From the Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, P. O. Box 94, Lund S-221 00, Sweden
Received for publication, June 16, 2000, and in revised form, November 8, 2000
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ABSTRACT |
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We have analyzed the content of
N-unsubstituted glucosamine in heparan sulfate from
glypican-1 synthesized by endothelial cells during inhibition of
(a) intracellular progression by brefeldin A,
(b) heparan sulfate degradation by suramin, and/or
(c) endogenous nitrite formation. Glypican-1 from brefeldin
A-treated cells carried heparan sulfate chains that were extensively
degraded by nitrous acid at pH 3.9, indicating the presence of
glucosamines with free amino groups. Chains with such residues were
rare in glypican-1 isolated from unperturbed cells and from cells
treated with suramin and, surprisingly, when nitrite-deprived. However,
when nitrite-deprived cells were simultaneously treated with suramin,
such glucosamine residues were more prevalent. To locate these
residues, chains were first cleaved at linkages to sulfated
L-iduronic acid by heparin lyase and released fragments
were separated from core protein carrying heparan sulfate stubs. These
stubs were then cleaved off at sites linking N-substituted
glucosamines to D-glucuronic acid. These fragments were
extensively degraded by nitrous acid at pH 3.9. When purified
proteoglycan isolated from brefeldin A-treated cells was incubated with
intact cells, endoheparanase-catalyzed degradation generated a core
protein with heparan sulfate stubs that were similarly sensitive to
nitrous acid. We conclude that there is a concentration of
N-unsubstituted glucosamines to the reducing side of the
endoheparanase cleavage site in the transition region between
unmodified and modified chain segments near the linkage region to the
protein. Both sites as well as the heparin lyase-sensitive sites seem
to be in close proximity to one another.
Most adherent cells produce cell-surface-located, heparan sulfate
(HS)1-substituted
proteoglycans (PG). Their core proteins are noncovalently inserted into
the plasma membrane via penetrating peptide segments or covalently
attached via a C-terminal glycosyl-phosphatidyl-inositol anchor as in
the glypican family (GLP). To date, six distinct GLP gene products have
been demonstrated in mammals and two in Drosophila
(1).2 Mammalian GLP-1 is the
only member that is widely expressed in the vascular system (2). The
three HS attachment sites in GLP-1 are situated close together and near
the membrane anchor within the sequence DDGSGSGSD. Biosynthesis of the
HS chains proceeds in a stepwise manner. The serine residues are first
substituted with Xyl, and then the common linkage-region,
GlcUA-Gal-Gal-Xyl is formed. HS assembly is initiated by a unique
Early structural studies on HS using degradations with nitrous acid or
specific enzymes revealed a common basic domain structure whereby
blocks of uninterrupted, consecutive GlcUA-GlcNAc repeats alternated
with more modified regions, containing both highly sulfated short
segments of IdoUA(SO4)-GlcNSO3 as well as mixed or alternating arrangements (4, 5). By using periodate oxidation and
alkaline scission of GlcUA in consecutive GlcUA-GlcNAc repeats, we
isolated oligosaccharides derived from the mixed regions, which seemed
to contain some GlcNH2 moieties (6). More recent studies using a monoclonal antibody directed against epitopes containing GlcNH2 have shown that these are located in regions of HS
composed of mixed N-sulfated and N-acetylated
disaccharide repeats (7). Because GlcNH2-GlcUA bonds appear
to be resistant to HS lyase degradation (8, 9), GlcNH2 can
be localized in the enzyme degradation products. Thus,
GlcNH2 moieties have been found both flanked by GlcUA
residues (8) and at the reducing side of IdoUA(2-SO4) (9).
Most HS chains analyzed to date seem to have the originally proposed
common basic design where N-sulfated regions are separated by intervening unmodified regions (see Fig. 1). Variations in HS
structure are due mainly to differences in the content and pattern of
O-sulfation (1, 3, 4-6, 10). Sulfated IdoUA residues
(boldface S in Fig. 1) are rare in HS from human vascular endothelial cells (11), but the number of such residues as well as the
length of the modified regions increase toward the nonreducing end of
the chain (12). It has been proposed (8) that GlcNH2 moieties are preferentially located on the proximal border (relative to
the linkage region) between high sulfated and low sulfated regions (see
Fig. 1).
Turnover of HSPG involves partial degradation of HS by endoheparanase,
which usually cleaves
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GlcNAc-transferase that adds the first GlcNAc. By the alternating
addition of GlcUA and GlcNAc, catalyzed by HS-copolymerases, the
extended, linear heparan backbone is formed. A unique step in HS
biosynthesis is the patchwise exchange of N-acetyl for
sulfate on GlcNAc catalyzed by various isoforms of
N-deacetylase/sulfotransferase. An intermediate in this
reaction is GlcNH2. Further modifications of the HS
precursor chain then take place, including epimerization of GlcUA to
iduronic acid (IdoUA) and O-sulfations at various positions.
A vast structural variation is generated by the presence of different
enzyme isoforms with a more or less extensive action in specific
structural contexts (3).
-D-glucuronidic linkages and is
inhibited by suramin (Refs. 13-17 and references therein). There are
indications for different cleavage sites; some are near the reducing
side, others are near the nonreducing side of the highly modified
regions (Fig. 1). The structural features
of a cleavage site (
) have been defined as
-GlcNR-GlcUA-
-GlcNR-HexUA(2-SO4)-GlcNR-, where R is an
unspecified substituent and HexUA could be IdoUA or GlcUA (14, 17).
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Fig. 1.
Schematic model of endothelial HS.
Unmodified stretches of GlcUA-GlcNAc (open) extend for
10-17 repeats from the linkage region ( , GlcUA-Gal-Gal-Xyl)
and then alternate with regions containing GlcNSO3
moieties, either in consecutive repeats (solid) or mixed
ones (hatched). The total length is generally from 50 to 100 disaccharides, but precursor forms may contain up to 200. Repeats
containing IdoUA(-SO4) are only found centrally within the
modified block regions (marked with boldface S). Proposed
sites for GlcNH2 are marked with +. Endoheparanase cleavage
sites are marked with arrows (open for those
acting on the nonreducing side, filled for those acting on
the reducing side). Sites for cleavage by HS lyase should be abundant
in the unmodified regions (except for those marked +) and also
sometimes in the mixed ones. Heparin lyase cleaves at boldface
S (see also Refs. 10-17).
We have recently shown (18) that the human vascular endothelial cell
line ECV 304 expresses a HSPG with a core protein of 60-70 kDa, which
is recognized by an anti-GLP-1 monoclonal antibody and by a polyclonal
antiserum raised against recombinant GLP-1 protein. By using metabolic
radiolabeling and compounds that inhibit intracellular progression of
secretory proteins or degradation of HS, effects on the turnover of
different GLP-1 glycoforms were studied. As summarized in Fig.
2 we have proposed a recycling of GLP-1
(18). In unperturbed cells, most of the radiolabeled GLP-1 carries
truncated HS chains and is accompanied by HS oligosaccharides. In
BFA-treated cells, a large GLP-1 PG with long HS chains accumulates and
the oligosaccharides disappear. The long HS chains contain scattered
and clustered GlcNH2 moieties. However, very little metabolic radiolabeling of the core protein of this PG could be obtained. Results of pulse-chase experiments indicated that the large-size PG is the precursor of a partly HS-degraded PG form that was
captured by suramin treatment. When cells pretreated with suramin were
chased in the presence of BFA, formation of large PG with long HS
chains is resumed with increased capacity. The latter process is
retarded by continued presence of suramin and completely blocked in
cells deprived of nitrite. Resynthesis of HS is restored when an NO
donor was supplied. Radiolabeling of GLP-1 core protein could only be
detected after pulse-labeling of protein in nitrite-deprived cells
followed by chase-labeling of the HS chains in BFA-treated cells
provided with an NO donor.
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Endogenous nitrite is formed by oxidation of NO (19), and endothelial
cells generate sufficient amounts to degrade exogenously supplied HS at
moderately acidic pH values (20). Cleavage at GlcNH2
moieties of endogenously formed HS during degradation and turnover is
thus a distinct possibility. In the present study we set out to examine
whether the content of GlcNH2 in the HS chains/stubs of
various GLP-1 glycoforms was affected by nitrite deprivation and where
in the HS chains these susceptible moieties were preferentially located.
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EXPERIMENTAL PROCEDURES |
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Materials--
Cells, culture media, sera, antiserum to
glypican-1, the various enzyme inhibitors and other inhibitory
compounds, radioactive precursors, enzymes, prepacked columns, and
other media or chemicals were the same as described previously (18). In
some experiments D-[6-3H]glucosamine with a
specific activity of 60 Ci/mmol (Amersham Pharmacia Biotech) was used,
and prepacked columns of Superdex peptide HR 10/30 and PD-10 were also
obtained from Amersham Pharmacia Biotech, Sweden. Centriplus 30 was
from Millipore, Sweden. Gal1-3GalNAcO[3H] was a gift
from the former BioCarb Company.
Cells, Media, and Radiolabeling-- ECV 304 cells were maintained in DMEM as described previously (18) and preincubated with the appropriate medium before radiolabeling. [35S]Methionine/cysteine labeling was performed in a low methionine DMEM, [35S]sulfate with DMEM containing MgCl2 instead of MgSO4, and M199 was used for preincubations in the presence of NOS inhibitors, neocuproine and sulfamate. Cell cultures were incubated with 50 µCi/ml of [35S]methionine/cysteine or with 20 µCi/ml D-[6-3H]glucosamine and 50 µCi/ml of [35S]sulfate. Treatments with BFA, suramin, NOS inhibitors, neocuproine, and sulfamate were carried out as described previously (18).
Extraction and Isolation of PG-- Cells were extracted with phosphate-buffered saline containing 0.1% (w/v) SDS, 0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate (RIPA) followed by immunoisolation of GLP-1 glycoforms using anti-GLP-1 antiserum in three consecutive steps performed as described previously (18). Regularly, PG and PG-derived material not reacting with the antiserum were recovered from the supernatants obtained after immunoisolation by passage over DEAE-cellulose as described (18). Separation into PG and degradation products was achieved by gel-permeation chromatography on Superose 6, by further purification of PG material by ion exchange chromatography on MonoQ (gradient elution), and sometimes by hydrophobic interaction chromatography on octyl-Sepharose (12, 18). In some experiments, material extracted with only Triton X-100-containing buffers (18) was directly subjected to the biochemical isolation procedure. Cells were sometimes released by trypsinization before extraction with Triton X-100. Trypsin digestion was carried out using 20 µg/ml for 5 min at 37 °C. The reaction was terminated by the addition of serum (10%, v/v) followed by centrifugation at 3000 rpm for 5 min. Cells and supernatants were analyzed separately.
Degradations and Separations--
HS chains and chain stubs were
released from the core protein by treatment with alkaline borohydride.
Enzymatic digestions of HS were performed with HS or heparin lyase and
deaminative cleavage with HNO2 either at pH 1.5 or 3.9. PG,
HS, and degradation products were separated by gel-permeation
chromatography. All procedures have been described in detail elsewhere
(12, 18). Buffer changes, concentration, and recovery of material were
performed using Centriplus 30 or PD-10 columns as described by the
manufacturer or by precipitations with 10 volumes of ethanol/0.4%
(w/v) sodium acetate. As carriers (25-100 µg/ml) we used HS chains
(18) and/or an oligosaccharide mixture consisting of equal parts of HS
degraded with HNO2 at pH 1.5, raffinose, sucrose, and
glucose. Radioactivity was measured by -scintillation.
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RESULTS |
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Biosynthesis and Characterization of GLP-1 with Long and Short HS Chains-- BFA can inhibit both transport of newly synthesized proteins to the Golgi (21) as well as endocytosis, recycling, and reprocessing of resident membrane proteins in polarized cells (18, 22). Therefore, we first assessed the proportions of extracellular and intracellular PG material in untreated or BFA-treated cells. ECV cells were incubated with [35S]sulfate for 24 h in the absence or presence of BFA. They were released from the culture plates by trypsinization and separated from solubilized material by centrifugation. The cell pellet was lysed in Triton X-100, and PG and PG-derived material were recovered separately from the trypsinate and the cell lysate by passage over DEAE-cellulose. In the case of untreated cells, 22% of total radiolabeled material was released by trypsin, and most of this was eluted as a small PG on Superose 6, whereas the inaccessible material consisted of HS-oligosaccharides (data not shown). In brefeldin A-treated cells, no radiolabeled material was released by trypsin. All of the radiolabeled material recovered from the cell pellet consisted of a large PG (18).
Previous studies have shown that the PG from BFA-treated cells
(comprising GLP-1) is substituted with long HS side-chains, whereas the
predominant glycoform from untreated cells has shorter HS chains (18).
In this study, we investigated whether the content of
GlcNH2 residues in the PG chains varied with the nitrite
level in the cells producing the PG. To obtain radiolabeled chains, [3H]glucosamine- and [35S]sulfate-labeled
GLP-1 glycoforms were isolated on a preparative scale by three
consecutive immunoisolations followed by gel-permeation chromatography
on Superose 6. In the case of untreated cells (Fig. 3), it was estimated that at least 75%
of all GLP-1 glycoforms had been isolated (A-C).
These profiles were also similar to those obtained previously with
total radiolabeled polyanionic material isolated from Triton X-100 cell
extracts by ion-exchange chromatography (18), except that no
accompanying HS chain fragments and oligosaccharides were obtained (see
dashed line without symbols in C). The
HS degradation products were recovered from the supernatant after
immunoisolation (data not shown). The HS chains of the immunoisolated
small GLP-1 glycoform from untreated cells (see bars in Fig.
3, A-C) were released by alkali and chromatographed on
Superose 6 (Fig. 3D). The chain size of the major pool (see
bar in D) was estimated to be 30-50 kDa (23).
Some smaller chain stubs were also present (fractions 45-55).
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The large-size radiolabeled PG from BFA-treated cells was much less reactive with the antiglypican antiserum. Only 5-10% was recovered after three consecutive immunoisolations. However, a 66-kDa-core protein, derived from the PG of BFA-treated cells by digestion with HS lyase, reacted strongly with monoclonal antibodies to GLP-1 upon SDS-polyacrylamide gel electrophoresis and immunoblotting (18). It is thus possible that the long HS side chains could shield epitopes in the core protein. Unfortunately, the PG from BFA-treated cells incorporates very little radioactivity from radiolabeled amino acids, because it appears to be primarily made from resident, unlabeled core protein precursors or HS-chain-truncated PG (18).
Because the yield of immunoreactive intact PG from BFA-treated cells
was low, the HS chains from immunoisolated as well as unreactive PG
material were analyzed separately. After gel-permeation chromatography
on Superose 6 (Fig. 4) large-size PG
material, eluting in the void volume, was obtained in both cases
(pool I in A and a corresponding pool in
B, see bar). Also a low molecular size
[3H]glucosamine- and [35S]sulfate-labeled
GLP-1 glycoform was obtained from the immunoisolated material
(pool II in A) but not from the unreactive
material (B). The peaks at 29-32 and 40-43 in Fig.
4B are probably glycoprotein contaminants.
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The elution profiles of the alkali-released HS chains from the large immunoreactive GLP-1 glycoform (pool I in Fig. 4A) and from corresponding material that was unreactive to the antiserum (see bar in Fig. 4B) as well as that of HS-chains from the small immunoreactive GLP-1 glycoform (pool II in Fig. 4A) are shown in Fig. 4, C, D, and G, respectively. The large PG forms contained long HS side chains in both cases (C and D) with an estimated average size of approximately 100 kDa (23). The small immunoreactive GLP-1 glycoform yielded a broad-size distribution on Superose 6 (Fig. 4A), suggesting a polydisperse HS chain composition. The chains from this PG separated into three major and one or two minor-size pools as judged from the distribution of the [3H]glucosamine label, which represents the backbone structure of the chains (Fig. 4G). Most of these, except for a minor portion, were of the same size or smaller than the PG (cf. pool II in Fig. 4A). However, the chains differed markedly in degree of sulfation. The smallest chains, eluting around fraction 50, were mostly nonsulfated, whereas some of the larger, minor ones, eluting around fraction 35, had a [35S]/[3H] ratio of approximately 4:1. Hence, the small GLP-1 glycoform from BFA-treated cells contained chains and chain stubs varying in size from 60 kDa down to less than 5 kDa (23). Apparently, the core protein simultaneously carried both short and long chains as the intact PG did not separate into discrete components. Moreover, each PG molecule should only contain one long chain. This chain would dominate the excluded volume of the PG, because the core protein is expected to have a rather compact structure (1). The presence of short HS chains may have facilitated its essentially quantitative recovery by immunoisolation. The small PG may be an immature form trapped in an early compartment of the secretory pathway.
Identification of GlcNH2 in Different GLP-1 Glycoforms-- The presence of GlcNH2 was indicated by selective deaminative cleavage at the reducing side of these residues in alkali-released HS chains using HNO2 at pH 3.9 (24) followed by gel-permeation chromatography on Superose 6. The small GLP-1 glycoform obtained from unperturbed cells (Fig. 3, A-C) yielded a major pool of HS-chains (see bar in Fig. 3D) with an estimated size of 30-50 kDa. These chains were rechromatographed before (Fig. 3E) and after (Fig. 3F) deaminative cleavage. The results showed that most of the chains were resistant, but a small proportion had been cleaved into medium-sized fragments (fractions 45-50).
The long HS side chains of the immunoisolated large GLP-1 glycoform from BFA-treated cells (Fig. 4C) were extensively depolymerized upon deaminative cleavage at pH 3.9 (Fig. 4E), indicating that GlcNH2 residues were present in all chains to a variable degree. The degradation products included fragments of a relatively uniform distribution (fractions 25-30). However, the major products were a series of intermediate-size (fractions 30-45, approximately 20-60 kDa) to small-size fragments (fractions 45-50, approximately 10 kDa) with gradually diminishing [35S]/[3H] ratios. The latter fragments should be derived from the unmodified regions (see Fig. 1). The HS chains of the material that was not immunoreactive (Fig. 4D) yielded essentially the same type of products after deaminative cleavage (Fig. 4F). Hence, any syndecan-derived HS that might be present in the PG pool (18) may also contain a similar amount of GlcNH2 residues. It should also be pointed out that nitrous acid causes N-desulfation and deaminative cleavage at GlcNSO3 residues under more acidic conditions, such as pH 1.5 (24). Release of free sulfate would be indicative of such a reaction. In all cases (Figs. 3F, 4E, and 4F) very little, if any, free sulfate could be detected (see below).
The [3H]glucosamine- and
[35S]sulfate-labeled GLP-1 glycoforms from
suramin-treated cells arise from the larger PG precursor by partial
endoglycosidic degradation of HS (Fig. 2, top right). These
truncated HS chains were also examined for the presence of
GlcNH2 residues. PG and PG-derived material not reacting
with the antiserum and immunoreactive material were separately
chromatographed on Superose 6 (Fig. 5,
A and B, respectively). The immunoisolated material (Fig. 5B) afforded a profile similar to that of
GLP-1 from unperturbed cells (Fig. 3, A-C). The supernatant
obtained after immunoisolation (Fig. 5A) contained some
unreactive material of the same size as the GLP-1 glycoforms and all of
the free heterogeneous, alkali-resistant (data not shown) HS chains,
chain fragments, and oligosaccharides (fractions 33-50, approximately
20-65 kDa with an average at 40 kDa). A comparison with the profile
obtained previously (18) with the total radiolabeled, polyanionic
material from suramin-treated cells (see dashed line in Fig.
5A) indicated that approximately half of the PG
material from these cells was immunoreactive.
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The alkali-released HS chains of the immunoisolated GLP-1 glycoform
from suramin-treated cells had approximately the same average size
(Fig. 5C) compared with those obtained from the major GLP-1
glycoform of untreated cells (Fig. 3D). Similarly, there was
only a slight shift in the profile when HS chains of GLP-1 from
suramin-treated cells were subjected to deaminative cleavage (Fig.
6D). Essentially the same
result was obtained with chains derived from the material that was not
immunoreactive (data not shown).
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The marginal effect of nitrous acid on the partially degraded HS side chains of GLP-1 from suramin-treated cells may be due to prior cleavage near the GlcNH2 residues by an endoheparanase not inhibited by suramin. Alternatively, endogenously formed nitrite may have consumed all available GlcNH2 sites. Therefore, we also examined the GLP-1 glycoforms obtained from nitrite-deprived cells as well as those from both suramin-treated and nitrite-deprived cells. The GLP-1 glycoforms from cells depleted of nitrite by treatment with NOS inhibitor, neocuproine, and sulfamate (Fig. 6A) were more size-heterogeneous than corresponding material from untreated (Fig. 3, A-C) or suramin-treated cells (Fig. 5B). Still, a substantial portion was recovered by immunoisolation (cf. Fig. 6A and bar in B). The HS chains derived from these glycoforms (Fig. 6C) were also more heterogeneous and somewhat larger (average size of approximately 50 kDa) than corresponding material from untreated (Fig. 3D) or suramin-treated cells (Fig. 5C). However, despite nitrite deprivation, very little cleavage was observed after treatment with HNO2 at pH 3.9 (Fig. 6D).
GlcNH2 residues and sites for endoheparanase cleavage could
be adjacent or very closely located on HS chains. Therefore, the HS
chains of GLP-1 glycoforms produced by nitrite-deprived cells may
indeed contain GlcNH2 residues, but cleavage nearby by
endoheparanase would make them difficult to detect with the present
approach. We therefore examined HS from glycoforms made by cells that
were simultaneously nitrite-deprived and treated with suramin to
inhibit endoheparanase. The GLP-1 glycoforms isolated from these cells were heterogeneous and included some large PG material (Fig.
7A). In the supernatant
obtained after immunoisolation, there were both unreactive material of
the same size as the reactive material (pool I in Fig.
7B) and HS chain fragments (pool II). When
material from the latter pool was recovered and treated with
HNO2 at pH 3.9, no effect was seen (data not shown).
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The immunoisolated glycoforms from nitrite-deprived and suramin-treated cells (Fig. 7A) were quantitatively bound to octyl-Sepharose. To remove any free HS chains and chain fragments from the unreactive PG material in pool I (Fig. 7B), this material was also passed over octyl-Sepharose. Pooled octyl-bound materials from both immunoreactive and unreactive material were treated with alkali, and released HS stubs were rechromatographed (Fig. 7C). The average size and size-distribution of these chains were essentially the same as for corresponding material from only nitrite-deprived cells (Fig. 6C). However, the HS-material derived from simultaneously suramin-treated and nitrite-deprived cells was more sensitive to HNO2 at pH 3.9 (Fig. 7, C and D) than were chains derived from cells treated with only suramin (Fig. 5, C and D) or only nitrite-depleted (Fig. 6, C and D). The HS chains derived from PG obtained after the combined treatment were therefore used to locate the GlcNH2 residues (see below).
Location of GlcNH2 Residues in HS Chains--
If HS
chains have the general domain structure depicted in Fig. 1, the fully
modified regions (filled boxes) should contain IdoUA(2-SO4) residues joined at their nonreducing side to
GlcNSO3 constituting sites for cleavage by heparin lyase
(marked with boldface S). Fragments generated from distal or
middle portions of an HS chain by cleavage with heparin lyase can be
separated from proximal HS stubs linked to the core protein by passage
over octyl-Sepharose, which adsorbs the latter material (12). Thus, octyl-Sepharose-bound glycoforms, obtained from cells subjected to
combined suramin treatment and nitrite deprivation, were digested with
heparin lyase and again passed over octyl-Sepharose. Approximately 20%
of the material (based on [3H]glucosamine) passed through
the column, indicating that heparin lyase-sensitive sites were
relatively rare. The released HS fragments were subjected to
gel-permeation chromatography on Superose 6 (Fig.
8A). Most of the material
consisted of fragments smaller in size (approximately 30 kDa on an
average) than those generated endogenously in suramin-treated cells
(Fig. 5A). The heparin lyase-generated HS fragments were
insensitive to nitrous acid at pH 3.9 (Fig. 8B), indicating
that GlcNH2 residues are rare distal to the heparin lyase
cleavage sites (Fig. 1).
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If a HS chain has the structure shown in Fig. 1, the octyl-Sepharose-bound heparin lyase degradation products should consist of the core protein substituted with more or less truncated HS chains. These chain stubs should consist of a major stretch of unmodified GlcUA-GlcNAc repeats (open box) immediately following the linkage region and sometimes extended with modified regions. The unmodified region should contain multiple sites for cleavage by HS lyase. Hence, the heparin lyase-degraded PG material was further degraded by treatment with HS lyase, and the products were again passed over octyl-Sepharose. More than 80% of the material passed through the column, indicating that nearly all of the radiolabeled HS chains had been released from the core protein. Because the released fragments should consist of small oligosaccharides, they were chromatographed on Superdex peptide (Fig. 8C). The material separated into one excluded fraction (pool I) and a series of included smaller-size saccharides (pool II). The former material, probably decasaccharides (approximately 2.5 kDa or larger), was treated with HNO2 at pH 3.9 and rechromatographed (Fig. 8D). The results showed extensive degradation into di-, tetra-, and hexasaccharides, indicating the presence of clustered GlcNH2 residues that had survived HS lyase treatment. Although the fragments generated by HS lyase had low [35S]/[3H] ratios, some sulfate could reside in surviving GlcNSO3 residues. To test this, materials from pools I and II were treated with HNO2 at pH 1.5. As shown in Fig. 8, E and F, respectively, they were completely resistant. Therefore, the saccharides containing GlcNH2 may also contain some O-sulfate. Taken together, the results obtained so far indicate that clustered GlcNH2 residues are concentrated to a proximal region between the core protein and the first heparin lyase-sensitive site (Fig. 1). To determine the placement of the endoheparanase cleavage sites relative to the clustered GlcNH2 residues, the following experiments were performed.
Large-size radiolabeled PG was isolated and purified from BFA-treated
ECV cells. Because the long radiolabeled HS chains produced in
BFA-treated cells appear to be made by extension of pre-existing, unlabeled HS stubs (18), cells were first incubated with
[3H]glucosamine in the absence of BFA to maximize
labeling of the HS chain stubs. Then the cells were incubated with
[35S]sulfate in the presence of BFA to radiolabel HS
chain segments made during accumulation of the large PG form. The
doubly labeled purified PG was then incubated with ECV cells to allow
cell-surface-located (if present) endoheparanase to degrade its HS
chains. As a control, incubations were also made in the presence of
suramin. The results showed (Fig. 9) that
exogenously supplied PG was degraded to medium-size PG-like (pool
II) and oligosaccharide-like (pool III) material (Fig.
9A). No degradation took place in the presence of suramin (Fig. 9B), indicating that the degradation was caused by
endoheparanase. The degradation products in pool II (Fig.
9A) were sensitive to alkali (Fig. 9C) confirming
their PG nature. The material in pool III (Fig.
9A) was alkali-resistant when analyzed on Superose 6, confirming their oligosaccharide nature (data not shown). They were
probably of decasaccharide size or larger as judged from the Superdex
peptide chromatogram (Fig. 9D). The HS chains derived from
the PG degradation product (Fig. 9C) were treated with
nitrous acid at pH 3.9 and rechromatographed on Superose 6 (Fig.
9E). The results showed extensive degradation into both
sulfate-rich and sulfate-poor fragments. When compared with the
profiles obtained after deaminative cleavage of the HS chains of the
substrate (Fig. 4, E and F) it is evident that
the longer chain fragments (eluting in fractions 20-30) were missing,
indicating that these were released by endoheparanase and should be
recovered in the oligosaccharide fraction (pool III in Fig.
9A). When the oligosaccharide material was subjected to
deaminative cleavage, no degradation could be observed upon
chromatography on Superose 6 (data not shown). To look for release of
very small saccharides, the oligosaccharides were also chromatographed
on Superdex peptide after treatment with nitrous acid at pH 3.9 (Fig.
9F). Again, no sign of degradation could be observed
(cf. Fig. 9D). These results thus indicate that the clustered GlcNH2 residues are located between the core
protein and the first endoheparanase cleavage site (Fig. 1).
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A PG substrate that was prepared from cells radiolabeled with
[3H]glucosamine and [35S]sulfate
simultaneously and in the presence of BFA was also used for incubations
with ECV cells. The HS chains of this PG should mainly be radiolabeled
in the peripheral regions (18). Using this PG substrate, only
radiolabeled HS oligosaccharides but no radiolabeled PG-type
degradation products were obtained (data not shown). This is consistent
with the idea that the large PG in BFA-treated cells is made by
extension of existing chains on a preformed PG and that the extension
starts somewhere near an endoheparanase cleavage site.
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DISCUSSION |
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Most GLP-1 glycoforms in unperturbed ECV cells contain HS chains that rarely exceed 50 kDa in size. However, some of the three available sites may contain shorter stubs indicating that elongation of HS chains on the different sites may not be synchronized. Moreover, because ECV cells should endogenously produce nitrite, it is perhaps not surprising that only a few GlcNH2 residues were detected. Hence, the short stubs may also be the result of deaminative cleavage.
The HS chains of the large PG from BFA-treated cells were approximately twice the size of those of the smaller PG, and GlcNH2 residues occurred in multiple places and were, surprisingly, more abundant than in chains obtained from nitrite-deprived cells. The reason for this could be that nitric oxide and subsequently nitrite were not produced in BFA-treated cells or that the PG was inaccessible to nitrite. Moreover, large GlcNH2-rich PG exogenously supplied to ECV cells suffered endoheparanase-catalyzed cleavage without substantial loss of GlcNH2 residues, probably because the substrate and/or nitrite concentration were not sufficiently high in this situation. In contrast, when endogenously produced PG was degraded by endoheparanase during pulse-chase experiments, most of the clustered GlcNH2 residues were lost, probably by deaminative cleavage.
Structural analysis of the HS chains from the PG produced in BFA-treated cells as well as of the HS stubs on the endoheparanase-generated PG product from exogenously supplied PG indicated that clustered GlcNH2 residues were concentrated to the region between the core protein and the first sites cleaved by endoheparanase and heparin lyase (Fig. 1). The analysis of the products also indicated that mainly the external portions of the HS chains are removed and replaced during recycling (Fig. 2). Similar observations were made with GLP from human fibroblasts (25, 26).
Recently, a mammalian endoheparanase with endo--glucuronidase
specificity (17) was cloned (27-31). Because only one gene was found
and the protein appeared unique, it was assumed that the same enzyme is
expressed in both normal and transformed cells. The enzyme, which
localizes to both endosomes and the cell-surface recognizes the
sequence -GlcUA-GlcNR-HexUA(2-SO4)-GlcNR (17). There are
two principal varieties of this sequence where HexUA is either GlcUA or
IdoUA. Therefore, the observations that there are multiple
endoheparanase activities and that some sites are more preferred (15,
16) could be ascribed to the presence of factors that bind to the
enzyme and modulate its specificity. Alternatively, basic compounds
such as polyamines and growth factors that bind to specific sequences
in HS may protect certain sites from cleavage (1, 3, 32).
Although endoheparanase is supposed to be inhibited by suramin (13), the GLP-1 glycoforms obtained from suramin-treated ECV cells contained truncated HS chains and were accompanied by HS degradation products indicative of some endoheparanase cleavage. It has been reported that human microvascular endothelial cells take up suramin via caveolae and that it is transported to the nucleus (33). It is thus possible that suramin does not have access to endoheparanase acting on HS when GLP-1 is recycling through, e.g. early endosomes. However, cell-surface-located enzyme was inhibited by suramin as shown in this study. Taken together, the results suggest that the potential endoheparanase site closest to the linkage region and near the clustered GlcNH2 residues (filled arrow in Fig. 1) is not cleaved.
The HS fragments released from peripheral regions of the chains by endoheparanase or heparin lyase did not seem to contain much GlcNH2, at least not in internal positions. However, the precursor of the suramin-arrested material, i.e. the large PG in BFA-treated cells, contained a heterogeneous population of HS chains, some of which must contain GlcNH2 residues also in more distal transition regions (Fig. 1). We had expected that the number of GlcNH2 residues should be significantly increased in HS produced in nitrite-deprived cells. However, this was not the case unless the cells were simultaneously treated with suramin. Taken together, these results suggest that endoheparanase cleavage sites and single GlcNH2 residues could be placed closely together (see Fig. 1, filled arrows). The finding that HS-chain fragments obtained after either endoglycosidic or deaminative cleavage had similar size distribution also supports this notion. Results of heparin lyase degradation indicated that also some of these sites were present in the near vicinity. This is in agreement with previous results (23), which indicated that heparin lyase and endoheparanase cleavage sites are juxtaposed. Hence, HS chains may contain "hotspots" where sites for cleavage by endoheparanase, heparin lyase, and HNO2 are close together as in -GlcNH2-GlcUA-GlcNSO3-IdoUA(2-SO4)-GlcNH2. The GlcNH2 residue at the reducing end is a potential target for 3-O-sulfation (9). GlcNH2 residues could also be present in segments not susceptible to heparin lyase, such as -GlcNH2/GlcNR-GlcUA-GlcNH2/GlcNR-GlcUA(2-SO4)-GlcNH2/GlcNR, where the glucosamine residue on the nonreducing side of GlcUA(2-SO4) is probably N-sulfated. There could also be consecutive GlcUA-GlcNH2 repeats or they could be alternating with GlcUA-GlcNAc repeats. Indications of the latter two arrangements were found in this study. The formation of GlcNH2 residues may be the result of insufficient N-sulfation after N-deacetylation, caused by a specific N-deacetylase/sulfotransferase isoform or by an endo-sulfamidase removing certain N-sulfate groups after completion of synthesis.
As mentioned above, the GlcNH2-containing repeats that were
clustered near the linkage region may inhibit further erosion by
endoheparanase (14, 17). This may give rise to HS stubs that are poor
primers for re-elongation (18). Formation of NO at relatively high
concentrations within the endothelial cell should result in rapid
conversion to nitrite before NO diffuses out of the cell (18, 19). It
is thus possible that NO indirectly, via nitrite, regulates recycling
by removing inhibitory "telosaccharides," thus facilitating
re-elongation. Recycling GLP-1 may be a vehicle for endo-/exocytosis of
growth factors and morphogens constituting an additional function of
HSPG in signaling and developmental patterning (1, 3, 32, 34).
Deaminative cleavage at GlcNH2 gives rise to reducing
terminal anhydromannose (24) in the released oligosaccharides and/or,
in combination with endoheparanase, free anhydromannose depending on
the structural context (see above). This unit and its reduced version
anhydromannitol have not yet been found in cells, neither in free form
nor at the reducing end of an HS oligosaccharide. However, exogenously
supplied anhydromannitol is phosphorylated by phosphofructokinase to
anhydromannitol-1-phosphate and -1,6-bisphosphate (Ref. 35 and
references therein). This causes inhibition of gluconeogenesis and
glycogenolysis and stimulation of glycolysis (35-37). The results
described in the present study thus invite inquiries into the
possibility of a connection between HS turnover and energy metabolism.
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ACKNOWLEDGEMENTS |
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The technical assistance of Birgitta Havsmark and Susanne Persson is greatly appreciated.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council, the Swedish Research Council for Engineering Sciences, the Cancer Fund, the Strategic Research Fund (Glycoconjugates in Biological Systems), and the WennerGren Foundation.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.
Contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 46-46-222-8573; Fax: 46-46-222-3128; E-mail: Lars-Ake.Fransson@medkem.lu.se.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M005238200
2 The first GLP gene product was originally termed dally (DglpA) and the second one glpK (DglpB), which was recently cloned by N. Khare and S. Baumgartner (personal communication).
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ABBREVIATIONS |
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The abbreviations used are: HS, heparan sulfate; BFA, brefeldin A; GlcNAc, N-acetylglucosamine; GlcNH2, N-unsubstituted glucosamine; GlcNR, glucosamine with unspecified N-substituent; GlcNSO3, N-sulfamidoglucosamine; GlcUA, D-glucuronic acid; GLP, glypican; HexUA, unspecified hexuronic acid; IdoUA, L-iduronic acid; NOS, nitric-oxide synthase; PG, proteoglycan; DMEM, Dulbecco's modified Eagle's medium; RIPA, radioimmune precipitation buffer.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bernfield, M., Götte, M., Park, P.-W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729-777[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Gengrinovitch, S.,
Berman, B.,
David, G.,
Witte, L.,
Neufeld, G.,
and Ron, D.
(1999)
J. Biol. Chem.
274,
10816-10822 |
3. |
Lindahl, U.,
Kusche-Gullberg, M.,
and Kjellén, L.
(1998)
J. Biol. Chem.
273,
24979-24982 |
4. | Linker, A., and Hovingh, P. (1975) Biochim. Biophys. Acta 385, 324-333[Medline] [Order article via Infotrieve] |
5. | Cifonelli, J. A., and King, J. A. (1977) Biochemistry 16, 2137-2141[Medline] [Order article via Infotrieve] |
6. | Fransson, L.-Å., Sjöberg, I., and Havsmark, B. (1980) Eur. J. Biochem. 106, 59-69[Abstract] |
7. |
van den Born, J.,
Gunnarsson, K.,
Bakker, M. A. H.,
Kjellén, L.,
Kusche-Gullberg, M.,
Maccarana, M.,
Berden, J. H. M.,
and Lindahl, U.
(1995)
J. Biol. Chem.
270,
31303-31309 |
8. | Toida, T., Yoshida, H., Toyoda, H., Koshiishi, I., Imanari, T., Hileman, R. E., Fromm, J. R., and Linhardt, R. J. (1997) Biochem. J. 322, 499-506[Medline] [Order article via Infotrieve] |
9. |
Liu, J.,
Shriver, Z.,
Blaiklock, P.,
Yoshida, K.,
Sasisekharan, R.,
and Rosenberg, R. D.
(1999)
J. Biol. Chem.
274,
38155-38162 |
10. | Lyon, M., and Gallagher, J. T. (1998) Matrix Biol. 17, 485-493[CrossRef][Medline] [Order article via Infotrieve] |
11. | Lindblom, A., and Fransson, L.-Å. (1990) Glycoconj. J. 7, 545-562[Medline] [Order article via Infotrieve] |
12. | Lindblom, A., Bengtsson-Olivecrona, G., and Fransson, L.-Å. (1991) Biochem. J. 279, 821-829[Medline] [Order article via Infotrieve] |
13. |
Nakajima, M.,
DeChavigny, A.,
Johnson, C. E.,
Hamada, J.-i.,
Stein, C. A.,
and Nicolson, G. L.
(1991)
J. Biol. Chem.
266,
9661-9666 |
14. |
Bai, X.,
Bame, K. J.,
Habuchi, H.,
Kimata, K.,
and Esko, J. D.
(1997)
J. Biol. Chem.
272,
23172-23179 |
15. |
Bame, K. J.,
and Robson, K.
(1997)
J. Biol. Chem.
272,
2245-2251 |
16. | Bame, K. J., Hassall, A., Sanderson, C., Venkatesan, I., and Sun, C. (1998) Biochem. J. 336, 191-200[Medline] [Order article via Infotrieve] |
17. |
Sandbäck-Pikas, D.,
Li, J.-P.,
Vlodavsky, I.,
and Lindahl, U.
(1998)
J. Biol. Chem.
273,
18770-18777 |
18. |
Mani, K.,
Jönsson, M.,
Edgren, G.,
Belting, M.,
and Fransson, L.-Å.
(2000)
Glycobiology
10,
577-586 |
19. |
Ignarro, L. J.,
Fukuto, J. M.,
Griscavage, J. M.,
Rogers, N. E.,
and Byrns, R. E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8103-8107 |
20. | Vilar, R. E., Ghael, D., Li, M., Bhagat, D. D., Arrigo, L. M., Cowman, M. K., Dweck, H. S., and Rosenfeld, L. (1997) Biochem. J. 324, 473-479[Medline] [Order article via Infotrieve] |
21. | Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080[Medline] [Order article via Infotrieve] |
22. | Hunziker, W., Whitney, A., and Mellman, I. (1991) Cell 67, 617-627[Medline] [Order article via Infotrieve] |
23. | Schmidtchen, A., and Fransson, L.-Å. (1994) Eur. J. Biochem. 223, 211-221[Abstract] |
24. |
Lindahl, U.,
Bäckström, G.,
Jansson, L.,
and Hallén, A.
(1973)
J. Biol. Chem.
248,
7234-7241 |
25. | Fransson, L.-Å., Edgren, G., Havsmark, B., and Schmidtchen, A. (1995) Glycobiology 5, 407-415[Abstract] |
26. | Edgren, G., Havsmark, B., Jönsson, M., and Fransson, L.-Å. (1997) Glycobiology 7, 103-112[Abstract] |
27. | Hulett, M. D., Freeman, C., Hamdorf, B. J., Baker, R. T., Harris, M. J., and Parish, C. R. (1999) Nat. Med. 5, 803-809[CrossRef][Medline] [Order article via Infotrieve] |
28. | Kussie, P. H., Hulmes, J. D., Ludwig, D. L., Patel, S., Navarro, E. C., Seddon, A. P., Giorgio, N. A., and Bohlen, P. (1999) Biochem. Biophys. Res. Commun. 261, 183-187[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Toyoshima, M.,
and Nakajima, M.
(1999)
J. Biol. Chem.
274,
24153-24160 |
30. | Vlodavsky, I., Friedmann, Y., Elkin, M., Aingorn, H., Atzmon, R., Ishai-Michaeli, R., Bitan, M., Pappo, O., Peretz, T., Michal, I., Spector, L., and Pecker, I. (1999) Nat. Med. 5, 793-802[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Dempsey, L. A.,
Plummer, T. B.,
Coombes, S. L.,
and Platt, J. L.
(2000)
Glycobiology
10,
467-475 |
32. | Belting, M., and Fransson, L.-Å. (1999) Biochem. J. 338, 317-323[CrossRef][Medline] [Order article via Infotrieve] |
33. | Gagliardi, A. R. T., Taylor, M. F., and Collins, D. C. (1998) Cancer Lett. 125, 97-102[CrossRef][Medline] [Order article via Infotrieve] |
34. | Perrimon, N., and Bernfield, M. (2000) Nature 404, 725-728[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Riquelme, P. T.,
Wernette-Hammond, M.,
Kneer, N. M.,
and Lardy, H. A.
(1984)
J. Biol. Chem.
259,
5115-5123 |
36. |
Hanson, R. L.,
Ho, R. S.,
Wiseberg, J. J.,
Simpson, R.,
Younathan, E. S.,
and Blair, J. B.
(1984)
J. Biol. Chem.
259,
218-223 |
37. | Stevens, H. C., and Dills, W. L. (1984) FEBS Lett. 165, 247-250[CrossRef][Medline] [Order article via Infotrieve] |