(Received for publication, August 26, 1996, and in revised form, October 11, 1996)
From the Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110
Once internalized, cell-associated heparan
sulfate proteoglycans are degraded to short glycosaminoglycans by the
action of endoglycosidases or heparanases. We have begun to address the question of how many heparanases are responsible for this process by
analyzing short heparan sulfate chains produced in vivo by Chinese hamster ovary (CHO) cell heparanases. Short heparan sulfate chains were purified from CHO cells and labeled at the reducing end
with [3H]NaBH4. Hydrolysis of the chains to
monosaccharides and analysis of the 3H-sugar alcohols
indicate that heparanase activities in CHO cells are
endo--glucuronidases. The modification state of the
heparanase-derived glycosaminoglycans was examined by treating the
[3H]heparan sulfate chains with nitrous acid or bacterial
heparin lyases, which cut the chain at specific sequences, and
analyzing the products by P2 gel filtration chromatography. Two
populations of short chains were identified that differ in the extent
of modification on the nonreducing side of the heparanase cleavage
site. One class of chains is unmodified for at least 9 residues from
the reducing end, while the other group has a modified domain within
3-7 residues from the heparanase cleavage site. Our results suggest a
model of heparanase action where the enzymes recognize differences in sulfate content between modified and unmodified regions and bind to
sites that encompass both domains. The enzymes then cleave the
glycosaminoglycan at junctions between the modified and unmodified sequences to produce the different populations of short heparan sulfate
chains.
Heparanases are a family of mammalian endoglycosidases that
degrade long heparan sulfate glycosaminoglycans to shorter chains (1-3). Some heparanases are secreted from cells where they degrade heparan sulfate proteoglycans in basement membranes and extracellular matrices (1). These enzymes are thought to play a role in remodeling basement membranes after injury or at inflammation sites (4-6). Other
heparanases are intracellular and are important for degrading cell
surface heparan sulfate proteoglycans once they have been internalized
(2). Inside cells, heparanases cleave long heparan sulfate
glycosaminoglycans on core proteins to short chains that have an
approximate Mr of 5000 (2, 3, 7-9). One obvious role of the intracellular enzymes is to generate additional nonreducing ends so heparan sulfate glycosaminoglycans can be efficiently degraded
by lysosomal exoglycosidases (10). However, these heparanases may have
other important functions. Cell surface proteoglycans have been shown
to be receptors for growth factors (11-13), enzymes (14), and viruses
(15), so intracellular heparanases may act to release bound ligands
from their proteoglycan receptors once the complex is internalized.
Short heparan sulfate chains generated by heparanases may also regulate
the interaction of a ligand with the proteoglycan in the endosome or,
if the chains are secreted, at the cell surface. Heparanases could play
a role in modulating the modification state of cell surface
proteoglycans. Recent evidence suggests that some proteoglycans are
internalized and recycled through the Golgi apparatus (16); thus,
heparanases could remove a portion of the heparan sulfate in endosomes
so that new chain polymerization and modification could occur when the
core protein is rerouted to the Golgi. Degradation of proteoglycans by
heparanases may also be an important mechanism to prevent long heparan
sulfate glycosaminoglycans from associating with molecules that would be deleterious to the cell. For example, heparan sulfate proteoglycans are components of senile plaques in Alzheimer's disease due to their
association with the -amyloid peptide (17). Heparanases may cleave
heparan sulfate chains from the core protein, thus preventing the
formation of the
-amyloid-proteoglycan complex deposited in amyloid
plaques.
Although heparanase activities have been described in a variety of
cells and tissues, not much is known about how the enzymes recognize
and cleave the glycosaminoglycan chain. Heparan sulfate glycosaminoglycans are linear chains of alternating uronic acid (GlcUA
or IdceA) and GlcN sugars (18). The GlcN residues may be either
N-acetylated or N-sulfated, and all four
monosaccharide species may be O-sulfated (18). Structural
studies of heparan sulfate indicate the N- and
O-sulfate groups are clustered in iduronic acid-rich domains
(modified domains), which are separated by regions of (GlcNAc1-4GlcUA)
disaccharide repeats that contain very little O-sulfate
(unmodified domains) (19). It has been postulated that heparanases
cleave within the unmodified regions, since, with the exception of the
small platelet enzyme that has been reported to be an
endoglucosaminidase (6), all of the mammalian enzymes examined are
endo--glucuronidases (7, 8, 20, 21). Inhibitor studies suggest
overall modification of the polysaccharide is more important for enzyme
recognition than a specific glycosaminoglycan sequence, because
N-sulfate groups are required for heparanases to cleave the
heparan sulfate substrate, but O-sulfate groups have little
effect on enzyme activity (22-25). Finally, most heparanases can only
act on heparan sulfate chains greater than or equal to 10 kDa (2, 3,
20, 25), indicating that the length of the glycosaminoglycan chain is
also important for substrate recognition.
One approach to address the substrate specificity of intracellular heparanases is to characterize the short heparanase-derived products for the cleaved glycosidic bond and the modification state of the chain at the cleavage site. However, to use this approach one needs microgram quantities of the glycosaminoglycan so that enough radioactivity can be incorporated into the reducing ends of the cleaved heparan sulfate for further experiments. In previous studies examining heparanase activities from placenta (20), platelets (6, 21), and B16 melanoma cell lines (7), commercially obtained heparan sulfate or heparin was cleaved with partially purified activity, and the newly formed reducing ends were labeled. While these studies yielded useful information about the glycosidic bond cleaved by the partially purified enzymes, they were not ideal for characterizing the sequences at and around the cleavage site for a number of reasons. First, most of the heparan sulfate commercially available is obtained from liver or kidney and may have been partially degraded by intracellular heparanases in the tissue before being purified. These chains may not be the best substrate, since they may no longer have the sequences recognized or cleaved by heparanases, or the chain itself may be too small. Another problem is that in these studies, large quantities of heparan sulfate were incubated with partially purified enzymes in vitro, and the conditions of the reaction may have forced the enzyme to cleave the chain at sequences it would not normally act at in vivo. Finally, it may be important to use heparanase activities and heparan sulfate glycosaminoglycans from the same cellular source. Studies have shown the modification state of heparan sulfate glycosaminoglycans is dependent on the cell that synthesizes it (26-28), and it is possible that intracellular heparanase specificities are fine-tuned for the modified glycosaminoglycan it normally encounters. By using a commercial source of glycosaminoglycans, one may be missing the correct sequences for efficient degradation by the heparanase of interest.
We have circumvented these problems by analyzing short heparan sulfate species produced by heparanases in vivo. For these studies we are using Chinese hamster ovary (CHO)1 cell heparan sulfate glycosaminoglycans. Lysosomes in CHO cells degrade heparan sulfate chains to monosaccharides inefficiently; thus, the short heparanase-derived glycosaminoglycans can be purified from the cells, and the reducing ends can be labeled with [3H]NaBH4. Analysis of the short [3H]heparan sulfate glycosaminoglycans shows there are distinct populations of heparanase-derived heparan sulfate chains in CHO cells and suggests that heparanases may recognize and cleave the heparan sulfate chain at junctions between modified and unmodified domains. Our results also indicate that there may be multiple heparanase activities in CHO cells.
Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (CCL-61; Rockville, MD). Cells were maintained in Ham's F-12 medium (Life Technologies, Inc.) supplemented with 5.0% (v/v) bovine serum (Life Technologies), 100 µg/ml streptomycin sulfate and 100 units/ml penicillin G (Sigma). Sulfate-free medium (defined F-12 medium) was used for all radioactive labeling experiments and was prepared as described previously (27). Cells were grown at 37 ± 0.2 °C in an atmosphere of 5% CO2 in air and 100% relative humidity. They were subcultured every 3-4 days with 0.125% trypsin, and after 15-20 passages, fresh cells were revived from frozen stocks stored in liquid nitrogen.
Time CourseConfluent CHO cells were incubated with 50 µCi/ml [35S]H2SO4 (DuPont NEN)
for 7 h at 37 °C. Radioactive medium was removed from the
plate, the cell layer was washed three times with phosphate-buffered saline, and trypsin was added to release the cells from the culture dish. Trypsinized cells were pelleted, resuspended in F12 media containing 1 mM Na2SO4, and
replated at approximately 2 × 105 cells/60-mm dish.
For the zero time point, a comparable aliquot of labeled cells was
treated with 0.1 M NaOH (final) for 15 min at room
temperature, neutralized with 10 M acetic acid (27), and
stored at 20 °C until the end of the chase. Plated cells were
allowed to grow for an additional 24, 48, or 72 h to allow for
further catabolism of intracellular glycosaminoglycans, and at that
time labeled heparan sulfate chains were isolated from cells as
described (27).
CHO cells were
grown to confluence in 150-mm diameter plates, rinsed three times with
cold phosphate-buffered saline, and scraped from the dish with 0.25 M sucrose, 5 mM HEPES, pH 7.6. Cells were
sedimented by centrifuging for 3 min in a clinical centrifuge,
resuspended in 5 mM HEPES, pH 7.6, and frozen at
20 °C. Once cells had been collected from approximately 100 150-mm
plates, the suspensions were thawed and treated with 0.1 M
NaOH (final) for 15 min at room temperature to break open the cells.
After the suspension was neutralized, DNase (Sigma)
and MgCl2 were added to final concentrations of 50 milliunits/ml and 40 mM respectively, and the mixture was
incubated overnight at room temperature to degrade the DNA released
from the cells. The next morning, Pronase (Boehringer Mannheim) was
added, and the mixture was incubated an additional 24 h at room
temperature to degrade cellular proteins. Following protease treatment,
the sample was diluted 5-fold with water, insoluble material was
pelleted by centrifugation in a clinical centrifuge for 3 min, and the
supernatant was applied to a 5-ml DEAE-Sephacel column to isolate the
cell-associated glycosaminoglycans. The column was washed with 60 ml of
0.25 M NaCl, 50 mM sodium acetate, pH 6.0, and
glycosaminoglycans were eluted with 25 ml of 1.0 M NaCl, 50 mM sodium acetate. The glycosaminoglycans in the 1 M NaCl eluate were concentrated on a new 1-ml DEAE-Sephacel column and then precipitated overnight at 4 °C with 95% ethanol. Precipitated glycosaminoglycans were treated with 10 milliunits/ml chondroitinase ABC (ICN Biochemicals, Costa Mesa, CA) for 16 h at
37 °C, and the heparan sulfate chains were separated from the newly
formed chondroitin sulfate disaccharides by ethanol precipitation. The
short intracellular heparan sulfate chains were purified further by a
Sepharose CL-6B column (104 × 1 cm), equilibrated in 0.2 M NH4HCO3. The elution position of
the short heparan sulfate chains was monitored by applying 5-50 µl
of each 1-ml fraction to Immobilon membranes (Millipore Corp., Bedford
MA) and staining the bound glycosaminoglycans with 0.2% Alcian blue in
50 mM NaCl and 50 mM MgCl2, and 50 mM sodium acetate, pH 5.7 (29). Fractions containing short
heparan sulfate species (3) were pooled, dialyzed against water, and
lyophilized to dryness. For these studies, two separate preparations of
short CHO cell heparan sulfate were made and labeled by borohydride
reduction (see below).
Five mCi of [3H]NaBH4 (American Radiolabeled Chemicals, St. Louis, MO) was resuspended in 80 µl of 50 mM NaOH, and aliquots were added to lyophilized heparan sulfate glycosaminoglycans (10 µg to 1.0 mg). The ratio of [3H]NaBH4 to heparan sulfate chains was approximately 2.5 mCi for 1.0 mg of glycosaminoglycan (dry weight), and the reaction volume was at least 40 µl. The mixtures were incubated at 45 °C for 24 h. The reduction was stopped by the addition of 10 M acetic acid and then neutralized with 1 M NaOH. Sodium acetate buffer (50 mM) was added to the reduction assay, and the mixture was applied to a 0.5-ml DEAE-Sephacel column to purify the [3H]heparan sulfate away from the majority of unincorporated radioactivity. The purified [3H]glycosaminoglycans were then precipitated twice with ethanol to remove residual 3H radioactivity that had not been incorporated in the polysaccharide. In some cases, carrier heparin was added to the [3H]heparan sulfate to facilitate the ethanol precipitation. Precipitated chains were lyophilized and stored at 4 °C.
Hydrolysis of Heparan Sulfate Chains to MonosaccharidesThe 3H-end-labeled heparan sulfate glycosaminoglycans were hydrolyzed to monosaccharides with 2 M trifluoroacetic acid and high pH nitrous acid following the procedures of Hook et al. (30). After hydrolysis the mixture was incubated with 1.5 M NH4OH (final) for 90 min at room temperature in order to convert any aldonic lactones to acids and then lyophilized and stored at 4 °C in the lyophilized form. To separate charged monosaccharide species from uncharged compounds, the base-treated hydrolysis mixture was applied to a 20-ml AGX18 column equilibrated in water (31). Once the sample was applied, the column was washed with 2.5% formic acid to elute any charged species. Fractions containing radioactivity were pooled, and they were lyophilized to remove the formic acid. In some cases, base-treated hydrolysis mixtures were applied to smaller AGX18 columns, equilibrated in 10 mM NH4OH. Uncharged 3H-species were eluted with 10 mM NH4OH, while charged 3H-residues were eluted with 5% formic acid.
Paper ChromatographyThe 3H-sugar alcohols were applied to cellulose phosphate paper (P-81, Whatman, Hillsboro, OR) and developed in ethyl acetate, pyridine, 5 mM boric acid (3:2:1 by volume) (32). Aldonic and uronic acids move very slowly in this system; therefore, in order to separate the aldonic acid species the chromatogram was developed for 48 h. Gulonic acid (Sigma) and idonic acid (graciously provided by Dr. T. Murphy, Pfizer, Inc.) were included in all chromatograms and were identified by a potassium permanganate stain (33). To ensure that the hydrolysis procedure did not affect the migration of the [3H]aldonic acid, both gulonic acid and idonic acid were hydrolyzed identically to the 3H-oligosaccharides. All samples were incubated with 1.5 M NH4OH for 90 min at room temperature before being applied to the cellulose phosphate paper to ensure that all of the 3H-labeled aldonic acids were in the acid form.
Gel Filtration ChromatographyGlycosaminoglycan chain size was examined by gel filtration chromatography on a TSK 3000 gel filtration column (7.5 × 30 mm, TosoHaas, Montgomeryville, PA) equilibrated in 0.1 M KH2PO4 (pH 6.0), 0.5 M NaCl, 0.2% Zwittergent 3-12 (3). The column was standardized with heparin, heparan sulfate, and chondroitin sulfate molecules of known molecular weight2 and with purified, low pH nitrous acid oligosaccharides (hexasaccharide to 14-mer) generated from [3H]glucosamine-labeled CHO heparan sulfate (27).
The size and proportion of 3H-oligosaccharides were examined by chromatography on a Bio-Gel P2 column (Bio-Rad; 90 × 1 cm) equilibrated in 0.5 M pyridinium acetate, pH 5.0, and run at a flow rate of 60-100 µl/min. One-ml fractions were collected. The column was standardized for even numbered oligosaccharides by determining the elution position of the products formed when [3H]glucosamine-labeled heparan sulfate was treated with low pH nitrous acid (27).
Glycosaminoglycan TreatmentsA portion of the 3H-reduced heparan sulfate (5,000-50,000 cpm) were lyophilized to dryness and treated with nitrous acid or the bacterial polysaccharide lyase, heparinase (EC 4.2.2.7) (Siekagaku America Inc., Ijamsville MD), to examine the modification of the glycosaminoglycan. Low pH nitrous acid-catalyzed deamination, which cleaves chains at N-sulfated GlcN residues, was performed according the low pH method of Shively and Conrad (35). High pH nitrous acid-catalyzed deamination, which cleaves chains at unsubstituted GlcN residues, was performed according to the method of Lindahl et al. (36). Heparinase (1.5 milliunits) was added to 3H-chains resuspended in 20 mM sodium acetate, pH 7.0, 1 mM calcium acetate (total volume of 30 µl) and incubated at 37 °C. After 16-24 h, the reaction mixture was cooled, and the volume was adjusted to 0.5 ml with 0.5 M pyridinium acetate. Low pH nitrous acid and heparinase samples were applied to the Bio-Gel P2 column to examine the size of the 3H-oligosaccharides generated by the treatment. High pH nitrous acid samples were examined on the TSK 3000 gel filtration column.
In order to
isolate chemical quantities of heparanase-derived CHO heparan sulfate
glycosaminoglycans, we needed to confirm that there were indeed short
chains inside cells. CHO-K1 cells were incubated with
35SO4 for 7 h to radiolabel newly
synthesized glycosaminoglycans and then trypsinized and replated in
fresh dishes containing chase media. At 0, 24, 48, and 72 h of
chase, [35S]heparan sulfate chains were isolated
separately from the cells and media. After 72 h, 75% of the total
[35S]heparan sulfate chains present at the beginning of
the chase were still precipitated by ethanol (data not shown),
indicating that CHO cells did not degrade the glycosaminoglycans to
monosaccharides and free sulfate very efficiently. Most of the
[35S]glycosaminoglycans were secreted from the cell
during the chase; however, even at 72 h over 30% of the
[35S]heparan sulfate synthesized in the 7-h pulse
remained cell-associated (data not shown). Initially, two species of
cell-associated [35S]heparan sulfate were resolved on a
TSK 3000 gel filtration column: long chains, which eluted at the void
volume of the column, and short, heparanase-derived glycosaminoglycans
that eluted with an average Mr of 5300 (Fig.
1A). By 24 h, the cell-associated chains
were degraded to smaller species that eluted with an average Mr of 3000 (Fig. 1A). The size of the
cell-associated [35S]heparan sulfate at 48 and 72 h
is similar to that of the 24-h chains, indicating that if shorter
species are produced during the subsequent chase, they may no longer be
precipitated by ethanol.
These results suggest that the short heparanase-derived heparan sulfate chains remaining in the cell are being degraded by the exoglycosidases in the lysosome, albeit not very efficiently. However, since lysosomal enzymes act at the nonreducing end of the polysaccharide (10), the intracellular heparan sulfate species should still have the reducing end that was generated by heparanases. Therefore, we isolated short heparan sulfate from CHO cells and labeled the reducing end with [3H]NaBH4. As expected, the 3H-reduced heparan sulfates were shorter than the chains initially produced by heparanase (Fig. 1B) but were similar to the size of the [35S]glycosaminoglycans found in the cell after a 24-72 h chase (Fig. 1A). These short [3H]heparan sulfate chains (Fig. 1B) were analyzed to examine the CHO heparanase cleavage sites
Heparanase Activities in CHO Cells Are EndoglucuronidasesBecause of the repeating disaccharide
structure of heparan sulfate, heparanases will cleave the
glycosaminoglycan chain so that either a uronic acid or a glucosamine
moiety is at the newly formed reducing end. Since the short heparan
sulfate chains were reduced in order to label the end sugar, the
3H-residues are actually the corresponding sugar alcohol.
When reduced, GlcUA and IdceA are converted to gulonic acid or idonic acid, respectively, while GlcNAc or GlcNSO3 residues are
converted to glucosaminitol. To determine the kinds of endoglycosidases present in CHO cells, the short 3H-reduced chains were
hydrolyzed to monosaccharides by a combination of trifluoroacetic acid
and high pH nitrous acid treatments. The hydrolysis mixture was treated
with base to convert any aldonic lactones to the acid form and then
applied to an AGX18 column to separate the neutral and acidic species.
Over 80% of the applied radioactivity required a decreased pH to elute
from the AGX18 resin (data not shown), indicating that the
3H-sugar alcohols contained a negatively charged functional
group and thus were reduced uronic acids. The acidic fractions from the
AGX18 column were pooled and analyzed by ascending paper chromatography on cellulose phosphate paper to identify the type of aldonic acid (Fig.
2). Except for a small amount of radioactivity that
remained at the origin, all of the 3H-labeled aldonic acids
migrated similarly to the gulonic acid standard, suggesting that they
were originally glucuronic acid residues (Fig. 2A). These
results indicate that the major heparanase activities in CHO cells are
endo--glucuronidases.
Characterization of Chain Modification at the Nonreducing Side of the Cleavage Site
Besides the glycosidic bond that is hydrolyzed, heparanases may be specific for the type of modified residues adjacent to the cleavage site. The modification state of a heparan sulfate chain can be examined by treating the glycosaminoglycan with various reagents that cleave the chain at different glycosidic bonds. Since the only label in the 3H-labeled short heparan sulfate is at the reducing end, the size of the [3H]oligosaccharides produced by these treatments indicates the distance of the first susceptible glycosidic bond from the cleavage site and gives an idea of the modification state on the nonreducing side of the sequence that is recognized and cleaved by heparanases.
Low pH nitrous acid cleaves heparan sulfate glycosaminoglycans on the
reducing side of GlcNSO3 residues (35) and thus is a useful
reagent for examining the position of the first N-sulfated residue on the nonreducing side of the heparanase cleavage site. When
3H-reduced heparan sulfate chains are incubated with low pH
nitrous acid and the deamination products were examined by a P2 gel
filtration column, several 3H-oligosaccharide peaks are
observed (Fig. 3, Table I). Approximately half of the 3H-chains are resolved by the column and elute
as odd numbered oligosaccharides. The remaining 3H-species
elute too near the void volume of the P2 column to determine chain
sizes; however, analysis of the voided material (fractions 32-40) on a
P10 gel filtration column shows that they range from 9 to 13 residues
(data not shown). Because nitrous acid cleaves heparan sulfate between
GlcNSO3 and uronic acid residues, the odd numbered
oligosaccharides confirm that a uronic acid is at the reducing end of
the heparanase-derived chains. The low pH nitrous acid results also
suggest that along with endoglucuronidase activities, CHO cells may
have a minor endoglucosaminidase activity, since a small amount of
3H-labeled material elutes as disaccharides.
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Over 90% of the 3H-oligosaccharides generated by low pH nitrous acid are trisaccharides or longer, suggesting that the residue adjacent to the [3H]gulonic acid must be GlcNAc. However, structural studies of heparan sulfate from endothelial cells have shown that on the chain there is a low level of unsubstituted GlcN residues (37) that are also resistant to cleavage by low pH nitrous acid (35). Therefore, it is possible that the residue next to the reducing end could be an unsubstituted GlcN. To explore this possibility, the 3H-reduced heparan sulfate glycosaminoglycans were treated with high pH nitrous acid, which cleaves the glycosaminoglycan at unsubstituted GlcN but not GlcNAc or GlcNSO3 residues (35, 36). Acid treatment did not change the elution position of the [3H]glycosaminoglycans on the TSK 3000 gel filtration column (data not shown), indicating that the glucosamine residues on the short CHO heparan sulfate chains are substituted with either acetyl or sulfate groups. These results confirm that CHO cell heparanase activities cleave the glycosaminoglycan so that, in nearly all instances, the disaccharide at the newly formed reducing end is (GlcNAc1-4GlcUA).
Nearly half of the heparanase-derived chains in CHO cells have a long unmodified sequence on the nonreducing side of the cleaved glycosidic bond, since the first GlcNSO3 is, on average, 10-14 residues from the cleavage site (Fig. 3). One explanation for this result is that these chains were originally attached to core proteins. Studies in fibroblasts and endothelial cells have shown that the glycosaminoglycan chain directly upstream from the linkage tetrasaccharide is unmodified (19), so it would be less sensitive to low pH nitrous acid than heparan sulfate derived from the middle or end of the glycosaminoglycan. However, heparan sulfate glycosaminoglycans are attached to core proteins through a xylose residue (18), so the sugar alcohol at the reducing end of short glycosaminoglycans derived from these sequences would be [3H]xylitol rather than the [3H]gulonic acid found at the reducing end of heparanase-derived heparan sulfates (Fig. 2A). Since over 80% of the short, 3H-reduced heparan sulfate glycosaminoglycans had gulonic acid at the reducing end (Fig. 2A), it is unlikely that the majority of the voided nitrous acid oligosaccharides came from the end of the chain attached to the protein core. In fact, 80% of the 3H-sugar alcohols generated from hydrolysis of the long oligosaccharides (Fig. 3, fractions 32-40) bound to the AGX18 resin (data not shown) and co-migrated with the gulonic acid standard on cellulose phosphate paper (Fig. 2B), confirming that nearly all of the nitrous acid oligosaccharides remaining at the void volume of the P2 column (Fig. 3) were derived from internal chain sequences.
The modification state of the glycosaminoglycans on the nonreducing
side of the cleavage site was further characterized by incubating the
3H-reduced chains with the bacterial enzyme, heparinase,
and analyzing the reaction products on the P2 column (Fig.
4A, Table I). Heparinase cleaves heparan
sulfate glycosaminoglycans between GlcNSO3 and IdceA2S
residues (38, 39), which are typically found in modified domains (19).
Therefore, the pattern of [3H]oligosaccharides resulting
from heparinase treatment will indicate the distance of this modified
sequence from the reducing end of the short heparan sulfates. Over half
of the 3H-reduced chains remain at the void volume of the
P2 column after incubation with heparinase (Fig. 4A, Table
I), indicating that they do not contain the
(GlcNSO31-4IdceA2S) sequence cleaved by the bacterial
enzyme. The remaining 3H-chains are converted by heparinase
to nona-, hepta-, and pentasaccharides (Fig. 4A, Table I).
Interestingly, incubation with the bacterial enzyme did not generate
any 3H-trisaccharides. This result suggests that none of
the CHO heparanase-derived chains have IdceA2S as the third residue
from the reducing end; however, a heparinase-susceptible bond directly
upstream of the reducing end may be a poor substrate, due to the
proximity of the reduced gulonic acid.
To look more closely at the properties of the chain populations that differ in their susceptibility to heparinase, both the [3H]oligosaccharides at the void volume (fractions 30-36) and the included 3H-species (fractions 37-53) were pooled and examined for their sensitivity to low pH nitrous acid (Fig. 4, B and C). The nitrous acid profile of the heparinase-voided chains is similar to the profile of the [3H]heparan sulfate treated with nitrous acid alone, except that the proportions of tri-, penta-, and heptasaccharides are reduced compared with the untreated glycosaminoglycans (compare Figs. 3 and 4B). This result shows there is a population of 3H-reduced heparan sulfate chains that have GlcNSO3 sugars close to the cleavage site, but they lack IdceA2S residues in the N-sulfated region. On the other hand, when the heparinase-susceptible 3H-chains are treated with nitrous acid, most of them are converted to trisaccharides (Fig. 4C, Table II), indicating that the nona-, hepta-, and pentasaccharides have at least one GlcNSO3 residue on the reducing side of the susceptible heparinase bond. This is not surprising, since IdceA2S sugars are normally present in regions of high N-sulfation (19). Thus, the combined use of heparinase and nitrous acid indicates that there is a population of heparanase-derived chains that have a modified sequence directly upstream of the cleaved glycosidic bond. Most of these chains have the sequence (GlcNSO31-4IdceA2S1- 4Glc NSO31-4HexA1-4GlcNAc1-4GlcUA).
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Our studies show that there are two populations of short,
heparanase-derived heparan sulfate glycosaminoglycans in CHO cells that
differ in the distance of the first modified domain from the glycosidic
bond cleaved by heparanases (Fig. 5). One population, class I, has relatively unmodified sequences for the first 9-13 residues from the heparanase cleavage site and remain as long oligosaccharides that elute at the void volume of a P2 gel filtration column after either low pH nitrous acid treatment (Fig. 3) or digestion
with heparinase (Fig. 4). The other population of short glycosaminoglycans, class II, has modified sequences that begin within
3-7 residues from where heparanases cleaves the chain. Class II chains
elute as shorter oligosaccharides after low pH nitrous acid treatment
(Fig. 3) and, based on their susceptibility to heparinase, may or may
not contain IdceA2S residues within the modified domain (Fig. 4). Both
chain populations are produced by endo--glucuronidase activities
that cleave a relatively unmodified sequence, since the reducing ends
of at least 90% of the heparanase products have the sequence
(GlcNAc1-4GlcUA). The two classes of heparanase products observed in
CHO cells may be a general feature of intracellular heparanase
catabolism, since similar results were seen with short heparan sulfate
chains isolated from rat hepatocytes (8) or produced by partially
purified placenta heparanase activities (20). In these earlier studies,
low pH nitrous acid converted the short heparan sulfate chains to
trisaccharides and oligosaccharides that eluted at the void volume of
Sephadex G-25 gel filtration columns (8, 20). Although the sizes of the
voided oligosaccharides were not identified in either study, it is
likely that they are comparable with the CHO class I products, while
the trisaccharide species are comparable with the class II
products.
Structural analysis of heparan sulfate shows that the glycosaminoglycan is composed of alternating modified and unmodified domains (19). Modified regions, made up of 3-7 disaccharides, are rich in GlcNSO3 and IdceA residues and are the primary sites for O-sulfation. They are separated by sequences of 14-25 (GlcNAc1-4GlcUA) disaccharides that contain few O-sulfate groups (19). Our results suggest that heparanases may recognize and cleave the heparan sulfate chain at the junctions between these domains (Fig. 5). Heparanases may recognize differences in the sulfate content between the modified and unmodified regions and bind to sites on the chain that encompass both domains. The enzymes then cleave the glycosaminoglycan in a relatively unmodified sequence to generate shorter chain products. If heparanases cleave the glycosaminoglycan on the nonreducing side of the modified domain, class I chains will be generated, while if the enzymes act on the reducing side of the modified domain, the products will be class II chains (Fig. 5). In either case, the heparan sulfate substrate may sit loosely in the catalytic site, since both class I and class II products have slight variations in the distance of the first GlcNSO3 from the cleaved glycosidic bond (Figs. 3 and 4). Our model of heparanase action is similar to the model proposed by Schmidtchen and Fransson (40). They analyzed the structure of heparan sulfate oligosaccharides from fibroblasts and found the chains to be rich in GlcUA residues, with IdceA2S sugars at the periphery of the molecules. From these findings, they proposed that the short glycosaminoglycans had been produced by heparanases that cleaved the chain at or near modified sequences (40).
A model where heparanases bind to both modified and unmodified regions is consistent with what is known about the substrate specificity of the enzymes. Modified domains are defined by the presence of N-sulfate groups, which have been shown to be essential for heparanases to recognize and cleave a heparan sulfate chain (22-25). Specific O-sulfate groups, found primarily in the modified regions, are not necessary for enzyme activity (22-25). This is not surprising if, as our model predicts, the enzyme only recognizes the difference in sulfate content between modified and unmodified domains. O-sulfation should promote the interaction of heparanase with the substrate, since it would increase the sulfate content of the modified domain, but binding should not be dependent on the type or arrangement of O-sulfate groups. In fact, our model would predict that the spacing and length of the modified domain within the substrate are more important for enzyme recognition than the actual sequence of uronic acid epimers and O-sulfate groups. The degree of IdceA residues in the modified domain may be important for enzyme recognition as well. Heparan sulfate chains are proposed to form a helix with 2-fold symmetry (34). Spectroscopic and molecular modeling studies indicate that IdceA residues may exist in two conformations that will determine how charged functional groups are oriented to the helix (34). The IdceA in the modified domains may orient the glycosaminoglycan so that the increased sulfate content of the region is emphasized and thus facilitate the binding of heparanase to the heparan sulfate substrate. Our model would also explain the size dependence of heparanase substrates. Studies in vivo and in vitro suggest that heparan sulfate chains must be at least 10 kDa in order to be cleaved by heparanases (2, 20, 25), which would be the minimum size of a heparan sulfate chain that is composed of one modified domain and one unmodified domain of average sizes.3
How many heparanases are required to generate class I and class II chains? Since our studies characterized short heparan sulfate chains formed in vivo, it is possible they were produced by multiple enzymes. Indeed, the simplest explanation for the formation of class I and class II products is that they were generated by separate heparanase activities. Each enzyme would recognize the substrate by differences in sulfate content between modified and unmodified regions but would bind the chain so that modified domains are oriented differently at the catalytic site. At the present time we do not know how many heparanases exist in CHO cells. We have preliminary evidence for two separate CHO-heparanase activities;4 however, further work is necessary to establish whether each activity catalyzes the formation of one or both classes of heparanase products.