount of the degraded FITC-HS was
calculated from the decrease in fluorescence intensity. One unit was
defined as the quantity of the enzyme that degraded 10 ng of
FITC-HS/min.
Enzymatic Digestion of FITC-Hep with Human
Heparanase--
Enzyme reactions were carried out in 100 µl of 0.1 M sodium acetate buffer, pH 4.2, containing 1 µg of
FITC-Hep, at 37 °C for 3 h and terminated by the addition of
100 µg of nonlabeled porcine intestinal Hep and subsequent heating at
100 °C for 5 min. The digests were centrifuged at 15,000 rpm for 5 min to precipitate the insoluble materials. The supernatant fluids were
analyzed by gel filtration HPLC on a TSK-GEL G3000SWXL
column (0.78 × 30 cm) preequilibrated with 50 mM
Tris-HCl, pH 7.5, containing 150 mM NaCl and 0.05% (v/w)
NaN3 at a flow rate of 1 ml/min.
Enzymatic Digestion of Structurally Defined Oligosaccharides with
Human Heparanase--
Structurally defined tetra- or hexasaccharide
(0.3 nmol each) isolated from porcine intestinal Hep or bovine kidney
HS was digested with 0.88 or 0.44 units of the purified human
heparanase in a total volume of 100 µl of 0.1 M sodium
acetate buffer, pH 4.2, at 37 °C for 21 h or the indicated
periods. The enzymatic reactions were terminated as described above. As
control experiments, each oligosaccharide was incubated with the
heat-inactivated heparanase under the same conditions. Each enzyme
digest was analyzed by HPLC on an amine-bound silica column as reported
previously (44). Eluates were monitored at 232 nm. The sensitivity of
each oligosaccharide to the enzyme was judged by the peak shift to
earlier elution positions. The area of the individual peaks was
compared before and after heparanase treatment, and the amount of the
degraded oligosaccharide was calculated from the decrease in the intact peak area using an integrator.
| |
RESULTS |
Expression and Purification of the Recombinant Human
Heparanase--
The pcDNA3/Hygro vector containing the full-length
human heparanase cDNA clone was expressed in human melanoma A375M
cells. The cell lysate prepared from the stable transfectant cells was subjected to affinity chromatographies using the matrices
Hep-Sepharose, concanavalin A-agarose, and anti-heparanase
antibody-immobilized Affi-Gel 10 to purify the recombinant enzyme.
Aliquots of fractions 1-17 eluted from the anti-heparanase antibody
column were subjected to the heparanase assay and SDS-PAGE followed by
silver staining (Fig. 1). The strong
catalytic activity toward FITC-HS was detected in fractions 6-8.
Heparanase is initially synthesized as an inactive 65-kDa glycoprotein
that is cleaved at the N terminus to generate an active 50-kDa enzyme
(for reviews, see Refs. 19 and 20). Being consistent with this, a
50-kDa band was detected evidently on SDS-PAGE in the fractions
containing HS-degrading activity. Silver staining showed a 30-kDa
protein band in fractions 6-8 and the faint 65-kDa protein band, which
corresponded to the IgG light chain, probably leaking from the
antibody-immobilized column and the enzyme precursor, respectively.
Fractions 6-8 were combined and used to investigate the substrate
specificity of the heparanase. The protein concentration and the
heparanase activity of the pooled fractions were measured. The specific
activity was 1.6 units/µg of protein.
View larger version (50K):
[in this window]
[in a new window]
|
Fig. 1.
SDS-PAGE of the fractions separated by
immunoaffinity chromatography using the anti-heparanase
antibody-immobilized column. An aliquot of each fraction eluted
from an anti-heparanase antibody-immobilized column was analyzed by
SDS-PAGE using a 4-20% gradient polyacrylamide gel. Three protein
bands are evident by silver staining and have molecular masses of
~30, 50, and 65 kDa, respectively. The 30-kDa band corresponds to the
IgG light chain leaked from the antibody-immobilized column. Fractions
6-8 were pooled and used for specificity studies.
|
|
Detection of the Human Heparanase Recognition Sites in Porcine
Intestinal Hep and Bovine Kidney HS--
The FITC-labeled Hep was
incubated with the purified recombinant heparanase to confirm the
enzyme activity and also the presence of the heparanase recognition
sequences in bovine kidney HS and porcine intestinal Hep chains. The
digest was analyzed by gel filtration HPLC on a column of TSK-GEL
G3000SWXL as shown in Fig. 2.
Although both FITC-HS and -Hep were degraded by the enzyme, the latter
appeared to be more susceptible to the heparanase-catalyzed cleavage
than the former. Since porcine intestinal Hep contains the heparanase
cleavable sites, experiments were undertaken to elucidate the substrate
recognition property of the heparanase using a series of structurally
defined sulfated oligosaccharides, which were isolated from the
repeating disaccharide region of porcine intestinal Hep and bovine
kidney HS (41, 45) and included 1 tri-, 28 tetra-, 1 penta-, 9 hexa-,
and 3 octasaccharides.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Gel filtration HPLC analysis of the
heparanase digests of FITC-labeled HS and Hep. Heparanase digests
of FITC-labeled HS (A) and Hep (B) were analyzed
by gel filtration HPLC on a column of TSK-GEL G3000SWXL and
were monitored by fluorescence intensity caused by FITC as described
previously (25) with excitation and emission wavelengths of 490 and 520 nm, respectively. The upper and lower
panels show the chromatograms before and after digestion,
respectively.
|
|
Development of the Assay Conditions and Analytical HPLC Assays of
the Recombinant Human Heparanase Using Structurally Defined
Oligosaccharides--
The substrate specificity of the human
heparanase was investigated using 8 hexa- and 12 tetrasaccharides
derived from Hep/HS (Tables I and
II). Their fine structures have been
established by 500-MHz NMR analysis (35-41). Eighteen contained GlcUA
residue(s) at the internal position of each oligosaccharide sequence,
whereas the other two contained only 
HexUA and IdoUA but not GlcUA
residues as uronic acid components. These 20 oligosaccharides (0.3 nmol each) were individually incubated with the purified recombinant heparanase under identical conditions (using 0.88 units of the enzyme,
at 37 °C for 21 h), and the reaction products from each digestion were analyzed by HPLC on an amine-bound silica column for
identification and quantification to compare the relative susceptibilities of the oligosaccharide substrates with the enzyme. Since the elution profile was monitored by absorbance at 232 nm of the
HexUA residue at the nonreducing terminus, only the intact substrate
and the product derived from the nonreducing side of each substrate
were detected, but the product from the reducing side was not due to
the lack of HexUA.
Upon heparanase digestion, Hexa-4 and -7 were almost completely
degraded (Fig. 3), and the peaks with UV
absorbance were shifted to the elution positions of less negatively
charged oligosaccharides. The major digestion product of Hexa-4 was
identified as HexUA(2S)-GlcN(NS,6S)-IdoUA-GlcNAc(6S)- GlcUA by
co-chromatography with the authentic tetrasulfated pentasaccharide (fraction 6-23 in Ref. 40), confirming that the cleavage had occurred at the glucuronidic bond in Hexa-4 (indicated by the arrow in Table I). Although Hexa-7 has two glucuronidic
bonds, which are the possible heparanase cleavage sites (indicated by the arrows in Table I), the major digestion product of
Hexa-7 was detected at around 29 min near the elution position of the authentic trisulfated disaccharide HexUA(2S)-GlcN(NS,6S) (at approximately 25 min; data not shown), suggesting that the major unsaturated oligosaccharide product is most likely the
trisulfated trisaccharide, HexUA(2S)-GlcN(NS,6S)-GlcUA, and not the
pentasulfated pentasaccharide,
HexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-GlcUA. The other
possible digestion product, the tetrasulfated trisaccharide GlcN(NS,6S)-GlcUA-GlcN(NS,6S), derived from the reducing side of the
Hexa-7 hexasaccharide, was not detected due to the lack of appreciable
UV absorption. It is most likely that the tetrasulfated trisaccharide
product GlcN(NS,6S)-GlcUA-GlcN(NS,6S) was further degraded into
GlcN(NS,6S)-GlcUA and GlcN(NS,6S) as discussed below. The very small
peak detected at ~45 min might be the presumably pentasulfated
pentasaccharide, HexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-GlcUA, produced by the cleavage at the glucuronidic linkage on the reducing side, and may represent a minor digestion product. The remaining peaks
at the elution position of the intact hexasaccharide substrates in Fig.
3, A and B, may suggest the incompletion of
digestion or be derived from minor contaminants in the original
samples.
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3.
HPLC analysis of the heparanase digests of
Hexa-4 and -7. Heparanase digests of Hexa-4 (A) and
Hexa-7 (B) (0.3 nmol each) were analyzed by HPLC on an
amine-bound silica column using a linear gradient of
NaH2PO4 from 213 to 902 mM over 80 min, as indicated by the dashed lines. The
upper and lower panels show the
chromatograms of the incubation mixtures using the heat-inactivated
heparanase and the active enzyme, respectively. The open
arrow in each lower panel indicates
the main peak of the heparanase digestion products. The peaks eluted at
around 5 and 10 min in each panel were derived from the incubation
buffer and the enzyme preparation.
|
|
Hexa-1, -8, -15, and -16 showed partial degradation, and Hexa-13S was
resistant to the enzyme action under the reaction conditions used
(Tables I and II). As representative chromatograms, the HPLC profiles
of Hexa-1, -15, and -16 are depicted in Fig.
4. The closed or
open arrow in each panel suggests the
elution position of the corresponding intact hexasaccharide
before digestion or the main peak of the digestion products,
respectively. About 26, 59, and 30% of each hexasaccharide was
converted to the corresponding product, respectively, under the
reaction conditions used. The peak, which remained at the original
position, being intact in each panel, was not a resistant
component, because these substrates were almost completely degraded
with a higher concentration of the enzyme (data not shown). The
percentage of the substrate conversion to products should reflect the
enzyme preference for the substrate recognition (46), although it is
conceivable that the enzyme may become thermally inactivated before
completion of the reaction.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
HPLC analysis of the heparanase digests of
Hexa-1, -15, and -16. Heparanase digests of Hexa-1 (A),
-15 (B), and -16 (C) (0.3 nmol each) were
analyzed by HPLC on an amine-bound silica column using a linear
gradient of NaH2PO4 from 213 to 803 mM over 80 min as indicated by the dashed
lines. The closed or open
arrow in each panel indicates the elution
position of the corresponding hexasaccharide before digestion or the
main product peak from the heparanase digestion, respectively. The
peaks eluted at around 5 and 10 min in each panel were
derived from the incubation buffer and the enzyme preparation.
|
|
Tetrasaccharides were also used to profile the substrate specificity of
the recombinant heparanase. Some of them were sensitive to the enzyme
despite their small sizes, suggesting that the minimum size for the
heparanase recognition is most likely a trisaccharide with the
-GlcN-GlcUA-GlcN sequence backbone. As shown in Fig. 5, the heparanase digestion resulted in
nearly complete degradation of Tetra-1,
HexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S), under the conditions used.
Tetra-6, -10, -28, and -29 were partially digested, and Tetra-3, -21, -22, -23, -25, -26, and -27 showed no degradation. These results are
summarized in Tables I and II. The different susceptibilities of these
tetrasaccharides to the heparanase are suggested to be attributable to
the differences in the modification patterns by sulfations. Thus, the
efficiency (percentage) of the hydrolysis percentage of each substrate
at the respective specific cleavage site obtained under these assay
conditions was used to investigate the structural requirements for the
heparanase action and to depict the substrate specificity of the
heparanase as discussed below.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
HPLC analysis of the heparanase digest of
Tetra-1. The heparanase digest of Tetra-1 (0.3 nmol) was analyzed
by HPLC on an amine-bound silica column using a linear gradient of
NaH2PO4 from 16 to 803 mM over 60 min as indicated by the dashed lines.
A, the chromatogram of the control incubation using the
heat-inactivated heparanase; B, the chromatogram of the
digest with the active enzyme. The closed arrow
in the upper panel and the open
arrow in the lower panel indicate the
elution positions of Tetra-1 before digestion and the main product peak
from the heparanase digestion, respectively. The peaks eluted at around
5 and 10 min in each panel were derived from the incubation
buffer and the enzyme preparation.
|
|
Endo-
-D-glucuronidase Activity of the Heparanase Is
Dependent on the Size and High Sulfation of the Oligosaccharide
Substrates--
Heparanase cleavage occurred only at glucuronidic
linkages but not iduronidic, unsaturated hexuronidic, or glucosaminidic linkage, confirming the endo--D-glucuronidase nature of
this enzyme as reported previously (19-21, 47). The percentages of the
heparanase-catalyzed conversion of the individual oligosaccharides to
products can be used to make a reasonable estimate of the sensitivity of the target glucuronidic linkages. At a cursory glance at Tables I
and II, it was noticed that the recombinant human heparanase acted
preferentially on the glucuronidic linkages in the highly sulfated
regions. The effects of chain lengths, sulfation degrees, and sulfation
positions of oligosaccharides on the heparanase action were evaluated below.
First, we noticed the size dependence of the heparanase cleavage of the
substrate oligosaccharides. The following four pairs of hexa- and
tetrasaccharides were compared in the susceptibilities to the enzyme;
Hexa-4 and Tetra-29, Hexa-7 and Tetra-1, Hexa-7S and Tetra-28, or
Hexa-15 and Tetra-25 share, around the heparanase cleavage site, the
identical tri- or tetrasaccharide sequences such as
-GlcNAc(6S)-GlcUA-GlcN(NS,6S),
HexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-, HexUA-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-, or -GlcNAc(6S)-GlcUA-GlcN(NS), respectively. In each pair, the hexasaccharide was more susceptible to
the enzyme than the tetrasaccharide counterpart (Tables I and II).
These findings suggest that longer oligosaccharides are most likely
better substrates for the human heparanase-catalyzed cleavage, although
the minimum size required for recognition is a trisaccharide.
Second, we evaluated for each oligosaccharide substrate the relation
between the cleavage efficiency (percentage) and the total number of
sulfate groups on the two GlcN residues flanking the GlcUA residue at
the heparanase cleavage site. It was found that at least two or three
sulfate groups in total on the two GlcN residues were required for the
heparanase-catalyzed cleavage of hexa- or tetrasaccharides,
respectively, suggesting that the high sulfation density confers on the
oligosaccharides the preferential recognition by the enzyme.
Since Hexa-4, -7, and -7S and Tetra-1 were almost completely degraded
under the digestion conditions used, they were next digested under
milder conditions to make a better comparison of their digestibility to
the enzyme; the incubation time was shortened to 1, 5, 10, or 23 h, and the amount of the recombinant enzyme was reduced to 50% (0.44 units). The results on the percentage of cleavage of individual
substrates during each incubation period are tabulated in Table
III. After 10 h of incubation,
Hexa-7 was almost completely converted to products, whereas Hexa-4,
Hexa-7S, and Tetra-1 were degraded by 21, 85, and 44%, respectively.
The relative rates of the degradation of these oligosaccharides suggest that the order of the substrate preference by the human recombinant heparanase is as follows: Hexa-7 > Hexa-7S > Tetra-1 > Hexa-4.
View this table:
[in this window]
[in a new window]
|
Table III
Comparison of the heparanase digestibility of highly sensitive
oligosaccharides
The oligosaccharides (0.3 nmol each) were digested with human
recombinant heparanase (0.44 units) for 1, 5, 10, or 23 h, and the
digests were analyzed by HPLC as described under "Experimental
Procedures."
|
|
The 6-O-Sulfated Group on the Nonreducing Side GlcN Residue of the
Target GlcUA Is an Important but Not Absolute Requirement--
Some
oligosaccharides that have the same degree of sulfation but distinct
sequences were degraded with different efficiencies, suggesting that
sulfation patterns on GlcN/GlcNAc residues adjacent to the target GlcUA
residue also significantly influence the heparanase action. Therefore,
the hierarchy of different sulfate groups in the human heparanase
recognition was inspected. When the trisaccharide structures around the
cleavage sites in Hexa-15, -GlcNAc(6S*)- GlcUA-GlcN(NS), and
Hexa-16, -GlcNAc-GlcUA-GlcN(NS,6S*), are compared, they are
different only in the location of the 6-O-sulfate group suggested by an asterisk. Since the human heparanase cleaved Hexa-15 more efficiently than Hexa-16 (59 versus 30%) (Table I),
the 6-O-sulfate group of the GlcN residue on the nonreducing
side of the cleavage site appears to be preferable for the enzyme
recognition over that on the reducing side. When the digestion
efficiencies of Tetra-6 and -29 were compared, the latter was more
efficiently degraded than the former (44 versus 56%) (Table
I). The structural difference between the two was the sulfation
positions on the GlcN residue on the nonreducing side of the heparanase
cleavage site; Tetra-6, HexUA(2S)-GlcN(NS*)-GlcUA-GlcN(NS,6S), has
an N-sulfate group on the GlcN residue, and Tetra-29,
HexUA(2S)-GlcNAc(6S*)-GlcUA-GlcN(NS,6S), contains a
6-O-sulfate group. However, the GlcNAc(6S) structure on the
nonreducing side of the GlcUA is not an absolute requirement for the
cleavage, since Hexa-16 and Tetra-6, which lack this structure, were
also susceptible to the enzyme action. It appears that the 6-O-sulfation on the GlcNAc residue on the nonreducing side
of the cleavage site can be replaced by sulfate groups on other
positions of the flanking GlcN residues or those on other residues in
the adjacent sequence (see below).
The GlcN(NS) Residue on Reducing Side of the Target GlcUA Is an
Essential but Not Sufficient Requirement--
Notably, the
oligosaccharides sensitive to the human heparanase always contained an
N-sulfated GlcN residue on the reducing side of the targeted
glucuronidic linkages, and hence this structure appears to be essential
for the heparanase action. Since, however, oligosaccharides that
contain a -GlcUA-GlcNAc- sequence and highly sulfated GlcN residues
adjacent to the GlcUA residue have never been isolated (12, 45, 48,
49), chemical synthesis of oligosaccharides that contain a unique
sequence, such as -GlcN(NS,6S)-GlcUA-GlcNAc(6S)-, will be desirable to
define whether the GlcN(NS) structure on the reducing side per
se is an absolute requirement for the substrate recognition by the
enzyme. However, the glucosamine residue on the immediate reducing side
of the target GlcUA in native HS and Hep chains is assumed to be always
N-sulfated, as will be discussed below. The enzyme failed to
act on Hexa-13S and Tetra-22, which contains only this
N-sulfate group on GlcN residues flanking the target
GlcUA residue (Table II), suggesting that this GlcN(NS) structure is not sufficient for the heparanase cleavage.
The GlcN(3S) Residue on the Reducing Side of the Target GlcUA
Exhibits a Promoting Effect in a Relatively Low Sulfated Sequence but
an Inhibitory Effect in a Highly Sulfated Sequence--
It has been
reported that heparanase catalyzed the cleavage of the
glucuronidic linkage in the antithrombin III-binding heparin octasaccharide,
IdoUA-GlcNAc(6S)-GlcUA-GlcN(NS,3S,±6S)-IdoUA(2S)-GlcN(NS,6S)-IdoUA(±2S)-2,5-anhydromannitol(6S) (33, 34). However, the susceptibility of the oligosaccharides lacking
the GlcN(3S) structure demonstrated in the present study suggests that
3-O-sulfation is not obligatory. Hexa-1, Hexa-8, or Tetra-10
comprises a portion of the antithrombin III-binding pentasaccharide
sequence containing the unique 3-O-sulfate group (35, 39,
40). The structures in Hexa-4, Hexa-15, and Tetra-25 lack this
particular 3-O-sulfate residing on the reducing terminal GlcN residue found in the corresponding structures of Hexa-1, Hexa-8,
and Tetra-10, respectively. Comparison of the data from Tetra-10 and
-25 suggested a promoting effect of the 3-O-sulfate group
(Tables I and II). Yet, there was no significant difference in
susceptibility to the heparanase action between Hexa-8 and -15 (Table
I). In contrast, when the data from Hexa-1 and -4 were compared, the
inhibitory effect of the 3-O-sulfate group was evident. In
summary, a 3-O-sulfate group appears to have a dual effect
on the heparanase action, exhibiting a promoting effect when it resides
in a relatively low sulfated sequence but an inhibitory effect when
located in a highly sulfated sequence.
The observation that the unique GlcN(3S)-containing oligosaccharides
served as poorer substrates is consistent with the concept that the
Hep-GAG fragments stored in cytoplasmic granules of mast cells have
been generated by the postbiosynthetic degradation of Hep-PG by the
heparanase action but still contain antithrombin III-binding sites
(50-52). Thus, it is reasonable to assume that the glucuronidic
linkage in the antithrombin III-binding pentasaccharide does not serve
as a good substrate for the heparanase under physiological conditions.
A IdoUA(2S) Residue Located Two Sugar Residues away from the Target
GlcUA Residue toward the Reducing Side Is Not an Absolute
Requirement--
Pikas et al. (34) suggested the
involvement of the 2-O-sulfate group on a hexuronic acid
residue located two monosaccharide units away from the cleavage site
toward the reducing end in the heparanase action and proposed a
minimally O-sulfated hexasaccharide sequence required for
the recognition by the heparanases purified from human hepatoma and
platelets (Fig. 6A). The panel
of the oligosaccharides used in the present study contained no such
2-O-sulfated IdoUA residue on the reducing side of the
cleavage site, suggesting that the IdoUA(2S) structure per
se is not an absolute requirement for the heparanase action.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Structures of the human heparanase cleavage
site in Hep/HS. A, the human heparanase cleavage site
proposed by Pikas et al. (34). B, the minimum
trisaccharide structure of the human heparanase cleavage site in
Hep/HS. The arrow indicates the glucuronidic linkage cleaved
by human heparanase. It appears that the highly sulfated structure is
critical for the enzyme action. The GlcN(2-N-sulfate)
structure on the reducing side and GlcN(6-O-sulfate)
structure on the nonreducing side of the cleavage site are considerably
important for the substrate recognition by the enzyme. These requisite
groups are shown in the largest font
size. The additional 2-N-sulfate group on the
nonreducing GlcN or 6-O-sulfate group on the reducing GlcN
appears to have a promoting effect on the heparanase action. These
effectual sulfate groups are shown in the middle
font size. The GlcN 3-O-sulfate
structure, which is enclosed in the rectangle,
causes inhibition of the enzyme in principle, although it also has
promoting effects through its negative charge. C, the
putative octasaccharide recognition sequence by the human heparanase in
native Hep chains deduced from the results obtained by the heparanase
digestion experiments of structurally defined oligosaccharides in the
present study and the mono- and oligosaccharides previously isolated by
bacterial heparinase/heparitinase digestions of porcine intestinal Hep
(36, 40, 53) and bovine lung and intestinal Hep (54).
|
|
We addressed the question of whether the HexUA(2S) at the
nonreducing terminus might substitute for such an IdoUA(2S) residue and
be recognized by the enzyme, suspecting the similarity of the steric
configuration between IdoUA(2S) and HexUA(2S) structures based on
our observation that the 2-O-sulfate group of HexUA(2S) of a disaccharide HexUA(2S)-GlcN(NS,6S) can be removed by the human
recombinant iduronate-2-O-sulfatase2 (see also
Ref. 55). Therefore, we investigated the
susceptibilities to the heparanase of the oligosaccharides with or
without a 2-O-sulfated HexUA residue. Tetra-28 has the
same structure as Tetra-1 but lacks the 2-O-sulfate group on
the nonreducing HexUA residue. When these tetrasaccharides were
compared, Tetra-28 was more susceptible than Tetra-1 (Table I),
supporting the promoting effect of the 2-O-sulfate group.
Heparanase digestion experiments on a pair of hexasaccharides, Hexa-7
and Hexa-7S, that was prepared by glycuronate-2-O-sulfatase digestion of Hexa-7 (Tables I and III) also suggest that the sulfate group on the HexUA residue augments the susceptibility of a
substrate to the heparanase. Since, however, neither Hexa-7 nor Hexa-7S contains IdoUA(2S) at the position two saccharides away from the enzyme
cleavage site toward the reducing side, the IdoUA(2S) structure is not
an absolute requirement for the cleavage by the human recombinant heparanase. However, the possibility cannot be excluded that the IdoUA(2S) residue has a promoting effect on the heparanase action. Since the heparanase preparations that Pikas et al. (34)
used were partially purified from human hepatoma and platelets, the contradictory specificities may be attributable to possible differences in the enzyme sources, glycoforms, and/or purities as discussed below.
Alternatively, the possibility that the IdoUA(2S) residue in question
may be critical for polymer HS and Hep chains to be cleaved by the
heparanase in the specific conformational orientation under
physiological conditions.
The Structure of the Predominant Cleavage Site of the
Heparanase--
Based on the results obtained in the present study, we
suggest that the minimum trisaccharide cleavage site structure in
Hep/HS recognized by the human recombinant heparanase is as illustrated in Fig. 6B. It appears that the highly sulfated structure in
the immediate vicinity of the targeted GlcUA residue is critical for the enzyme action. The preference of the heparanase for the GlcN(NS) structure on the reducing side and the GlcN(6S) structure on the nonreducing side was demonstrated. However, the GlcN(NS) on the reducing side and the GlcN(6S) on the nonreducing side are not sufficient, since Tetra-25 was not cleaved by the enzyme. An additional 2-N-sulfate group on the nonreducing GlcN,
6-O-sulfate group on the reducing GlcN, or an additional
sulfate group on the adjacent sequence (see Hexa-15) appears to have a
promoting effect on heparanase action. The GlcN 3-O-sulfate
structure has dual effects as discussed above.
| |
DISCUSSION |
In the present study, the substrate recognition by the homogenous
recombinant human heparanase was examined in detail for the first time
using a series of structurally defined oligosaccharides. The findings
of the present study demonstrated the importance of the highly sulfated
structure, which is located on both sides of the targeted GlcUA
residue. Notably, the preferred sulfation positions by the heparanase
were revealed; the enzyme cleaves in principle the glucuronidic linkage
in the -GlcNAc(6S)-GlcUA-GlcN(NS)- sequence, although this sequence is
not sufficient, and an additional sulfate group on this or adjacent
sequence (see Hexa-15) appears to be required.
The elucidated sequence requirement was in good agreement with the
cleavage sequence, suggested based on the structures of the remnant
cleavage sites found in the penta- and trisaccharides isolated from
porcine intestinal native Hep-GAG after digestion with bacterial
heparinase/heparitinases (36, 40, 53). Among a series of sulfated
oligosaccharides generated by bacterial lyases (45), the
unsaturated pentasaccharide,
HexUA(2S)-GlcN(NS,6S)-IdoUA-GlcNAc(6S)-GlcUA (fraction
6-23 in Ref. 40 and fraction VI in Ref. 53), and the saturated
trisaccharide, GlcN(NS)-IdoUA(2S)-GlcN(NS,6S) (fraction III-5b
in Ref. 36), are of particular interest in view of the recognition
sequence by porcine or mammalian heparanase. They are odd-numbered
unlike other even-numbered oligosaccharides and appear to be
derived from the reducing and nonreducing ends of the parent Hep-GAG
chains as discussed previously (36, 40), since they do not possess GlcN
at the reducing terminus or HexUA at the nonreducing terminus,
respectively. The above mentioned oligosaccharides were most
likely generated from the sequence of
-HexUA(2S)-GlcN(NS,6S)-IdoUA-GlcNAc(6S)-GlcUA*-GlcN(NS)-Ido- UA(2S)-GlcN(NS,6S)-
by the action of endogenous heparanase on the glucuronidic bond
indicated by an asterisk. Although the unsaturated pentasaccharide is a
major digestion product (40, 53), the above mentioned trisaccharide was
a minor product. In this context, it is noteworthy that Nader et
al. (54) identified free GlcN(NS,6S) as one of the major products
among other unsaturated di- and tetrasaccharides in the
Flavobacterium heparinum haparinase digest of bovine lung and intestine Hep preparations. Based on these findings, the structural characteristics of the predominant saccharide sequence required for the
substrate recognition by the heparanase in native Hep polysaccharides
could be summarized as shown in Fig. 6C. It is conceivable
that human, porcine, and bovine heparanases are similar in their
specificity. The proposed structure is consistent with the previous
finding by Oldberg et al. (47) for human platelet heparanase
and the recent finding by Podyma-Inoue et al. (56) for rat
parathyroid heparanase that the predominant GlcN residue on the
nonreducing side of the cleavage site GlcUA of was
N-acetylated and also overlaps with the structure proposed
for human hepatoma and platelet heparanases by Pikas et al.
(34) (Fig. 6A), although the present results are
apparently contradictory to the previously claimed indispensability of
the IdoUA(2S) and GlcN(3S) structures (33, 34, 57). It should also be
noted that rat parathyroid heparanase is strikingly different from our
enzyme in that it works at neutral and slightly acidic pH values and
does not cleave heparin (56).
The presence of a 2-O-sulfate group on a HexUA residue,
which is located two residues away from the cleavage site toward the reducing end, turned out to be not obligatory for the enzyme action, which is incompatible with the previous proposal that
2-O-sulfated IdoUA residues in HS are essential for the
heparanase action (34, 57). In the previous studies, which demonstrated
the importance of IdoUA(2S), partially purified enzymes from human
hepatoma and platelets (34) and cultured CHO cells were used (57).
Therefore, the partially purified enzyme preparations might contain
unidentified regulatory factors that interact with the
2-O-sulfate group and modulate the enzyme activity. The
purified platelet heparanase was reportedly a 137-kDa protein (58),
which may suggest aggregation or complex formation with other
protein(s). The 2-O-sulfate group in question appears to be
nonessential for the enzyme action, as suggested previously (59), but
may facilitate the enzyme action. Although the formation of
intermediate HS breakdown products characteristic of the heparanase
action was prevented in the HS 2-O-sulfotransferase-deficient CHO mutant cells, the
heparanase transcript was not detected in the purified CHO cell
mRNAs (19). Therefore, the major enzyme responsible for degrading
HS in CHO cells may be a distinct heparanase(s) (60, 61).
Structural changes in HS in animal models of malignancy have been
reported, showing a transformation-associated reduction in charge
density of HS chains (for a review, see Ref. 62). In metastasizing
tumor cells, heparanase is highly expressed and degrades the ECM and
vascular basement membranes (22-26), whereas less sulfated HS chains
on the tumor cell surface may evade the attack of the heparanase
secreted by the tumor cells themselves. Alternatively, heparanase plays
critical roles in tumor growth and metastasis by releasing bioactive HS
fragments from the tumor cell surface HS-PGs, which can serve as potent
promoters of tumor progression. Being consistent with this concept, HS
fragments generated from tumor cell surface HS-PGs by the treatment
with bacterial heparinase promoted tumor cell growth mediated through basic fibroblast growth factor signaling pathways (63). In the transition to malignancy in human colon adenoma cells, the overall molecular organization of HS is preserved but distinctly modified in
the sulfated domains, and it may contribute to the aberrant behavior of
the cancer cells (15).
Tetra-1, HexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S), wasone of the
best substrates among the oligosaccharides tested in the present study
(Table I). This is the major tetrasaccharide product generated from
commercial Hep preparations by the treatment with the bacterial
heparinase (3.6 µmol from 100 mg of heparin) (38, 64). Previous
heparanase assay methods used heterogeneous Hep/HS polysaccharides as
substrates (19, 20). Since the number of the heparanase cleavage sites
varies among different Hep/HS preparations, it has been difficult to
make a precise comparison of the activities. The use of structurally
defined Tetra-1 as a substrate makes it possible to develop a simple
and quantitative assay to measure the heparanase activity, which will
provide a powerful diagnostic method to assess the invasive state of
the cancer.
The findings of the present study also provided clues for developing
specific inhibitors for the heparanase. The minimum heparanase recognition sequence contained neither GlcN(3S) nor IdoUA(2S), which
are imperative elements for Hep/HS to interact with antithrombin III or
basic fibroblast growth factor, respectively (for reviews, see Refs. 5
and 12). Therefore, it should be possible to design oligosaccharide
analogs for developing potent heparanase-specific inhibitors by
avoiding GlcN(3S) and IdoUA(2S) and hence harmful side effects that may
be induced by inhibition or acceleration of those biologically active
proteins. Thus, the findings of the present study will lead to a
framework toward the development of HS-based anti-cancer or
anti-inflammation therapeutics.
§,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe
658-8558, Japan. Tel.: 81-78-441-7570; Fax: 81-78-441-7569.
