From the ¶ Department of Pathology and Section
of Comparative Medicine, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157-1040 and the
National
Institute for Biological Standards and Control, Blanche Lane, South
Mimms, Potters Bar, Harts, En6 3QG, United Kingdom
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ABSTRACT |
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Laminin-1 is a basement membrane glycoprotein
implicated in tumor-host adhesion, which involves the cell-binding
domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are
necessary for binding to laminin-1 have not been characterized. To
identify this laminin-binding oligosaccharide sequence,
GlcNSO4-rich oligosaccharides terminating with
[3H]2,5-anhydromannitol (AManR) residues were
isolated from human breast cancer cell (MCF-7)-derived HS through
hydrazinolysis/high pH (4.0) nitrous acid
treatment/[3H]NaBH4 reduction. These
oligosaccharides were chromatographed on a laminin-1 affinity column. A
high affinity dodecasaccharide was isolated and characterized.
Disaccharide analysis yielded IdoA(2-SO4) AManR(6-SO4) as the only disaccharide upon
treatment of this dodecasaccharide with nitrous acid at low pH (1.5).
The sequence of laminin-binding high affinity oligosaccharide is
therefore [IdoA(2-SO4)
GlcNSO4(6-SO4)]5[IdoA(2-SO4)
AManR(6-SO4)]. Low affinity
dodecasaccharides composed of [IdoA(2-SO4)
GlcNSO4(6-SO4)]5, [IdoA(2-SO4)
GlcNSO4] were also isolated
by laminin-1 affinity chromatography. Molecular modeling studies
indicate that a heparin-binding peptide sequence corresponding to amino
acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of
laminin-1, modeled as a right-handed
-helix, carries an array of
basic residues well placed to bind to clusters of sulfate groups on the
high affinity dodecasaccharide.
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INTRODUCTION |
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Heparan sulfate (HS)1 is a glycosaminoglycan polymer consisting of sequences of uronic acid-glucosamine disaccharides (1) in which uronic acid may be either glucuronic or iduronic acid and the glucosamine residue may be either N-acetylated (GlcNAc) or N-sulfated (GlcNSO4). Structural studies (2) indicate that blocks of GlcNSO4 containing sulfate-rich disaccharides ("heparin-like" domains) are interspersed with blocks of sulfate-poor domains containing GlcNAc disaccharides. The latter domains might also be composed of unsubstituted glucosamine-containing domains (3). Thus, heparan sulfate is a multidomain polysaccharide. These domains, with different structural modifications including O-sulfation of uronic acid residues and N- and O-sulfation of glucosamine residues, impart specific properties to heparan sulfate. For example, 3-O-sulfated glucosamine and 2-O-sulfated hexuronic acid residues have been implicated in antithrombotic (1) and mitogenic activities of bFGF-induced cell proliferation (4). Heparin-like domains of HS are also implicated in binding to a number of biologically important molecules such as lipoprotein lipase (5, 6), hepatocyte growth factor (7), and platelet factor 4 (8). Heparin as well as HS is also known to bind to laminin-1 (9, 10), a basement membrane glycoprotein postulated to be involved in tumor-host adhesion in metastasis. It is likely that sulfate groups of heparin/HS might be involved in binding to a region(s) of basic amino acids in laminin-1 with high affinity. However, no information is available on the nature of the laminin-binding oligosaccharide sequences in tumor cell HS. In the present study, we report the identification of a distinct oligosaccharide structure in human breast cancer cell (MCF-7) HS that exhibits strong affinity for laminin-1.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Human breast cancer cells (MCF-7) were purchased from American Type Culture Collection (Rockville, MD). Cells were cultured in MEM with Earle's salts containing non-essential amino acids, 2 mM glutamine, 10% fetal bovine serum, 1 mM sodium pyruvate, bovine insulin (10 µg/ml), and penicillin (100 units/ml)/streptomycin (100 µg/ml) in 225-cm2 flasks, incubated at 37 °C with 5% CO2.
Preparation of Laminin-1 Affinity Column-- Mouse laminin-1 (nidogen-free) was purchased from ICN. Laminin-1 was coupled to Reacti-Gel (6×) Support according to manufacturer's instructions (Pierce), in the presence of heparin (Sigma, Grade I) (laminin:heparin ratio = 1:30, by weight). The efficiency of coupling was approximately 80%. The laminin affinity column prepared in this manner contained 0.8 mg of laminin/ml of gel.
Preparation of MCF-7 Cell-associated HS--
MCF-7 cells were
biosynthetically radiolabeled (11) with 20 µCi/ml carrier-free
[35S]Na2SO4 (~43 Ci/mg of
sulfur) (ICN Pharmaceuticals Inc.) for 48 h. The medium was
removed, and cells were washed twice with phosphate-buffered saline.
The cell layer was solubilized with cold 0.1 M NaOH for
15-20 min. An aliquot of the cell fraction was assayed for protein
content (12), and the remainder was adjusted to pH 8.0 by adding 50%
(v/v) acetic acid. After adjusting to 5 mM
CaCl2, the material was digested with 2% w/w Pronase
(Calbiochem) at 37 °C for 48 h. The digest was diluted by 5 volumes with water and subjected to purification by DEAE-Sephacel
(Amersham Pharmacia Biotech) filtration (13). The sample was applied to
the column (5 ml bed volume), which was equilibrated with 20 mM sodium acetate buffer, pH 6.5, containing 0.25 M NaCl. After washing the column with 20 volumes of the
equilibrating buffer, bound glycosaminoglycan was eluted with 10 column
volumes of 2 M NaCl in 20 mM sodium acetate
buffer, pH 6.5. The glycosaminoglycan material was recovered by
extensive dialysis using Spectraphore Membrane
(Mr cut off 3500) against water, followed by
lyophilization. The purified glycosaminoglycan was digested with
chondroitinase ABC lyase (Seikagaku America Inc.) (14). The digest was
fractionated on a DEAE-cellulose column (3 ml bed volume) in
pyridine-formate buffers (15). After removal of chondroitin
sulfate-derived disaccharides by washing with 0.5 M
pyridine acetate, pH 5.0, [35SO4]HS peptides
were eluted with 2.5 M pyridine acetate, pH 5.0. [35SO4]HS peptides were subsequently
subjected to -elimination (1 M NaBH4, 50 mM NaOH, 45 °C, 20 h) to release HS chains (16). Excess borohydride was destroyed by addition of ice-cold 2 M acetic acid, and HS chains were recovered after passage
through AG-50W-X8 (200-400 mesh H+ form) (Bio-Rad).
35SO4 activity was monitored during HS
isolation only, whereas 3H activity was monitored during
the subsequent fractionation of 3H-oligosaccharides.
Preparation of MCF-7 Cell-associated HS Oligosaccharides by Hydrazinolysis-- Hydrazinolysis of HS chains was carried out by treatment with anhydrous hydrazine, 1% hydrazine sulfate for 9 h at 100 °C as described previously (5, 17). The deacetylated HS chains were treated with high pH (4.0) nitrous acid reagent (0.5 M H2SO4, 5.5 M NaNO2, 2:5) for 30 min at room temperature (18). The pH of the oligosaccharide products was adjusted to 8.5 with 1 M Na2CO3 and reduced with 0.5 mCi of [3H]NaBH4 (1.5 Ci/mmol) (ICN) at 50 °C for 30 min (5). The labeled oligosaccharides were recovered by passage through AG-50W-X8 (200-400 mesh, H+form), as described previously.
Chromatography of MCF-7 HS Oligosaccharides on Laminin-1 Affinity Column-- 3H-Labeled HS oligosaccharides were loaded onto a 1-ml laminin-1 affinity column, which was equilibrated with 2 mM sodium acetate buffer, pH 6.5, 5 mM CaCl2, 5 mM MgCl2. The flow-through was reloaded onto the column three to four times to achieve maximal binding. The column was washed with 30 ml of equilibrating buffer containing 140 mM NaCl. Bound oligosaccharides were eluted in a stepwise manner with the buffer containing 0.4 and 1.5 M NaCl. Fractions of 1 ml were collected, and the radioactivity in an aliquot of each fraction was determined.
Gel Chromatography-- Gel chromatography of laminin-bound 3H-labeled HS oligosaccharides was performed on a Bio-Gel P-10 (Bio-Rad) column (1.5 × 85 cm) in 0.5 M pyridine acetate, pH 5.0 (13); 1-ml fractions were collected.
Disaccharide Analysis-- The laminin-bound oligosaccharides were converted to disaccharides by low pH (1.5) HNO2 treatment (19) and reduced with [3H]NaBH4. The disaccharides were further purified by passage through Darco-Celite columns (20) and then analyzed on a 4.6 × 250 mm Partisil 10 SAX column (Whatman) using a stepwise gradient of 12 mM, 26 mM, and 200 mM KH2PO4 to elute nonsulfated, monosulfated, and disulfated disaccharides, respectively. The sulfated 3H-disaccharide mixture standard, from heparin, was purchased from Chirazyme Inc., Urbana, IL.
Molecular Modeling-- Molecular models were built (5, 21) and visualized using the program Insight (MSI, San Diego, CA).
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RESULTS |
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Laminin Affinity Fractionation of MCF-7 Cell-associated HS Oligosaccharides-- Isolated MCF-7 HS chains were N-deacetylated by hydrazinolysis and reacted with nitrous acid at high pH (4.0) (18) to cleave the glycosidic linkages following N-unsubstituted D-glucosamine residues. The resulting oligosaccharide mixture, terminating at 2,5-anhydromannose residues, was reduced with [3H]NaBH4 to produce 3H-oligosaccharides, which were fractionated on a laminin-1 affinity column. Approximately 97% of the oligosaccharides did not bind to the column. Bound oligosaccharides, constituting 3% of the total radioactivity loaded, were eluted from the column in the 0.4 and 1.5 M NaCl fractions (Fig. 1).
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Disaccharide Composition of Laminin-1-bound
Oligosaccharides--
Low pH (1.5) nitrous acid treatment was employed
to degrade high affinity oligosaccharides into disaccharides, which
were further reduced with [3H]NaBH4.
3H-Disaccharides were subsequently analyzed by ion-exchange
HPLC on a Partisil 10 SAX column eluted with a
KH2PO4 gradient. By comparison with standard
disaccharides from commercial heparin (Fig.
3A) and with published data
(18), ISMS was found to be the only major disacccharide (Fig.
3B) of the laminin-1 high affinity oligosaccharide. Based on
these findings, the probable structure for laminin-1 high affinity
oligosaccharide is [IdoA(2-SO4) GlcNSO4(6-SO4)]5[IdoA(2-SO4)
AManR(6-SO4)]. The low affinity dodecasaccharide gave rise to two disaccharides, ISMS and ISM at
the ratio of 5:1 (data not shown).
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Molecular Modeling of Laminin-1 High Affinity Dodecasaccharide and
Its Interaction with the G Domain of Laminin-1--
A molecular model
of a heparin dodecasaccharide, corresponding with the high affinity
structure described above, was obtained by using the atomic coordinates
of an NMR-derived solution conformation of heparin (21). Heparin
binding activity has been localized to the G domain of recombinant
laminin-1 (22), and a peptide sequence in this region, amino acids
3010-3031 (KQNCLSSRASFRGCVRNLRLSR), has been suggested as a
heparin-binding site (23). A three-dimensional structure has not been
reported for the G domain of laminin-1, and the above sequence does not
show sufficiently strong similarity to any peptide segment of known
structure to allow modeling on the basis of homology. However, the
sequence does contain several basic residues spread along its length,
and when the sequence is modeled as a right-handed -helix all four
internal arginine residues are found on one side of the helix (Fig.
4A). This configuration of the
peptide would allow the flexible arginine side chains to interact with
clusters of sulfate groups along the length of the high affinity
oligosaccharide (Fig. 4B).
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DISCUSSION |
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The present investigation shows that a sulfate-rich
oligosaccharide fraction in MCF-7 tumor cell HS, composed of a sequence of six disaccharides, binds strongly to laminin-1. This oligosaccharide was generated by employing a hydrazinolysis/deamination procedure (18),
which causes the cleavage of deacetylated
N-acetylglucosaminic bonds at pH 4.0 and releases HS
fragments rich in N-sulfated glucosamine residues. This high
affinity oligosaccharide (Fig. 1) consists of six repeating
disaccharide units of the structure [IdoA(2-SO4) GlcNSO4(6-SO4)] (Figs. 2 and
3B). Therefore, the structure for laminin-bound
high affinity oligosaccharide is [IdoA(2-SO4)
GlcNSO4(6-SO4)]5[IdoA(2-SO4)
AManR(6-SO4)]. A slightly less sulfated
dodecasaccharide of the composition [IdoA(2-SO4)
GlcNSO4(6-SO4)]5[IdoA(2-SO4)
GlcNSO4] was also isolated. While the homogeneity of
this oligosaccharide remains to be ascertained, it binds to laminin-1
with low affinity (Fig. 1) compared with the high affinity
oligosaccharide, even though both of these oligosaccharides are
approximately the same size (Fig. 2).
The strong interaction of laminin-1 with heparin or HS has been known (9) for quite some time. Indeed, heparin-agarose chromatography has been routinely employed (24) to separate laminin-1 subunits. Despite reports suggesting a strong binding between laminin-1 and heparin or heparan sulfate, to our knowledge, this is the first report of a specific extended oligosaccharide sequence in HS that has high affinity for laminin-1.
A number of heparin-binding proteins, comprised of different
biologically important macromolecules, including bFGF (25), hepatocyte
growth factor (26), platelet factor 4 (8), and lipoprotein lipase (5)
interact with heparan sulfate via specific oligosaccharide sequences
enriched in IdoA(2-SO4) GlcNSO4(6-SO4). Along with
N-sulfation of glucosamine residues,
2-O-sulfation of iduronic acids and/or
6-O-sulfation of glucosamine residues are the necessary
structural requirements for these heparin-binding proteins to interact
and elicit their biological activities. In the present study, a similar
oligosaccharide sequence was also involved in the binding of HS to
laminin-1. Experiments are in progress to examine the minimal sulfation
requirements of HS for binding to laminin-1, through chemical
modification of laminin affinity oligosaccharide. Alternatively,
Chinese hamster ovary cell mutants defective in heparan sulfate
biosynthesis (13, 27) could be employed to study the role of sulfation
in interaction and adhesion to laminin-1.
Heparan sulfate is also known to bind to a number of biologically
important molecules (28) including antithrombin (1), bFGF (25),
hepatocyte growth factor (26), and lipoprotein lipase (5). The binding
sites, or the percent of oligosaccharides with affinity for these
proteins, constitute less than 5% of the total oligosaccharides (1,
5). Thus, in the present study it is not surprising that laminin-1
bound to highly specific oligosaccharide sequences, which comprise only
3% of the total MCF-7 oligosaccharides. It is possible that even
though present in low proportions, the strategic locations of these
binding regions and concentration on the tumor cell surface would favor
their interaction with laminin-1. Highly sulfated, iduronate-rich
heparan sulfate oligosaccharide sequences such as these can be expected
to adopt the characteristic heparin conformation (21), in which
clusters of sulfate groups are formed and are favorable to bind to
basic amino acids on the protein surface. Modeled as an -helix, the
heparin-binding sequence in the G domain of laminin-1 (Fig.
4A) offers a linear array of basic residues well-placed to
interact with these sulfate clusters (Fig. 4B). Amphipathic
-helical peptides with high heparin affinity have previously been
described in other proteins (29). This model of the heparin/laminin
interaction remains tentative, and it is insufficiently detailed to
explain the difference in affinity between the fully sulfated and
slightly undersulfated dodecasaccharide fragments. The full
heparin-binding site of laminin may also be more extensive, involving
discontinuous segments of peptide sequence (30).
A family of cell surface transmembrane HS proteoglycans, called
syndecans (syndecan I through IV), have been reported to be synthesized
by a variety of cells (31, 32). In syndecans, HS chains are generally
composed of a series of conserved sulfate-rich (mainly
N-sulfated) domains, containing IdoA(2-SO4) GlcNSO4 ± (6-SO4) disaccharide units,
interrupted by sulfate-poor (mainly N-acetylated) domains,
with the relative proportions of these domains varying between
different syndecans (33). Laminin-1 high affinity dodecasaccharide is a
sulfate-rich oligosaccharide, containing repeating disaccharide units
of IdoA(2-SO4)
GlcNSO4(6-SO4), and is derived from the cell surface of MCF-7 cells. Therefore, it is
possible to conclude that this oligosaccharide is part of the
sulfate-rich domains of syndecan HS. Syndecan I particularly, has been
shown to bind to laminin-1 via heparan sulfate chains (34). However,
even though MCF-7 cells synthesize heparan sulfate proteoglycans, it is
unclear which of these syndecans carry the laminin-1-binding
oligosaccharide in their HS chains.
Aside from their central role in tumor-host adhesion in metastasis,
laminin-HS interactions may have biological importance in basement
membrane assembly due to the ubiquitous occurrence of HS proteoglycans
and laminin in basement membranes and the fact that laminin-1 contains
putative heparin-binding sites (9). Perlecan, the predominant
proteoglycan of basement membrane, is a HS proteoglycan and has been
shown to bind to laminin-1 via its HS chains (35). In addition,
Engelbreth-Holm-Swarm tumor heparan sulfate, most likely derived from
perlecan, was shown to contain at least 80% of its sulfate residues in
N-sulfated form (36). Furthermore, the disaccharide
IdoA(2-SO4) GlcNSO4(6-SO4) constitutes approximately 50% of the total disaccharide in
Engelbreth-Holm-Swarm tumor heparan sulfate. It is possible that
laminin-perlecan interactions could involve the laminin high affinity
dodecasaccharide, composed of IdoA(2-SO4)
GlcNSO4(6-SO4) repeating disaccharide units, and the G domain of laminin-1. Such ionic interactions could contribute to the macromolecular assembly of basement membrane (37).
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FOOTNOTES |
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* This work was supported in part by grants from the North Carolina Biotechnology Center (to N. P.) and an intramural research grant (to N. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Section of Comparative Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157-1040. Tel.: 336-716-2699; Fax: 336-716-6279; E-mail: npartha{at}wfubmc.edu.
The abbreviations used are:
HS, heparan sulfate; GMS, GluA AManR(6-SO4)IMS, IdoA
AManR(6-SO4)ISM, IdoA(2-SO4)
AManRISMS, IdoA(2-SO4)
AManR(6-SO4)GSM, GluA(2-SO4)
AManRHPLC, high performance liquid chromatographybFGF, bovine fibroblast growth factorMEM, minimum essential medium.
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REFERENCES |
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