From the Craniofacial Developmental Biology and
Regeneration Branch, NIDCR, National Institutes of Health, Bethesda,
Maryland 20892-4370 and the ¶ Graduate School of Environmental
Earth Science, Hokkaido University,
Sapporo 060-0810, Japan
Received for publication, January 26, 2001, and in revised form, March 28, 2001
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ABSTRACT |
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AG73 (RKRLQVQLSIRT), a peptide from the G domain
of the laminin Heparan sulfate proteoglycans
(HSPGs)1 are abundant in both
the extracellular matrix and on cell surfaces. The diverse biological activities of HSPGs largely depend on interactions between specific oligosaccharide sequences of the glycosaminoglycan (GAG) side chains and protein sequences. Modifications of the GAG side chains provide structural heterogeneity and a basis for the exquisite specificity of its interactions (1-4). Heparin is used to study GAG
interactions with cells and is the most negatively charged GAG, whereas
heparan sulfate and chondroitin sulfates are the biological GAG ligands
present on epithelial cell surfaces. The sulfation patterns of GAGs
vary in different sites, cells, and tissues and at various times in
development, allowing for protein- and cell-specific interactions
(5-7). For example, extensive studies on the role of heparin and
fibroblast growth factor receptor function have revealed important size
and sulfation requirements of heparin for different tissue-specific
FGF-mediated biological activities (8-10).
Laminins are a family of heparin-binding, biologically active
glycoproteins found in basement membranes. Currently, there are five
We have identified a biologically active sequence, AG73 (RKRLQVQLSIRT),
from the laminin Here, we compare the activity of AG73 with the homologous sequences in
the other laminin Cell Culture--
The HSG cell line (30) was cultured in
Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12),
containing 5% fetal bovine serum (Biofluids, Rockville, MD), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies,
Inc.). B16F10, a mouse melanoma cell line (31), was cultured in DMEM
containing minimal essential medium with nonessential amino acids (Life
Technologies, Inc.), 100 units/ml penicillin, 100 µg/ml streptomycin,
and 10% fetal bovine serum. Both cell types were maintained at
37 °C in a humidified, 5% CO2, 95% air atmosphere.
Preparation of Peptides and Beads--
All peptides were
manually synthesized using the 9-fluorenylmethoxycarbonyl (Fmoc)-based
solid-phase strategy with a C-terminal amide form and purified by
reverse phase high performance liquid chromatography as described
previously (32). The peptide-resin beads were also synthesized as
described previously (25).
Cell Attachment Assay with Peptide Beads--
Cell attachment to
peptide beads was assayed in 48-well dishes. The dishes and peptide
beads were blocked with 3% BSA in DMEM/F-12 for 1 h at 37 °C.
After washing with 0.1% BSA in DMEM/F-12, 3.0 × 104
cells were added and incubated for 2 h at 37 °C. The beads were washed, stained with DiffQuik (Baxter, Miami, FL), and photographed.
Cell Attachment Assays--
Peptides were coated onto
round-bottomed 96-well plates (Immulon 2B, Dynex Technologies,
Chantilly, VA) by either drying overnight in 50 µl of distilled
H2O or by incubating in 50 µl of PBS for 2 h at
37 °C. The wells were then blocked with 3% BSA in DMEM/F-12 for
1 h at 37 °C and then washed twice with 0.1% BSA in DMEM/F-12. 3.5 × 104 HSG cells in 100 µl of 0.1% BSA in
DMEM/F-12 or 3.0 × 104 B16F10 cells in 50 µl of
0.1% BSA in DMEM were added per well for 30 min at 37 °C. The
medium was gently removed from the wells, and the cells were stained
with crystal violet for 10 min. After washing twice with water, the
cells were lysed with 50 µl of 10% SDS and the optical density (600 nm) measured.
Biotinylated Heparin Binding Assay--
Peptide-coated plates,
prepared as above, were blocked with 3% BSA in PBS for 30 min at
37 °C and washed with 0.1% BSA in PBS. Biotinylated, sized heparin
(Heparin-BH with an average mass of 12.5 kDa, Celsus Laboratories,
Inc., Cincinnati, OH), 20 ng/well in 0.1% BSA in PBS, was added to the
wells and incubated for 30 min at 37 °C. The heparin was gently
removed, the plate was washed twice with 0.1% BSA in PBS, and the
bound biotinylated heparin was detected with streptavidin-alkaline
phosphatase (Pierce). After incubation with the enzyme substrate, the
optical density was measured at 405 nm. Initial dose-response
experiments with biotinylated heparin binding to a fixed amount of
peptide determined that 20 ng/well resulted in 50% of the maximal
binding detected with streptavidin-alkaline phosphatase.
Inhibition of Cell Attachment--
GAGs, modified heparin, and
heparin fragments were added to the cell attachment assay. GAGs (5 µg/ml) were incubated with the peptide-coated wells for 15 min, and
then the cells were added. Heparin, heparan sulfate, chondroitin
sulfates A, B, and C, keratan sulfate, hyaluronic acid (Sigma),
de-N-sulfated-N-acetylated heparin (DNSNAc),
completely desulfated-N-acetylated heparin (CDSNAc), and
completely desulfated-N-sulfated heparin (CDSNS) (Seikagaku, Rockville, MD) were used. Sized heparin fragments (a gift from Dr. A. Marolewski, RepliGen Corp., Needham, MA) were prepared by alkaline
depolymerization of heparin with an average mass of 5 kDa, (Enzyme
Research Laboratories, South Bend, IN) that was fractionated on a
Sephadex G50 column using ammonium bicarbonate buffer (33). Fractions
were dried and weighed. Size was determined by gradient electrophoresis
with comparison to known standards that had been prepared by capillary
electrophoresis (34).
Inhibition of cell attachment by competition with peptides was used to
determine IC50 values of competitor peptides. Decreasing
amounts of substrate peptides AG73-A5G73 were used to determine the
amount of peptide coating resulting in half-maximal cell attachment.
The cells were preincubated in solution with different amounts of the
competitor peptide for 10 min at 37 °C. Then the cells and
competitor peptide were added to the wells, and cell attachment was
quantitated as above. HSG and B16F10 cell attachment to AG73 was also
competed with a series of peptides containing amino acid substitutions and truncations. The amount of peptide used to compete was the same as
the amount of AG73 used to compete itself.
B16F10 Network Formation on Matrigel--
B16F10 cells (1.5 × 104 cells/well) were cultured in serum-free DMEM with
200 µg/ml peptides in 48-well flat-bottomed plates (Costar Corp.,
Cambridge, MA) coated with 133 µl of Matrigel (Becton Dickinson,
Bedford, MA). After 18 h, the cells were fixed, stained with
DiffQuik, and photographed.
Ex Vivo Salivary Gland Organ
Culture--
Submandibular/sublingual salivary gland rudiments
dissected from embryonic day 13 (E13) ICR mice were cultured on Whatman Nucleopore Track-etch filters (13 mm, 0.1-µm pore size, VWR, Buffalo Grove, IL) at the air/medium interface. The filters were floated on 240 µl of DMEM/F-12 in 50-mm glass-bottomed microwell dishes (MatTek,
Ashland, MA). The medium was supplemented with 100 units/ml penicillin,
100 µg/ml streptomycin, 150 µg/ml vitamin C, and 50 µg/ml
transferrin. Six E13 gland rudiments were cultured on each filter at
37 °C in a humidified 5% CO2, 95% air atmosphere.
Glands were photographed after ~1, 24, and 48 h, and the number
of end buds was counted at each time point. Various peptide
concentrations (26) were added to the medium at the beginning of the experiment.
Surface Plasmon Resonance--
Biotinylated, sized heparin
(Heparin BH, Celsus Laboratories Inc., Cincinnati, OH) was prepared by
oxidative cleavage with periodate, and the terminal aldehyde at the
reducing end of the heparin was labeled with biotin hydrazide. Thus,
the heparin would attach by its reducing end when immobilized to a
streptavidin-coated sensor chip (Sensor Chip SA, BIAcore, Inc.,
Piscataway, NJ). Heparin attached to a sensor ship by the reducing end
interacted more with protein than if attached at its midpoint (35). The
heparin, at 40 µg/ml in 25 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.005% surfactant P20 was
immobilized on the sensor chip at 10 µl/min for 4 min to an
immobilization level of 300 resonance units. Peptides (20 µM in 25 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.005% surfactant P20) were injected on the
heparin-coated surface at 30 µl/min in a BIA-coreTM 1000 instrument.
The association rates (ka) and dissociation
rates (kd) were registered (2 min each), and the
KD was calculated from the equation
KD= kd/ka. The
streptavidin-heparin surface was regenerated between each run by two
successive injections of 30 µl of 20 mM NaOH containing 1 M NaCl. In control experiments, the peptides were run over
a blank streptavidin chip. The sensorgrams were analyzed by non-linear least square curve fitting using BIAevaluation 2.1 software assuming single-site association and dissociation models.
Structural Alignment of the AG73 Peptide on the Crystal Structure
of the Cell Attachment to Laminin HSG and B16F10 Cell Attachment to AG73 Is Competed by AG73, A2G73,
and A3G73--
The specificity of HSG and B16F10 cell attachment to
AG73 was determined by competing attachment with the other homologous peptides. Increasing amounts of peptides in solution were added to both
HSG and B16F10 cell attachment assays to AG73, and the IC50
for each peptide was determined (Table
II). AG73 inhibited HSG and B16F10 cell
attachment to AG73 with IC50 values of 15 and 27 µg/ml,
respectively, and A2G73 inhibited HSG and B16F10 cell attachment to
AG73 with IC50 values of 24 and 119 µg/ml, respectively.
A3G73 inhibited HSG and B16F10 cell attachment with IC50
values of 13 and 277 µg/ml, respectively. However, A4G73 (IC50 > 400 µg/ml for both cell lines) could not inhibit
HSG or B16F10 cell attachment to AG73, and A5G73 could not inhibit
B16F10 cell attachment to AG73 but could compete HSG cell attachment with an IC50 of 241 µg/ml. These data show that HSG cell
attachment to AG73 can be inhibited by AG73, A2G73, and A3G73 with
similar IC50 values, suggesting they recognize a similar
receptor. In contrast, B16F10 cell attachment to AG73 is more specific,
in that A2G73, A3G73, A4G73, and A5G73 do not compete cell attachment with an IC50 similar to that for AG73 and may bind with
lower affinity or recognize different cell surface receptors.
Biotinylated Heparin Binds to AG73, A2G73, and A3G73--
We used
an enzyme-linked immunosorbent assay-type assay to investigate heparin
binding to the peptides. Biotinylated, sized heparin (12.5 kDa) was
incubated in peptide-coated 96-well plates and detected with
streptavidin-horseradish peroxidase. The biotinylated heparin also
inhibited HSG cell attachment to the peptides (data not shown). The
biotinylated heparin bound in a saturable, dose-dependent manner to AG73, A2G73, and A3G73 with KD values
(50% maximal binding) all in the nanogram range: 38 ng for A3G73, 107 ng for A2G73, and 121 ng for AG73 (Fig.
2). Biotinylated heparin bound to A4G73
and A5G73 with KD values in the microgram range, 4.4 µg for A4G73 and 21 µg for A5G73. AG73T showed some heparin binding
when >30 µg of peptide/well were used, suggesting nonspecific
trapping of heparin occurred at these doses. Thus, biotinylated heparin
binding to the peptides follows a similar pattern of activity as the
cell attachment to the peptides, in that AG73, A2G73, and A3G73 are
more active than A4G73 and A5G73, although A3G73 binds to heparin with
a higher affinity than AG73.
Inhibition of Cell Attachment to the Peptides with GAGs and
Modified Heparin--
Interestingly, different GAGs inhibited HSG and
B16F10 cell attachment to the peptides, suggesting cell type-specific
interactions with the peptides (Fig. 3).
Various amounts of the GAGs were used to inhibit cell attachment to the
peptides (data not shown). The concentration shown (5 µg/ml)
highlights the differences (Fig. 3, A and B). HSG
cell attachment to all five peptides was inhibited by heparin. Although
cell attachment to A3G73 was only inhibited ~50% by the dose shown,
it was completely inhibited at higher doses. HSG cell attachment to the
peptides was inhibited by heparan sulfate, with the exception of
attachment to A3G73, which was not inhibited even at higher doses.
Chondroitin sulfate B inhibited HSG cell attachment to A4G73 and A5G73,
and DNSNAc inhibited cell attachment to A5G73. Thus, cell attachment to
A4G73 and A5G73 is inhibited by less sulfated GAGs. HSG cell attachment
to the AG73 homologues was not inhibited by less sulfated GAGs, such as
chondroitin sulfate-A and -C, keratan sulfate, CDSNAc, or CDSNS.
B16F10 cell attachment to the peptides was inhibited with different
GAGs than HSG cells (Fig. 3B). Generally, B16F10 cell attachment was inhibited by less sulfated GAGs, including chondroitin sulfate B. Heparin and heparan sulfate inhibited B16F10 cell attachment to the AG73 homologues. Chondroitin sulfate B and DNSNAc heparin inhibited cell attachment to AG73, A4G73, and A5G73, but not to A2G73
and A3G73. Thus, a more sulfated region on the GAG mediates cell
attachment to A2G73 and A3G73 and a less sulfated region not requiring
an N-sulfate mediates cell attachment to AG73, A4G73, and
A5G73. CDSNS heparin inhibited attachment to AG73, suggesting the
interaction between B16F10 cells and AG73 requires only one sulfate/disaccharide. Chondroitin sulfates A and C and CDSNAc heparin
did not inhibit cell attachment to any of the peptides. Taken together,
these data indicate that different GAGs or similar GAGs with different
patterns of sulfation potentially mediate the interactions between HSG
and B16F10 cells with the peptides. A2G73 and A3G73 may bind to regions
of higher sulfation than AG73, and A4G73 and A5G73 may bind to less
sulfated regions or to chondroitin sulfate.
Inhibition of Cell Attachment with Sized Heparin
Fragments--
The interaction of heparin with growth factors and
their receptors is dependent on a critical size and sulfation pattern
of the heparin chain (37). The inhibition of HSG cell attachment to the
laminin peptides was also dependent on oligosaccharide size. Heparin
oligosaccharides with a degree of polymerization of 10 saccharides
(dp10) inhibited HSG cell attachment to AG73, A2G73, and A3G73 by
~50%, to A4G73 by ~90%, and to A5G73 by ~75% (Fig.
4A). Heparin oligosaccharides
with dp12 inhibited HSG cell attachment to AG73, A2G73, and A3G73 by
60-70% and to A4G73 and A5G73 by 90%. HSG cell attachment to A4G73
was also inhibited ~85% by an oligosaccharide with dp8. The trend
was similar for B16F10 cell attachment; heparin oligosaccharides with
dp10 inhibited B16F10 cell attachment to AG73, A3G73, A4G73, and A5G73
by >50% but only inhibited cell attachment to A2G73 by ~25% (Fig.
4B). Interestingly, B16F10 cell attachment to A2G73 was not
inhibited with dp20. Taken together with Fig. 3, the data further
indicate that different types of cell surface GAGs may recognize the
peptides with different sized areas of charge distribution or sulfation patterns.
Branching Morphogenesis of Mouse Embryonic Day 13 (E13) Salivary
Glands and B16F10 Network Formation on Matrigel Are Inhibited Only by
AG73--
In cell attachment and heparin binding assays, AG73, A2G73,
and A3G73 gave similar, although not identical results, suggesting they
may bind a similar receptor. Therefore, we compared their biological
activities in more complex assays. When E13 mouse embryonic salivary
gland rudiments are cultured on filters, they undergo branching
morphogenesis. When the homologous peptides from the other laminin
When B16F10 cells are cultured on basement membrane (Matrigel), they
form networks within 18 h. These cell-cell and cell-matrix interactions are important in the invasive phenotype exhibited by
malignant tumor cells on Matrigel (38). When the peptides were added to
this assay, only AG73 inhibited network formation and the cells grew as
a monolayer (Fig. 5B). Thus, AG73 was able to competitively
inhibit specific cell-matrix interactions that the other peptides could
not. Although the five homologous peptides are similar, based on the
number of identical and conserved residues in their sequences (Table
I), our data suggest that there are specific residues or motifs in AG73
that are responsible for its biological activity.
Amino Acid Substitutions and Truncations Identify Residues in AG73
Important for Its Biological Activity--
Peptides with a series of
alanine substitutions, conserved and non-conserved substitutions in
residues Ile10 and Arg11, and N-terminal
truncations were synthesized and tested in various assays (Table I).
Cell attachment assays to these peptides with both HSG and B16F10 cells
showed similar results; no single amino acid substitution inhibited
cell attachment. Truncations with less than 8 amino acids, AG73D and
AG73G, however, did not support cell attachment.
The peptides with substitutions and truncations were tested in solution
for their ability to compete HSG and B16F10 cell attachment to AG73.
Alanine substitutions that resulted in the peptide with decreased
ability to compete HSG cell attachment to AG73 were Arg3
Biotinylated heparin binding assays identified four peptides with
alanine substitutions that resulted in decreased heparin binding:
Leu4
The series of peptides with substitutions and truncations were also
tested for their ability to inhibit salivary gland branching morphogenesis to identify amino acids critical for AG73 function (Fig.
6, A and B).
Alanine substitutions in AG73 at Leu4
The peptides with amino acid substitutions and truncations were also
used to define the sequence that inhibited B16F10 cell networks on
Matrigel. Alanine substitution Arg1 Surface Plasmon Resonance Analysis of Peptide Binding to
Biotinylated Heparin--
Biotinylated, sized heparin was immobilized
on a streptavidin sensor chip, and the association rates
(ka), dissociation rates (kd), and the dissociation constants
(KD) of a series of peptides with alanine
substitutions in the AG73 sequence (Table I) were measured in solution
(Table III). There was an absence of
detectable binding of Thr12 Molecular Modeling Indicates That AG73 Is Located in a Laminin-1 contains multiple heparin-binding sites, including AG73,
a peptide from the G4 module that binds the heparan sulfate side chains
of syndecan-1 (15). Here, we have defined the functional interactions
of this peptide and the homologous sequences from the other laminin
Peptides generally do not have well defined secondary
structure but are flexible and so assume random conformation in
solution. We see differences in the biological activity of a particular peptide depending on what assay it is used in and whether it is bound
to a plate or in solution. When the 12 peptides with a single alanine
substitution are bound to plates, they all support HSG cell adhesion,
but only 6 are able to compete HSG cell adhesion to AG73 when they are
added in solution (Table I). These data could suggest that peptides
bound to plastic have a fixed or distorted conformation and thus less
selective activity, whereas in solution the peptides assume a
conformation with a more selective biological activity that may mimic
the site on laminin HSG and B16F10 cell attachment to the AG73 homologues was inhibited to
various extents by different GAGs and by heparins with modified sulfate
groups, which provides important information about the potential
receptors for the peptides on the cell surface. Typically, in
vitro studies use heparin to analyze the putative heparin-binding
sites on laminins; however, the cell surface receptors are heparan
sulfates and, as our data suggest, potentially chondroitin sulfates.
The specificity of interactions with GAGs is not determined by total
sulfation but by unique sulfation patterns on the GAGs. Our data
suggest that HSG and B16F10 cell attachment is dependent on specific
sulfation patterns or different cell surface GAGs (Fig. 2) and a
specific size of GAG (Fig. 4). The sulfation patterns on different GAGs
are known to be clustered in regions of higher and lower sulfation (6,
43). The cell surface ligand for AG73 on HSG cells was identified as
the heparan sulfate side chains of syndecan-1, whereas the cell surface
ligand for B16F10 cells may be both the heparan and chondroitin sulfate
side chains.
GAGs do not act nonspecifically, but have unique functions in signaling
pathways during cell differentiation and morphogenesis (1). The
interaction of heparin with growth factors and their receptors is
dependent on a critical size and sulfation pattern of the heparin
chain, resulting in both binding and activation of the cell surface
receptor (9). Recently, heparin hexasaccharides were reported to have
dual roles of FGFR binding and dimerization, and
6-O-sulfation played a pivotal role in both processes (37). Others showed that heparin dodeca- (dp12) and deca- (dp10) saccharide fractions were mitogenic, and a clear correlation between total sulfate
content and receptor activation activity was demonstrated (8, 44). The
subtle differences in the HSG and B16F10 cell GAG composition may
enable different biological responses to the laminin-derived peptides
in a cell-dependent manner.
When the different AG73 homologues were tested for their activity in
more complex in vitro assays, AG73 was active but the homologues displayed little activity (Fig. 5). Branching morphogenesis of submandibular glands was only inhibited by AG73 and not by the other
homologues. The developing mouse submandibular gland basement membrane
contains the laminin An important paradigm has emerged in the FGF field for the role of GAGs
in regulating tissue-specific functions of FGFs (2). The amino acid
residues in the heparan sulfate-binding region of the FGFs are not
completely conserved, suggesting that the affinity or specificity of
different FGFs for their receptors depend on specific heparan sulfate
sequences. The comparison of heparan sulfates from various tissues and
cell types reveals differences in sulfation patterns (6).
Tissue-specific differences in the structure of heparan sulfate modify
the activity of FGFs. For example, tissue-specific heparan sulfate
fragments differentially activate FGFs 1, 2, and 4 (47). The sulfation
patterns of the heparan sulfates also regulate FGF activity, with
2-O-sulfation being required for FGF binding to the FGF
receptor and 6-O-sulfation required for receptor activation.
Therefore, tissue-specific patterns of O-sulfation and the
local concentration of heparan sulfate regulate the activity and
specificity of FGFs (2). This model may provide a framework to
understand the function of the multiple heparin-binding sites that have
been described in laminins, particularly in their G domains. Sites that
are identified in vitro as heparin-binding, in fact, bind to
heparan sulfate and potentially chondroitin sulfates in
vivo. Laminin expression is developmentally regulated in tissue- and site-specific locations. Variation in the amino acid sequences of
heparin-binding sites would potentially modulate the affinity and
specificity of the interaction, thus providing a mechanism for
regulating different functions of a laminin isoform within a tissue or
between different tissues. The heparan sulfate-containing receptors for
laminins include dystroglycan, syndecans, agrin, and perlecan, and cell
type-specific interactions involving these GAG-containing receptors and
the multiple integrin receptors could be modified by differences in the
amino acid sequences of the heparin-binding sites on laminin isoforms.
Laminin isoforms bind to various integrins, but different laminin
isoforms can bind to the same integrins yet have different functions
(12). Similarly, the interaction with heparan sulfates could provide a
mechanism to modulate the functions of laminin isoforms. Syndecans, a
family of transmembrane proteoglycans, bind a wide range of components, including the AG73 peptide, through their heparan sulfate side chains.
All cells and tissues except B-stem cells express syndecans in cell-,
tissue-, and developmentally specific patterns (48, 49). Specific
interactions between different laminin isoforms and syndecans have not
been reported and could provide a mechanism for regulating cell-,
tissue-, or developmentally specific interactions with laminin isoforms.
The biological activity of a peptide sequence within a molecule is
affected by the conformation of the surrounding protein. Previously, we
reported that AG73 inhibits HSG cell attachment to the E3 fragment,
which contains the AG73 sequence and partially inhibits cell attachment
to laminin-1, suggesting it is a functional sequence in the intact
molecule (15). When we align the sequence of AG73 using structure-based
sequence alignment, it forms In conclusion, multiple levels of regulation control the interactions
between laminins and their cell surface and extracellular matrix
receptors, as evidenced by the broad range of biological activities of
laminins. We have investigated one biologically active heparin-binding
sequence from the G domain of the laminin 1 chain, has diverse biological activities
with different cell types. The heparan sulfate side chains of
syndecan-1 on human salivary gland cells were previously identified as
the cell surface ligand for AG73. We used homologous peptides from the
other laminin
-chains (A2G73-A5G73) to determine whether the
bioactivity of the AG73 sequence is conserved. Human salivary gland
cells and a mouse melanoma cell line (B16F10) both bind to the
peptides, but cell attachment was inhibited by glycosaminoglycans,
modified heparin, and sized heparin fragments in a cell type-specific
manner. In other assays, AG73, but not the homologous peptides,
inhibited branching morphogenesis of salivary glands and B16F10 network formation on Matrigel. We identified residues critical for AG73 bioactivity using peptides with amino acid substitutions and
truncations. Fewer residues were critical for inhibiting branching
morphogenesis (XKXLXVXXXIRT) than
those required to inhibit B16F10 network formation on Matrigel
(N-terminal XXRLQVQLSIRT). In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be
important for heparin binding. Structure-based sequence alignment
predicts AG73 in a
-sheet with the N-terminal K
(Lys2) and the C-terminal R (Arg10) on
the surface of the G domain. In conclusion, we have determined that
differences in cell surface glycosaminoglycans and differences in the
amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, three
, and three
chains, which form 15 different heterotrimers (11, 12). Laminin isoforms are present at different times
during development with tissue-specific expression patterns. Cell
type-specific interactions between laminin isoforms and multiple integrins and heparan sulfate-containing receptors provide mechanisms for regulating the broad range of biological activities of laminins (13). Several heparin-binding sites have been identified on laminin
chains and interactions with the heparan sulfate proteoglycans, including dystroglycan, syndecans, agrin, and perlecan, have been demonstrated (14, 15). The elastase fragment of the laminin
1 chain,
E3, which contains the C-terminal LG4 and LG5 modules, is an important
heparin-binding site (16, 17). Recent studies have identified other
heparin-binding sites in the homologous LG4-5 regions of laminin
1
(14, 18),
2 (14),
3 (19),
4 (20, 21), and
5 (22, 23).
Different heparin-binding sequences have been identified depending on
the laminin isoform and the purified ligand, cell, or tissue type tested.
1 G4 module using a synthetic peptide approach
(24). AG73 promotes attachment of multiple cell types (24, 25), induces
salivary acinar cell differentiation (15), inhibits branching
morphogenesis of embryonic salivary glands (26), stimulates neurite
outgrowth (27), stimulates matrix metalloproteinase secretion by PC12
cells (28), and promotes liver metastasis by melanoma cells (29). AG73
inhibits human submandibular gland (HSG) and B16F10 melanoma cell (29)
attachment to the E3 fragment of laminin-1. The receptor on HSG cells
for AG73 was identified as syndecan-1, and the interaction occurs via
the heparan sulfate side chains (15). Recent data from our laboratory
with B16F10 cells suggest a similar AG73 receptor, with related but
different GAG side
chains.2
-chains (A2G73-A5G73, Table
I) to determine whether the active
sequence is specific to the
1 chain. We show that cell type-specific
interactions with the peptides involve different GAGs. We identify the
amino acids in AG73 that mediate its specific biological activity in
different assays. Our data indicate that cell-specific activities of
AG73 are mediated by interactions with cell surface GAGs and that
specific amino acids in AG73 modulate different biological activities
in a cell-specific manner.
Summary table of results
-chains are highlighted.
The other amino acid substitutions and truncations (light shading) are
also highlighted.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 LG5 Module--
AG73 was mapped on the crystal structure
of the
2 LG5 module (Protein Data Bank code 1qu0) using the RasMol
program (36). The AG73 sequence aligns to
Gly2955-Thr2966 of the
2 sequence.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Chain Homologues of AG73--
Resin
beads with peptides synthesized directly on the beads were initially
used to compare HSG cell attachment to AG73 and to the homologous
sequences from the other laminin
-chains. Cell attachment after
2 h to AG73, A2G73, and A3G73 was greater than to A4G73 and A5G73
(Fig. 1A). AG73T, a scrambled
version of AG73, showed no cell attachment activity. Different amounts
of peptides were then dried onto 96-well plates, and HSG and B16F10
cell attachment was compared. Both cell types bound to the AG73
homologues but not to AG73T. For both cell types, the relative amount
of cell attachment activity of the peptides was similar: AG73 > A3G73 > A2G73 > A5G73 > A4G73 (Fig. 1, B
and C). These data demonstrate that homologues of the
laminin
1 chain peptide AG73 are active for cell attachment, to
varying degrees, when coated on culture plates.
View larger version (50K):
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Fig. 1.
HSG and B16F10 cells adhere to AG73 and the
homologous peptides from the laminin
-chains. A, cells were incubated
with peptide beads for 2 h at 37 °C, and the beads were washed
and stained with DiffQuik. There are similar levels of HSG cell
attachment to AG73, A2G73, and A3G73 peptide beads and less cell
attachment to beads with A4G73 and A5G73. Cells do not adhere to the
scrambled peptide AG73T beads. B and C, cell
adhesion assays are described under "Materials and Methods." Cells
were incubated for 30 min at 37 °C in plates coated with increasing
amounts of peptides. After washing, the cells were stained, lysed, and
the A600 nm measured. HSG cells adhere to
plates coated with AG73 > A3G73 > A2G73 > A4G73 > A5G73 > AG73T. B16F10 cell attachment to plates coated with
increasing amounts of peptides. B16F10 cells adhere to plates coated
with AG73 > A3G73 > A2G73 > A5G73 > A4G73 > AG73T. All experiments were repeated at least three times,
graphs in B and C represent three
wells/dose, and S.E. are indicated.
IC50 values of competitor peptides (µg/ml) during HSG and
B16F10 cell adhesion to AG73
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Fig. 2.
Biotinylated heparin shows increased binding
to wells coated with AG73, A2G73, and A3G73 compared with binding to
wells coated with A4G73 and A5G73. Biotinylated heparin was
incubated for 30 min at 37 °C in peptide-coated dishes and detected
with streptavidin-horseradish peroxidase. Heparin binds to plates
coated with A3G73 > A2G73 > AG73 > A4G73 > A5G73 > AG73T. The KD values (50% of maximal
binding) for the peptides are listed. Triplicate wells were used for
each condition, graphs are representative of at least three
experiments, and S.E. are indicated.
View larger version (32K):
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Fig. 3.
Glycosaminoglycans and modified heparin
inhibit HSG and B16F10 cell attachment to wells coated with the
homologous peptides from the laminin -chains
in a cell type-specific manner. Cell adhesion assays are described
under "Materials and Methods." Peptide-coated wells were incubated
with GAGs (5 µg/ml) for 15 min, and then cells were added.
A, inhibition of HSG cell attachment. B,
inhibition of B16F10 cell attachment. Triplicate wells were used for
each condition, graphs are representative of at least three
experiments, and S.E. are indicated.
View larger version (39K):
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Fig. 4.
Sized heparin fragments inhibit HSG and
B16F10 cell attachment in a cell type-specific manner. Cell
adhesion assays are described under "Materials and Methods."
Peptide-coated wells were incubated with sized heparin fragments (5 µg/ml) for 15 min, and then cells were added. A,
inhibition of HSG cell attachment. B, inhibition of B16F10
cell attachment. Triplicate wells were used for each condition,
graphs are representative of at least three experiments, and
S.E. are indicated.
-chains were added to the assay, they had no inhibitory effect on
branching (Fig. 5A).
Therefore, AG73 activity is specific; even though the other peptides
compete HSG cell attachment to AG73-coated wells in a more complex
assay, they do not have the same activity as AG73.
View larger version (100K):
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Fig. 5.
The biological activity of AG73 is specific
compared with the other -chain
homologues. AG73 inhibits branching morphogenesis of embryonic day
13 submandibular gland culture and B16F10 melanoma cell network
formation on Matrigel. A, salivary gland organ culture is
described under "Materials and Methods." AG73 (200 µg/well)
inhibits branching morphogenesis, whereas the homologous peptides were
similar to the control, scrambled peptide, AG73T. Each assay contained
at least five salivary glands, the assays were repeated at least three
times, and a representative gland is shown. B, B16F10
melanoma cells cultured on Matrigel form networks within 18 h.
When peptides are added to the media, only AG73 inhibits the network
formation. Triplicate wells were used for each condition, the
experiments were repeated at least three times, and a representative
well is shown.
Ala, Leu4
Ala, Val6
Ala,
Leu8
Ala, Ile10
Ala, and
Thr12
Ala. Truncated peptides that were unable to
compete HSG cell attachment were AG73D and AG73G, which are both
shorter than 8 amino acids, and AG73H, which was only missing the
N-terminal Arg1 and the C-terminal Thr12.
Ala, Val6
Ala, Leu8
Ala, and Ile10
Ala (Table I). These substitutions
resulted in lower heparin binding than AG73 but not as low as measured
for A4G73 and A5G73. We compared the four residues in AG73 that are
important for heparin binding to see if they are identical or conserved
in A2G73, A3G73, A4G73, and A5G73 (Table I). In A2G73, which bound
heparin similar to AG73 (Fig. 4), the two leucines (Leu4
and Leu8) are identical and the other two residues are
conserved. Comparing A3G73, which had slightly better heparin binding
(KD 38 ng), to AG73 (KD 121 ng),
Leu4, Leu8, and Ile10 are identical
and the other residue, Val6, is not conserved. However,
when comparing these to A4G73, which had very low heparin binding, none
of them are identical, two are conserved, and in A5G73, only one is
identical and two conserved.
Ala,
Val6
Ala, and Ile10
Ala showed no
inhibition of branching. An alanine substitution at Thr12
Ala, or a non-conserved substitution, Arg11
Glu,
resulted in peptides that only partly inhibited branching. Peptide
truncations resulting in loss of AG73 activity were AG73B, a deletion
of Lys2 from AG73A (AG73A still inhibited branching), and
AG73H, which is a truncation of the C-terminal Thr12. These
truncations were important to define the minimal sequence length that
inhibits branching morphogenesis as AG73A (KRLQVQLSIRT). Taken together
with the amino acid substitutions, the residues critical for function
(i.e. inhibiting branching of salivary glands) are
XKXLXVXXXIRT.
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Fig. 6.
The amino acids in the AG73 sequence
responsible for the inhibition of branching morphogenesis were
identified using amino acid substitutions and deletions.
A, salivary gland organ culture is described under
"Materials and Methods." After 48 h of culture, the end buds
were counted. AG73 (200 µg/well) inhibited branching morphogenesis,
and the other peptides were added at a similar dose. Five glands were
used for each condition, the graph is representative of at
least three similar experiments, and S.E. are indicated. B,
representative example of two glands after 48 h of culture with
Val6 Ala and Gln7
Ala.
Hematoxylin/eosin-stained sections of the glands highlight the ducts
and the terminal lobules that form the branching structure of the
gland. C, the peptides were also used to inhibit B16F10
network formation on Matrigel. The data are presented in Table I, and
two representative examples are shown after 18 h of culture with
Arg1
Ala and Leu4
Ala.
Ala and
Lys2
Ala inhibited and Arg3
Ala
partially inhibited network formation (Table I and Fig. 6C).
AG73A (Arg1 truncation) inhibited and AG73B
(Arg1/Lys2 truncation) partly inhibited B16F10
network formation. Taken together these results suggest the N-terminal
RK- residues are not required for AG73 function in this assay, but any
other truncation or substitution results in loss of AG73 activity.
Thus, RLQVQLSIRT is the minimal sequence required for AG73 to inhibit
B16F10 cell network formation. It also suggests that HSG and B16F10
cell surface GAGs recognize different amino acids in the AG73 sequence.
Ala and the scrambled
peptide AG73T. Thr12
Ala and AG73T both lack the
biological activity of AG73; they do not inhibit branching
morphogenesis of submandibular glands, do not inhibit B16F10 network
formation on Matrigel, and are unable to compete attachment of HSG
cells to AG73 (Table I). Many substitutions altered the
ka by a few fold, which in this type of assay is
not considered significant, but two substitutions, Ile10
Leu and Arg11
Lys, decreased the
ka by greater than 5-fold. Ile10
Leu partly inhibited HSG cell attachment to AG73, and Arg11
Lys partly inhibited branching morphogenesis of submandibular glands; however, both peptides were unable to inhibit B16F10 network formation on Matrigel. Peptide substitutions only affected the kd by a few fold, except for Arg11
Lys (kd = 1.5 × 10
3 M
1 × s
1), which resulted in a 5-fold decrease
in kd compared with AG73 (kd = 8.2 × 10
3
M
1 × s
1). Interestingly, the KD
values of the peptide substitutions were all in the low micromolar
range. However, Ile10
Leu (KD = 79 µM) had a 12-fold decrease in affinity compared with AG73
(KD = 6.4 µM). Therefore, surface plasmon resonance analysis suggests that the C-terminal
Ile10, Arg11, and Thr12 are
important for the interaction of AG73 in solution with heparin and
mediate part of the biological activity of AG73. Interestingly, these
three residues are not identical in A2G73-A5G73 and only A2G73 and
A3G73 have two identical residues (Table I), which may explain their
inability to inhibit branching morphogenesis of submandibular glands
and to inhibit B16F10 network formation on Matrigel.
Surface plasmon resonance analysis of AG73 and peptides with amino acid
substitutions; binding to heparin immobilized on the sensor chip
-Sheet in
the G4 Module of the Laminin
1 Chain--
The crystal structure of
the laminin
2 LG5 module and the
2 LG4-LG5 module pair has been
determined (39, 40). Using the structure of the laminin
2 LG5 module
and structure-based sequence alignment, the LG4 of laminin
1 can be
modeled and the location of AG73 predicted (Fig.
7). Based on this prediction, three of
the residues (Lys2, Arg11, and
Thr12) important for branching morphogenesis are predicted
to be on the surface of the structure at either end of a
-sheet. The
other amino acids, Leu4, Val6, and
Ile10, are internal in the structure. Five amino acids
required for inhibition of B16F10 cell networks (Arg3,
Val5, Lys7, Arg11, and
Thr12) were surface-exposed on the predicted model.
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Fig. 7.
Mapping of the AG73 peptide on the crystal
structure of the 2 LG5 module shows it is
located in a
-sheet. View of peptide AG73
(shaded gray) mapped on the crystal structure of
the
2 LG5 module (Protein Data Bank code 1qu0) (39) using the RasMol
program (36). The AG73 sequence aligns to
Gly2955-Thr2966 of the
2 sequence. The
first three residues (RKR) of the AG73 sequence are located spatially
close to a calcium-binding site (shaded black)
that has been implicated in
-dystroglycan binding to the laminin
2 chain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chains. Our data demonstrate that the cellular responses to this
sequence vary and are dependent on the cell type and assays used as
well as on the environment of the cells. When the homologous peptides
from the other laminin
-chains are coated on dishes, their
biological activities are similar with both salivary gland and melanoma
cells in cell attachment assays and in enzyme-linked immunosorbent
assay-type heparin-binding assays. The highest heparin-binding peptide
did not correspond to the peptide with highest cell attachment
activity, indicating that something other than charge is involved
(i.e. something specific about the AG73 sequence or
something specific to the heparan sulfate on the cell surface).
Furthermore, the biological activity of AG73 is specific; in assays
where the peptide is in solution (i.e. inhibiting branching
morphogenesis of submandibular glands and inhibiting B16F10 cell
network formation on Matrigel), the homologous peptides from the other
laminin
-chains have no activity. Although it may seem surprising
that such a small peptide has such specificity, there is precedent in
the literature. RGD-containing peptides have been widely used to
investigate integrin-dependent cell adhesion and have
exquisite biological specificity (41, 42).
1 in vivo. This is supported by our
recent mutational analysis of the AG73 sequence in recombinant LG4-5
of laminin
1, which indicates specific residues are critical for
specific biological
activities.3
1,
2,
3, and
5 chains (26, 45, 46).
Our results suggest that the AG73 sequence in the laminin
1 chain
has a specific role in the morphogenesis of the gland. Likewise,
melanoma cells plated on basement membrane Matrigel form a complex
network, which was only blocked by AG73 and not by the homologues.
Matrigel contains only laminin-1; therefore, the interaction between
B16F10 cells and the AG73 sequence on the laminin
1 chain is
specific and is not disrupted with the homologous peptides. In these
more complex assays, the peptides were tested in solution, so changes
in conformation could explain differences in activity. Both assays
demonstrate a high degree of specificity for the AG73 sequence.
Therefore, we determined which amino acids were required for the
activity of AG73 in different assays. These data confirm that
functional differences in AG73 activity depend on the region of the
peptide recognized by the particular cell type and are in accordance
with previous studies with PC12 cells. Although the amino acids
required for PC12 cell attachment and neurite outgrowth were different,
the amino acids required for neurite outgrowth and matrix
metalloproteinase secretion were identical (28).
strand C in the crystal structure of
the
2 LG5 module. Our data here suggest that of the six
surface-exposed residues, Lys2, Arg3,
Val5, Lys7, Arg11, and
Thr12, three are critical to inhibit branching
morphogenesis, and five are necessary to inhibit B16F10 cell network
formation on Matrigel. Mutation of these residues in a recombinant
molecule would be necessary to confirm the sequence is active in
vivo. It is possible that AG73 binds to heparan sulfate and blocks
the interaction with other heparin-binding sites on laminin.
Interestingly, the basic residues Arg1, Lys2,
and Arg3 are solvent-exposed and located spatially close to
a calcium-binding site and several basic residues that were previously
implicated in
-dystroglycan and heparin binding of both the
1 LG4
and
2 LG5 modules (17, 39). Alanine mutagenesis of basic residues mapped heparin- and
-dystroglycan-binding residues to two sites in
the
1 LG4 module, with the sequences GKGRTK (K3 mutant) and EYIKRK
(K1 mutant) (17). A minor site, HYARARN (R1 mutant), was also found to
be critical for binding of
-dystroglycan but not for heparin. It is
interesting to note that the three heparin- and/or
-dystroglycan-binding sites are identical to AG78
(HFMFDLGKGRTK), AG81 (AHVKTEYIKRK), and AG86
(LGGLPSHYRAR), three cell-binding sequences identified
earlier by systematic screening of synthetic peptides (24).
1 chain and determined
that cell type-specific differences in cell surface GAGs modulate the
biological activity of this peptide in both in vivo and
in vitro experimental systems. It remains to be determined
whether cell and tissue type-specific sulfation patterns and GAG
composition will effect the interactions of other recently identified
heparin-binding sites on the different laminin isoforms and thereby
modulate their function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Melinda Larsen and Harry Grant for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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: NIDCR, National Institutes of Health, 30/430, 30 Convent Dr., MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-1660; Fax: 301-402-0897; E-mail: mhoffman@mail.nih.gov.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M100774200
2 J. A. Engbring, unpublished data.
3 P. K. Nielsen, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HSPG, heparan sulfate proteoglycan; GAG, glycosaminoglycan; DNSNAc, de-N-sulfated-N-acetylated heparin; CDSNAc, completely desulfated-N-acetylated heparin; CDSNS, completely desulfated-N-sulfated heparin; HSG, human submandibular gland; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; E, embryonic day; FGF, fibroblast growth factor; dp, degree of polymerization.
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