From the
Differentiated human neuroblastoma LA-N1 cells that were exposed
to dibutyryl adenosine 3`,5`-cyclic monophosphate for 5 days (primed
cells) showed increased adhesion to laminin-, fibronectin-, and
collagen type I-coated plates as compared to unprimed cells. Moreover,
primed cells seemed to adhere best to laminin. The binding site in
laminin, mediating cell attachment, was identified as containing the
YIGSR sequence, a known cell binding motif, located in the short arm of
the B1 chain of laminin. The synthetic peptide amide,
C(YIGSR)
Laminin, the major glycoprotein of basement membrane is the
first extracellular matrix (ECM)
Certain clones of human and mouse
neuroblastoma (NB) cell lines acquire differentiated properties when
treated with a variety of inducing agents such as cAMP analogs,
retinoic acid, nerve growth factor, interferon-
In recent years, the number of
identified laminin-binding proteins (LBPs) has increased rapidly,
although the function of each is still unclear. The LBPs include the
integrins, a complex family of cell-surface receptors that mediate cell
interactions with various components of the
ECM
(41, 42, 43, 44, 45, 46) .
In addition, several non-integrin LBPs have been identified that range
in size from 32 to 180 kDa. The most studied of these proteins is a
67-kDa LBP that is present in many cell types but is not always
identical in its
properties
(16, 47, 48, 49, 50) .
Using monoclonal antibodies directed against the 67-kDa LBP, human and
mouse cDNA clones were generated that encode a 32-kDa but not a 67-kDa
protein, suggesting the existence of a 32-kDa precursor of the laminin
receptor
(5, 51, 52) . In many cell types, one of
the binding sites of the 67-kDa protein is defined by the peptide
sequence YIGSR located on the B1 chain of laminin
(16) . Other
LBPs bind to a site defined by the sequence IKVAV found on the A-chain
of laminin. These include 55-, 88-, and 100-kDa
proteins
(53, 54) . One more LBP is a 35-kDa protein that
has very high homology to murine galactose-binding lectin, the
macrophage antigen Mac-2, suggesting the importance of
galactose-containing side chains in mediating the binding to laminin
(55). The 35-kDa protein is also related to the 67-kDa LBP, which may
have more than one binding motif
(56) . Here we report the
identification and purification of a 67-kDa YIGSR-binding protein from
cultured human NB cells that appears to be down-regulated during the
differentiation process.
Laminin is a multidomain protein that modulates many cell
functions including promotion of cell attachment, migration, growth,
differentiation, neurite outgrowth, and tumor metastases. Different
sites on laminin have been shown to interact with various cell surface
receptors in different cell
types
(1, 2, 3, 15) . Neuroblastoma cells
were also found to synthesize and adhere to
laminin
(38, 65) . The mechanisms by which NB cells
transduce the information inherent in the structure and molecular
organization of laminin are not fully understood. We have, therefore,
studied the interaction of these cells with the ECM components to
identify cell surface-binding proteins that play a role in
neuroblastoma-matrix adhesion. We have shown that priming of human NB
cells with Bt
We have further endeavored to identify cell surface recep-tors
interacting with the YIGSR sequence of laminin using several different
techniques: biotinylation of surface proteins in intact neuroblastoma
cells, the use of an affinity column conjugated to a repeat of the
YIGSR sequence (C(YIGSR)
A 67-kDa receptor has
been isolated by laminin affinity chromatography from other cell types,
among them, carcinoma
(47) , skeletal muscle (48), mouse
neuroblastoma
The observation that the expression of the 67-kDa protein is
dramatically reduced in differentiated cells was unexpected, since
adhesion of the cells to laminin was increased upon induction of
differentiation (Fig. 1) and was almost completely inhibited by
the C(YIGSR)
We thank Dr. Hynda K. Kleinman for the laminin and the
initial supply of C(YIGSR)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-NH
, containing a repeat of this
binding motif, inhibited the attachment of neuroblastoma cells to
laminin in a competitive manner, and its inhibitory activity was
inversely dependent on laminin concentrations. Affinity chromatography
of membrane-extracted proteins over an Affi-Gel 10 column conjugated to
C(YIGSR)
-NH
, revealed a major YIGSR-binding
protein with an apparent molecular mass of 67 kDa. The 67-kDa surface
membrane protein was specifically eluted from the column with the
soluble C(YIGSR)
-NH
peptide, but not with an
unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was
recovered from an unrelated peptide matrix with the free
C(YIGSR)
-NH
peptide. Ligand blot overlay assays
with biotin-labeled C(YIGSR)
-NH
peptide
demonstrated that the 67-kDa receptor is indeed a YIGSR-binding
protein. This 67-kDa laminin-binding protein appeared to be
down-regulated upon differentiation of LA-N1 cells, as indicated by the
level of this protein and its mRNA.
(
)
protein to
appear during embryogenesis, and is known to promote cell adhesion,
growth, migration, and neurite
outgrowth
(1, 2, 3) . Laminin is involved in
cancer metastases, as well as tumor invasiveness
(4, 5) ,
and malignant cells often display aberrations in their relationship to
laminin (6-10). Laminin isolated from the Engelbreth-Holm-Swarm
mouse sarcoma is a large multidomain glycoprotein composed of three
polypeptides (A, B1, and B2) held together in a cross-like structure by
disulfide bonds (2) and belongs to a family of proteins with several
genetically distinct subunit
chains
(11, 12, 13) . Various sites in the
laminin molecule, responsible for distinct functions, have been
identified using both proteolytic fragments and synthetic
peptides
(14, 15) . For example, the YIGSR and PDSGR
sequences on the B1 chain, as well as the RGD-containing sequence on
the A chain, were found to promote cell adhesion and
migration
(16, 17, 18) . Induction of neurite
outgrowth, cell migration, and adhesion appear to be affected by
another site containing the IKVAV sequence on the A chain of
laminin
(19, 20) .
, tumor necrosis
factor, dimethyl sulfoxide, and hexamethylene bisacetamide. These
compounds cause NB cells to extend neurites, increase the expression of
sodium channels and plasminogen activator, enhance the activity of
neurotransmitter-synthesizing enzymes, and change the spatial
organization of cytoskeletal elements. The inducing agents were also
found to modulate the levels of surface glycoproteins and class-I
histocompatibility
antigens
(21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) .
Due to these properties NB cells have been suggested as an excellent
model system for the study of neuronal maturation. Cultured human NB
cells have been shown to synthesize the ECM proteins such as laminin,
fibronectin, and type IV collagen
(36) . Moreover, the adhesion
of NB cells to the substratum and their ability to extend neurites have
been shown to depend on the composition of the ECM
proteins
(37, 38, 39, 40) . An apparent
difficulty in studying the differentiation process of human NB cells is
that only a limited number of cells respond to the inducers. However,
it was found in our laboratory that over 90% of the cells extend
neurites if the human NB cells are first primed with Bt
cAMP
or other inducers and then replated on laminin-coated
plates
(32) . Thus, during the priming period certain changes
have been induced in these cells that increase the adhesion to laminin
and lead to their differentiation.
Reagents
Laminin was extracted from the
Engelbreth-Holm-Swarm mouse sarcoma and generously supplied by Dr. H.
K. Kleinman. Collagen type I was extracted from rat tails and kindly
supplied by Dr. R. Reich. Mouse fibronectin and bovine serum albumin
fraction V were obtained from Sigma, Affi-Gel 10 was purchased from
Bio-Rad, NHS-LC-biotin was from Pierce, and streptavidin conjugated to
horseradish peroxidase was from Amersham International. The synthetic
peptide amide C(YIGSR)-NH
as well as the
peptide GRGDS and the unrelated peptide CYKNVRSKIGSTENLKHQPGGGKV were
synthesized on a model 430A peptide synthesizer (Applied Biosystems)
and further purified by high performance liquid chromatography. The
synthetic peptide C(YIGSR)
-NH
or the unrelated
peptide were coupled to Affi-Gel 10 according to the
manufacturer's instructions and contained 10 mg of peptide/ml of
resin. Biotin-labeled peptide was prepared by incubating the
C(YIGSR)
-NH
peptide (5 mg/ml) in 100
mM sodium bicarbonate buffer, pH 8.0, with 1 mg/ml
sulfosuccinimidyl 6-(biotinamido)hexanoate (NHS-LC-biotin) for 1 h at
room temperature.
Cell Cultures
Human neuroblastoma cell lines LA-N1
and CHP-134 were cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal calf serum, 2 mML-glutamine,
and 50 µg/ml gentamycin (Biological Industries, Kibbutz Beth
Haemek, Israel) at 37 °C, in a humidified atmosphere of 5%
CO/95% air. The medium was replaced every second day. For
the induction of differentiation, the cells were replated at a
concentration of 1
10
cells in 75-cm
Nunc tissue culture flasks with the same medium containing 1
mM Bt
cAMP for 3-5 days.
Detection of Laminin in Differentiated Human
Neuroblastoma Cells
Human neuroblastoma LA-N1 cells were
cultured in a 24-well plate for 5 days in the presence of 1 mM
BtcAMP. The cells were washed twice with serum-free medium
and fixed with methanol for 2 h at -20 °C. They were then
incubated at room temperature for 1 h with serum-free medium containing
2.5 mg/ml bovine serum albumin followed by two washes with serum-free
medium. The cells were subsequently incubated with rabbit polyclonal
antibody (1:100) directed against laminin (BioMakor, Rehovot, Israel)
for 18 h at 4 °C and then for additional 2 h at 37 °C. The
cells were then washed twice with serum-free medium, incubated with
horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:100),
and stained with 3,3`-diaminobenzidine/H
O
.
Cell Adhesion Assay
Various amounts of laminin,
fibronectin, or collagen type I were diluted in serum-free RPMI 1640,
and aliquots of 0.1 ml were added to each well in a 96-well microtiter
plate. The plates were incubated overnight at 4 °C, and the
solutions were then removed. Serum-free RPMI 1640 containing 2.5 mg/ml
bovine serum albumin was added, and the plates were incubated for an
additional 2 h at 37 °C in a 5% CO/95% air. Wells were
washed twice with serum-free RPMI 1640 and then were ready to be plated
with cells. Neuroblastoma cells were harvested by centrifugation and
washed twice with serum-free RPMI 1640 (with or without 1 mM
Bt
cAMP), and 0.1 ml aliquots of these cells were added to
each well. After 2 h of incubation with LA-N1 cells or 3.5 h with
CHP-134 cells at 37 °C in a 5% CO
/95% air, the
supernatants were removed and the wells were washed once with
serum-free RPMI 1640. The adherent cells were fixed with 3.7%
paraformaldehyde for 30 min at 20 °C, washed once with
phosphate-buffered saline (PBS), and stained overnight with 0.3% cresyl
violet. The wells were then washed extensively with water, and the
absorption of the colored cells was measured at 540 nm. In experiments
involving inhibition of cell adhesion, the NB cells were primed with 1
mM Bt
cAMP for 5 days, harvested by centrifugation,
and resuspended in serum-free RPMI 1640 with the indicated
concentrations of synthetic peptides. The cell suspension was incubated
for 30 min at 37 °C, and 0.1-ml aliquots were added to laminin- or
fibronectin-precoated wells. Cell adhesion was determined as described
above.
Labeling of Cell Surface Receptors by
Biotinylation
Undifferentiated and differentiated cells (5
10
) were washed three times with PBS and incubated
in 2 ml of PBS containing 1 mg/ml NHS-LC-biotin at 4 °C for 1 h.
Thereafter, cells were washed three times with cold PBS containing 15
mM glycine to quench the reaction. The packed cells were
suspended in homogenization buffer containing 20 mM Tris-HCl,
pH 7.6, 130 mM NaCl, 1 mM MnCl
, 1
mM MgCl
; 1 mM CaCl
, 10
µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml soybean
trypsin inhibitor, 1 mM benzamidine, 5 µg/ml pepstatin A,
and 1 mM phenylmethylsulfonyl fluoride. The cells were
subjected to three cycles of freezing and thawing followed by
homogenization in an all-glass homogenizer. The suspension was
centrifuged at 12,000
g for 15 min at 4 °C, and
the precipitated particulate fraction was washed three times with the
homogenization buffer. Membrane proteins were extracted from the
particulate fraction with homogenization buffer containing 1% Triton
X-100 for 1 h at 4 °C. Detergent-insoluble material was removed by
centrifugation at 12,000
g for 15 min at 4 °C. The
supernatant fraction was either subjected immediately to affinity
chromatography or stored at -70 °C.
Affinity Column Chromatography
The
C(YIGSR)-NH
-Affi-Gel 10 matrix was equilibrated
with wash buffer containing 100 mM Tris-HCl, pH 7.6, 150
mM NaCl, 1 mM MnCl
, 1 mM
CaCl
, 1 mM MgCl
, 0.1% Triton X-100, 1
mM phenylmethylsulfonyl fluoride and incubated overnight with
0.1 ml of the detergent-extracted membrane fraction (200 µg of
protein) under gentle agitation at 4 °C. The beads were then packed
into a column and warmed to 20 °C. The column was washed overnight
with wash buffer at 20 °C followed by 5 ml of wash buffer lacking
NaCl. The bound proteins were then eluted with 3 ml of elution buffer
containing 100 mM Tris-HCl, pH 7.6, 0.1 mg/ml free
C(YIGSR)
-NH
, 0.1% Triton X-100, and 1
mM phenylmethylsulfonyl fluoride. The eluate was concentrated
in Centricon-30 (Amicon Inc.), subjected to 7.5% SDS-PAGE, and blotted
onto nitrocellulose sheets. The sheets were blocked with 10% low fat
milk in PBS for 30 min and then rinsed three times with PBS containing
0.5% Tween 20 (PBST). To visualized the resolved proteins, the sheets
were incubated with streptavidin conjugated to horseradish peroxidase
(1:750) in PBST for 30 min. Nitrocellulose sheets were washed five
times in PBST for a total of 35 min and developed using the ECL
(enhanced chemiluminescence) reagents supplied by Amersham.
Biotinylated Peptide Overlay
Assay
Detergent-extracted membrane proteins were isolated from
NB cells that were not labeled with biotin and purified by affinity
chromatography, as described above. The proteins were resolved by gel
electrophoresis and blotted onto nitrocellulose sheets as described
above. An overlay assay with biotinylated C(YIGSR)-NH
peptide was employed to detect YIGSR-binding proteins. The
nitrocellulose sheets were incubated in buffer containing 250 µg/ml
biotin-labeled peptide, 100 mM Tris-HCl, pH 8.0, 1 mM
CaCl
, 1 mM MgCl
, and 0.1% Tween 20 for
16 h at 4 °C. The nitrocellulose sheets were washed once with the
binding buffer without the peptide for 5 min by gentle agitation and
then incubated with streptavidin conjugated to horseradish peroxidase
(1:750) in PBST for 30 min and subsequently washed four times for 5 min
each with the same buffer. Detection of labeled proteins was carried
out with the ECL reagents as described above.
Immunoprecipitation of Human Neuroblastoma 67-kDa LBP by
Anti-67-kDa Receptor Antibody
Detergent-extracted membrane
proteins (200 µg/0.1 ml) from LA-N1 cells were loaded onto a
C(YIGSR)-NH
affinity chromatography column and
eluted, as described above. The eluate fraction containing the 67-kDa
LBP in a volume of 0.25 ml was incubated overnight at 4 °C either
with 46 µg preimmune serum or with 3 µg of anti-67-kDa laminin
receptor antibody. This antibody was raised against a bacterial fusion
protein encoded by the
-galactosidase gene plus 0.9 kb from the
carboxyl end of the 1.1-kb cDNA encoding a 32-kDa protein that was
found to recognize 32-, 42-, and 67-kDa proteins, it has been
designated anti-67-kDa
(57) and was a gift from H. K. Kleinman.
Antibody-protein complexes were absorbed to Sepharose-protein A beads
and washed three times with elution buffer. Precipitated proteins were
eluted with sample buffer, resolved by means of 7.5% SDS-PAGE, and
blotted onto nitrocellulose membranes. The nitrocellulose sheets were
incubated with the anti-67-kDa fusion protein antibody (2 µg/ml) in
a buffer containing 20 mM Tris-HCl, pH 7.6, 150 mM
NaCl, and 1 mg/ml bovine serum albumin, for 16 h at 4 °C. The
nitrocellulose sheets were washed three times with a buffer containing
20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.02% Tween
20 and then incubated with horseradish peroxidase-conjugated goat
anti-rabbit F(ab`)
antibody (1:20,000) for 1 h at 20 °C
followed by the ECL reagent as described above.
Extraction of Total RNA and Blot
Hybridization
Total cellular RNA was extracted from NB LA-N1
cells employing guanidinium thiocyanate
(58) . Equal amounts of
total cellular RNA (20 µg) from undifferentiated and differentiated
cells were loaded onto 1.0% agarose-formaldehyde gel. Following
electrophoresis the RNA in the gel was transferred onto nylon Hybond
filters (Amersham). The probe consisted of a 680-base pair cDNA insert
(number 1485) derived from the 1.1-kb cDNA that encodes a 32-kDa LBP.
This cDNA was cloned from a mouse F9 teratocarcinoma cell library and
found to be related to the 67-kDa LBP
(59) . Plasmid DNA was
purified and digested with EcoRI. After electrophoresis on a
low melting point gel, the cDNA insert was cut out, melted for 5 min at
65 °C, and diluted with three volumes of HO. This
insert was
P-labeled by a random primer using the
Fermentas kit. Specific activity of the DNA probe was about 2
10
cpm/µg of DNA. Blotted filters were hybridized
overnight at 42 °C to
P-labeled DNA insert in 5
SSC, 50% formamide, 5
Denhardt's solution, 20
mM sodium phosphate buffer, pH 6.8, 0.1% SDS, and 250 µg
of sonicated denatured herring sperm DNA. The filters were washed with
2
SSC, 0.1% SDS, and 0.1% sodium pyrophosphate, and then
exposed to x-ray films (Kodak X-Omat AR) at -70 °C.
Adhesion of Human Neuroblastoma Cells to Extracellular
Matrix Components
Adhesion of adrenergic LA-N1 cells was
measured after 2 h of incubation on dishes precoated with ECM
components. The cells were fixed and stained with cresyl violet, and
the absorption of the stained cells determined. Fig. 1shows that
the LA-N1 cells adhered to laminin, fibronectin, and collagen I in a
concentration-dependent manner. Differentiated LA-N1 cells that were
primed with 1 mM BtcAMP for 5 days showed a large
increase in their capacity to adhere to all substrata, as compared to
undifferentiated cells. It was also noted that in the concentration
range of 5-80 ng/well, adhesion of cells to laminin was greater
than that observed with fibronectin or collagen I. The preferential
adhesion of the cells to laminin was already evident at a concentration
of 5 ng of laminin/well, indicating that LA-N1 cells may have potent
cell surface proteins that promote attachment to laminin. Similar
results were obtained with the cholinergic CHP-134 cells (not shown).
These cells had to be incubated for 3.5 h before cell attachment took
place. It was also observed that, at longer periods of incubations,
laminin promotes neurite outgrowth in Bt
cAMP-primed NB
cells. The neurites were more branched and complex with increasing
laminin concentrations. Undifferentiated cells developed only short,
thick extensions, which were mostly unbranched, regardless of the
laminin concentrations, as we have shown previously
(32) .
Moreover, differentiated human NB cells were able to synthesize a
considerable amount of laminin. The cells were incubated with rabbit
polyclonal antibodies directed against laminin and then with
horseradish peroxidase-conjugated to goat anti-rabbit antibodies and
stained with 3,3`-diaminobenzidine/H
O
. Both the
cell bodies and the neurites were stained under these conditions (not
shown). We have therefore concentrated our efforts on the
adhesion of human NB cells to laminin, in order to detect both the
binding sites on the laminin molecule and the cell-binding proteins
that are involved in the process of neuroblastoma-laminin adhesion.
Figure 1:
Adhesion of human neuroblastoma LA-N1
cells to extracellular matrix components. Human neuroblastoma LA-N1
cells that were either primed to differentiate with 1 mM
BtcAMP for 5 days or not treated were plated at 8
10
cells/well in a 96-well microtiter plate precoated with
laminin, fibronectin, or collagen I at the indicated concentrations.
Undifferentiated (
) and differentiated (
) cells were then
incubated at 37 °C for 2 h, washed, and fixed with 3.7%
paraformaldehyde. The cells were stained with 0.3% cresyl violet, and
the color was read at 540 nm. Data points represent the mean of
quadruplicates ± standard error of the mean (S.E.). In some
cases S.E. values are low and are obscured by the
symbols.
Inhibition of Neuroblastoma Cell Adhesion to Laminin by
C(YIGSR)
One of the first laminin
binding sites to be described for many cell types is located in domain
III of the B1 chain of laminin and is defined by the sequence YIGSR
corresponding to amino acid positions 929-933. The YIGSR peptide
has been shown to promote cell adhesion and migration and inhibit
melanoma lung colonization
(60, 61) . Moreover, polymeric
and cyclic forms of YIGSR are more active than the
pentapeptide
(61, 62) . We have therefore used a
synthetic peptide amide C(YIGSR)-NH
-NH
, containing
a repeat of the sequence from this binding domain of laminin. The
ability of this peptide to block laminin-mediated adhesion is shown in
Fig. 2
. For these experiments Bt
cAMP-primed LA-N1
cells were incubated for 30 min with various concentrations of the
peptide and then plated on wells that were precoated with laminin. The
C(YIGSR)
-NH
peptide inhibited the attachment of
these cells to laminin in a competitive manner, and the inhibition was
inversely dependent on the laminin concentrations. Significant
inhibition (88%) was already observed at a concentration of 20
µg/ml C(YIGSR)
-NH
peptide. The ability of
the C(YIGSR)
-NH
peptide to inhibit cell
attachment to laminin suggests that the YIGSR motif is a major binding
site for LA-N1 cells.
Figure 2:
Inhibition of cell adhesion to laminin by
C(YIGSR)-NH
. Human neuroblastoma LA-N1 cells
were primed to differentiated with 1 mM Bt
cAMP for
5 days and then incubated for 30 min at 37 °C in serum-free RPMI
1640 medium supplemented with the indicated concentrations of the
C(YIGSR)
-NH
peptide. The cells were then plated
in a 96-well microtiter plate (4.2
10
/well),
precoated with laminin at 40 ng/well (
), 80 ng/well (
), or
160 ng/well (
). Cells were further fixed and stained as described
in legend to Fig. 1. Data points represent the mean of quadruplicates
± standard error of the mean (S.E.). In some cases S.E. values
are low and are obscured by the symbols. The absorbance values at 540
nm for 100% adhesion were 0.257, 0.234, and 0.331 for 40, 80, and 160
ng/well laminin, respectively.
Purification of YIGSR-binding Proteins by Affinity
Chromatography
The process of cell attachment and spreading on
laminin has been thought to be mediated by a number of laminin-binding
proteins (LBP) or receptors. To explore the potential role for such a
receptor, NB LA-N1 cells were surface-labeled with NHS-LC-biotin. This
technique specifically labeled the surface membrane proteins, while
cytoplasmic proteins were left intact. The cells were homogenized and
the biotinylated membrane proteins were detergent-extracted from the
particulate fraction. The solubilized proteins were then incubated
overnight with C(YIGSR)-NH
conjugated to
Affi-Gel 10. Bound proteins were eluted with a buffer containing 0.1
mg/ml free C(YIGSR)
-NH
peptide and resolved by
SDS-PAGE. The blotted proteins were detected with streptavidin
conjugated to horseradish peroxidase. Fig. 3shows that
incubation of the affinity column with as little as 0.1 mg/ml free
C(YIGSR)
-NH
peptide, eluted one major
YIGSR-binding protein with an apparent molecular mass of 67 kDa
(Fig. 3, laneE). The specificity of the
affinity chromatography technique was indicated by the observation that
only traces of the 67-kDa protein were detected upon elution with an
unrelated peptide (Fig. 3, laneC). Further
incubation of the column with 1 mg/ml free
C(YIGSR)
-NH
peptide eluted relatively small
amounts of the 67-kDa protein (not shown), indicating that the elution
of the 67-kDa LBP with the free C(YIGSR)
-NH
peptide is specific. It was also observed that the 67-kDa LBP
protein did not bind to an unrelated peptide conjugated to Affi-Gel 10,
as it was not detected when this column was eluted with 1 mg/ml
C(YIGSR)
-NH
peptide (Fig. 3, laneF).
Figure 3:
Affinity purification of a 67-kDa
YIGSR-binding protein from LA-N1 cells. Human neuroblastoma LA-N1 cells
were primed with 1 mM BtcAMP for 4 days and then
labeled with NHS-LC-biotin as described under ``Materials and
Methods.'' Detergent-extracted membrane proteins (200 µg) were
incubated overnight with 0.6 ml of
C(YIGSR)
-NH
-Affi-Gel 10 at 4 °C. The beads
were then packed into a column, warmed to 20 °C, and washed
extensively. The bound proteins were eluted at 20 °C as described
under ``Materials and Methods.'' The protein solutions in the
various column fractions were concentrated and subjected to 7.5%
SDS-PAGE. The blotted proteins were incubated with streptavidin
conjugated to horseradish peroxidase and visualized by the ECL method.
LaneA, input cell membrane extract; laneB, unbound proteins; lane C, elution with 0.1
mg/ml soluble unrelated peptide CYKNVRSKIGSTENLKHQPGGGKV; laneD, wash; a single major protein was eluted with 0.1 mg/ml
soluble C(YIGSR)
-NH
peptide (laneE) and elution with 0.1 mg/ml soluble
C(YIGSR)
-NH
peptide from a separate Affi-Gel 10
column linked to the unrelated peptide, CYKNVRSKIGSTENLKHQPGGGKV
(laneF).
Identification of YIGSR-binding Proteins by an Overlay
Assay
Detergent-extracted surface membrane proteins were
isolated from LA-N1 cells that were not labeled with biotin. Following
affinity chromatography and SDS-PAGE, the blotted proteins were
incubated with a buffer containing
biotinylated-C(YIGSR)-NH
peptide. The overlay
assay allowed the detection of the 67-kDa LBP in eluates from the
C(YIGSR)
-NH
-Affi-Gel 10 column (Fig. 4,
laneB), suggesting that this protein indeed binds to
the YIGSR sequence. In addition, a fainter band of 60 kDa was also
evident and may represent a proteolytic degradation product of the
67-kDa LBP. The overlay assay did not reveal the presence of any
protein in the column wash fraction (Fig. 4, laneA); neither did the biotinylated peptide interact with
bovine serum albumin, which was used as a control (Fig. 4,
laneC).
Figure 4:
Binding of biotinylated
C(YIGSR)-NH
peptide to affinity-purified
proteins. Human neuroblastoma LA-N1 cells were primed to differentiated
with Bt
cAMP for 4 days; these cells were not labeled with
biotin. Membrane proteins were detergent-extracted (200 µg) and
incubated overnight with 0.6 ml of
C(YIGSR)
-NH
-Affi-Gel 10 at 4 °C. The last
1-ml fraction was saved separately and concentrated in Centricon-30.
Bound proteins were eluted from the column with 3.0 ml of 0.1 mg/ml
soluble C(YIGSR)
-NH
and then concentrated in
Centricon-30. Proteins were resolved by means of 7.5% SDS-PAGE and
blotted onto nitrocellulose sheets. The YIGSR-binding proteins were
detected by the overlay procedure with biotinylated
C(YIGSR)
-NH
peptide (250 µg/ml) as
described under ``Materials and Methods.'' Binding proteins
were visualized with streptavidin conjugated to horseradish peroxidase
followed by the ECL method. LaneA, last wash
fraction; laneB, C(YIGSR)
-NH
eluate; laneC, 50 µg of bovine serum
albumin.
Effect of the Differentiation Process on the Level of the
67-kDa LBP in Neuroblastoma Cells
We then investigated whether
the expression of the 67-kDa LBP is altered during cell
differentiation. The level of the receptor in membrane extracts from
undifferentiated cells was compared to that found in
BtcAMP-differentiated cells using the overlay assay. Equal
amounts of membrane proteins from each extract were resolved by
SDS-PAGE, blotted onto nitrocellulose membranes, and subjected to the
overlay assay with biotinylated-C(YIGSR)
NH
.
Fig. 6B shows that under these conditions the labeled
peptide was also bound to the 67-kDa LBP. It also appears that the
level of the 67-kDa LBP is decreased during the differentiation
process. Similar results were obtained with fractions purified by
affinity chromatography (Fig. 5C). Thus, assays of
membrane extracts and affinity purified proteins, indicate that the
67-kDa LBP is down-regulated during Bt
cAMP-induced
differentiation. As a control for the assay, the biotinylated peptide
was replaced with free biotin. Fig. 5A shows that under
these conditions the 67-kDa LBP could not be detected, suggesting that
the binding of the biotinylated-C(YIGSR)
NH
peptide is mediated through the peptide moiety itself.
Figure 6:
Immunoprecipitation of 67-kDa LBP from
human neuroblastoma cells. Detergent extracts of membrane proteins were
purified by affinity chromatography as described in the legend to Fig.
3. The C(YIGSR)-NH
eluted fraction containing
the 67-kDa LBP (250 µl) was incubated overnight at 4 °C with 46
µg of preimmune serum (laneA) or with 3
µg/ml anti-67-kDa receptor antibody (laneB).
Following precipitation of the immune complexes, the precipitates were
washed three times with elution buffer and dissolved in sample buffer,
and proteins were resolved by means of 7.5% SDS-PAGE. Proteins were
blotted onto nitrocellulose sheets and subsequently incubated with 2
µg/ml of the same anti-67-kDa fusion protein antibody. The filters
were washed and then incubated with horseradish peroxidase conjugated
to goat anti-rabbit F(ab`)
antibody (1:20,000) for 1 h at
20 °C, and the proteins were visualized by the ECL
method.
Figure 5:
Effect of differentiation on the level of
the 67-kDa LBP. Undifferentiated and BtcAMP-differentiated
human neuroblastoma LA-N1 cells were cultured for 4 days; these cells
were not labeled with biotin. Detergent extracts of membrane proteins
(100 µg) were either resolved by SDS-PAGE (B) or were
incubated overnight with 1.2 ml of
C(YIGSR)
-NH
-Affi-Gel 10 column at 4 °C. The
67-kDa protein was then eluted from the column with free
C(YIGSR)
-NH
and resolved by SDS-PAGE
(C).The blotted proteins were then detected by the overlay
assay with biotinylated C(YIGSR)
-NH
as
described in the legend to Fig. 4. Detergent extracts of membrane
proteins from undifferentiated and differentiated cells were overlaid
with free biotin alone (A).
Immunoreactivity of the 67-kDa LBP
The
immunological relationship between the 67-kDa LBP purified from human
NB LA-N1 cells and that from mouse F9 teratocarcinoma was examined.
This was achieved by the use of anti-67-kDa teratocarcinoma LBP
antibody that was raised against a bacterial fusion protein encoded by
the -galactosidase gene plus 0.9 kb from the carboxyl end of the
1.1-kb cDNA
(5, 64) . As shown in Fig. 6(laneB), the antibody immunoprecipitated the affinity-purified
human NB 67-kDa LBP. The ability of the anti-67-kDa mouse
teratocarcinoma receptor antibody to recognize a similar human NB LBP
suggests that these two proteins are either identical or belong to a
closely related family of 67-kDa proteins
(57) .
RNA Blot Hybridization
To examine whether the
decline in the level of the 67-kDa LBP observed in differentiated LA-N1
cells was due to a decrease in the level of the LBP mRNA, total RNA
samples were examined by blot hybridization. Equal amounts of total RNA
(20 µg) from undifferentiated and BtcAMP-primed NB
LA-N1 cells were hybridized with
P-labeled cDNA insert.
The cDNA probe encodes part of a 32-kDa LBP, which was found to be
related to the 67-kDa LBP, and was cloned from a mouse F9
teratocarcinoma cell library
(59) . Fig. 7shows that the
expression of the NB mRNA was significantly lower in differentiated
cells (laneB) as compared with undifferentiated
cells (laneA), suggesting that the differentiation
process involves a down-regulation of the mRNA for the 67-kDa LBP.
Figure 7:
Blot
hybridization analysis of mRNA from undifferentiated and differentiated
human neuroblastoma cells. Equal amount of total RNA (20 µg) from
undifferentiated (lanesA and C) and
differentiated (lanesB and D) LA-N1 cells
were subjected to chromatography on a 1.0% agarose gel, blotted and
hybridized with P-labeled cDNA probe (lanesA and B).The 18 and 28 S rRNA were stained with methylene
blue and are indicated as a loading control (lanesC and D).
cAMP increases their capacity to be attached
to ECM components (Fig. 1). It has been previously noted that NB
differentiation can be divided into two distinct phases. The first
phase involves priming of the cells for several days with inducing
agents, while the second phase involves adhesion of the primed cells to
laminin. Thus, during the priming period, certain changes have been
induced in human NB cells that increase their capacity to adhere to
laminin and lead to their differentiation
(32) . One of the first
laminin binding sites to be described for many cell types is located in
domain III of the B1 chain of laminin and is defined by the peptide
sequence YIGSR. The YIGSR peptide has been shown to promote cell
adhesion, migration and inhibit melanoma lung
colonization
(60, 66) . Taken together, we speculated
that YIGSR may inhibit attachment of NB cells to laminin as well. From
our peptide inhibition studies, it appears that the YIGSR motif is a
major binding site for human NB cells, since over 96% inhibition of
cell adhesion was observed with the synthetic peptide containing a
repeat of the YIGSR sequence, C(YIGSR)
-NH
(Fig. 2). Various other cell lines, including HT-1080
(fibrosarcoma), MCF-7 (breast carcinoma), A431 (epidermoid cells), and
G-8 (muscle), are attached as readily to the YIGSR
sequence
(60) . On the other hand, neuronal cells are known to
recognize the long arm of laminin
(67) and NG108-15 mouse
neuroblastoma
rat glioma hybrid cells show reduced attachment
to YIGSR in comparison to laminin
(60) , suggesting that these
cells may bind to multiple sites on laminin through various receptors.
-NH
), elution of
specific proteins with the free C(YIGSR)
-NH
peptide rather than with salt, and blot overlay assays with
biotinylated C(YIGSR)
-NH
. We selected the
synthetic peptide C(YIGSR)
-NH
, containing a
repeat of the YIGSR sequence from the binding domain of laminin, for
determining the inhibition of cell adhesion as well as for the affinity
chromatography studies. This was prompted by the observation that
polymeric and cyclic forms of YIGSR exhibit more cell adhesion activity
than the pentapeptide, suggesting that tertiary structure may be an
important factor in receptor recognition
(61, 62) . The
use of the C(YIGSR)
-NH
peptide in the affinity
chromatography procedure facilitated the purification of a 67-kDa YIGSR
binding receptor from NB cells. The identification of the 67-kDa LBP is
based on the following evidence. (a) Upon cell surface
biotinylation the 67-kDa protein was biotinylated, suggesting that it
is exposed to the extracellular milieu. (b) It is bound to
C(YIGSR)
-NH
Affi-Gel column and eluted
specifically by the free C(YIGSR)
-NH
peptide.
(c) The interaction of the 67-kDa protein with YIGSR
sequences is specific since an unrelated peptide eluted only traces of
this protein from the affinity column. (d) It was not detected
in eluates from unrelated peptide-Affi-Gel column. (e) Ligand
blot overlay assays demonstrated that the 67-kDa LBP present in NB
extracts is indeed a YIGSR-binding protein. (f) The almost
complete inhibition of cell binding to laminin by the free
C(YIGSR)
-NH
peptide suggests that this
interaction does not involve cryptic sites.
rat glioma NG108-15 hybrid
cells
(49, 50) , PC12 cells, chick dorsal root ganglion,
chick spinal cord, and rat astrocytes
(50) . These receptors are
similar in their properties; they all have an apparent molecular mass
of 67 kDa and bind laminin with a K
of
about 10
M. The human neuroblastoma 67-kDa
LBP appears to belong to this family of receptors, as it is
immunoprecipitated with an antibody raised against a fusion protein
encoding a 32-kDa LBP that is related to the 67-kDa receptor. This
antibody was found to recognize 32-, 42-, and 67-kDa LBPs and has been
designated anti-67-kDa
(57) . In our experiments the 32- or
42-kDa proteins were not detected by the ligand blot overlay assay with
biotinylated C(YIGSR)
-NH
peptide. Thus, these
two proteins, if they exist in human NB cells, appear to have a low
affinity with the YIGSR sequence or are missing the YIGSR recognition
site.
-NH
peptide in a competitive manner
(Fig. 2). There are several possible explanations for these
phenomena. For example, the low level of the residual 67-kDa receptor
found in differentiated cells may undergo posttranslational
modifications that increase its affinity to laminin. An alternative
hypothesis would suggest that during cell differentiation there is an
up-regulation of other proteins acting in a synergistic manner with the
67-kDa receptor, to facilitate the increased cell attachment to
laminin. The decline in expression of the 67-kDa is paralleled by the
low level of the 67/32 mRNA in differentiated cells (Fig. 7),
although it is not clear whether the cDNA used codes for the
neuroblastoma 67-kDa LBP or is only related to it. The marked reduction
in the expression of the 67/32 mRNA in differentiated neuroblastoma
cells correlates with recent blot hybridization studies that have
indicated that the level of the mRNA in poorly differentiated colon
carcinoma is higher than that of their normal
counterparts
(51, 63) . It therefore appears that the
level of the 67-kDa LBP and the 67/32-kDa mRNA may serve as a
prognostic marker for human neuroblastoma cancer and its progression.
cAMP,
dibutyryl cyclic AMP; LBP, laminin-binding protein; NB, neuroblastoma;
PBS, phosphate-buffered saline; PAGE, polyacrylamide gel
electrophoresis; kb, kilobase(s).
-NH
peptide, Dr.
Yoshihiko Yamada for the 32-kDa LBP cDNA, and Ruth Marx-Rattner and
Einat Sadot for help in the blot hybridization experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.