©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Down-regulation of a 67-kDa YIGSR-binding Protein upon Differentiation of Human Neuroblastoma Cells (*)

Ilana Bushkin-Harav, Nira B. Garty, and Uriel Z. Littauer (§)

From the (1) Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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)-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.


INTRODUCTION

Laminin, the major glycoprotein of basement membrane is the first extracellular matrix (ECM)() 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) .

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-, 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 BtcAMP 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.

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.


MATERIALS AND METHODS

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 BtcAMP 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/HO.

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 BtcAMP), 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 BtcAMP 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.


RESULTS

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 BtcAMP-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/HO. 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)-NH

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, 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 BtcAMP-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 BtcAMP 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 BtcAMP 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 BtcAMP-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).




DISCUSSION

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 BtcAMP 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.

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)-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.

A 67-kDa receptor has been isolated by laminin affinity chromatography from other cell types, among them, carcinoma (47) , skeletal muscle (48), mouse neuroblastoma 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 10M. 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.

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)-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.


FOOTNOTES

*
This work was supported in part by the collaborative program between the Children's Hospital of Philadelphia and the Weizmann Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-8-343641; Fax: 972-8-465260.

The abbreviations used are: ECM, extracellular matrix; BtcAMP, dibutyryl cyclic AMP; LBP, laminin-binding protein; NB, neuroblastoma; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Dr. Hynda K. Kleinman for the laminin and the initial supply of C(YIGSR)-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.


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