Fibulin-2 Binds to the Short Arms of Laminin-5 and Laminin-1 via Conserved Amino Acid Sequences*

(Received for publication, September 3, 1996, and in revised form, October 25, 1996)

Atsushi Utani , Motoyoshi Nomizu and Yoshihiko Yamada Dagger

From the Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Epithelial cell-specific laminin-5, consisting of three chains, alpha 3, beta 3, and gamma 2, is a component of the anchoring filament that traverses the lamina lucida beneath the hemidesmosomes of epidermal cells and functions to link these cells to the basement membrane. We have studied the molecular interaction between laminin-5 and extracellular matrix proteins using recombinant proteins and synthetic peptides. Affinity chromatography assays with recombinant fragments of the laminin gamma 2 short arm identified a 195-kDa binding protein in the conditioned media from the mouse epidermal cell line Pam 212 and from primary dermal fibroblasts. This molecule was identified by Western blotting as fibulin-2, a recently identified extracellular matrix protein. Using deletion mutants and various synthetic peptides in competition assays, the 9-amino acid sequence SADFSVHKI (residues 199-207) in domain IV of the gamma 2 chain was defined as a critical site for fibulin-2 binding. An anti-gamma 2 antibody co-immunoprecipitated fibulin-2 from the conditioned media, further confirming the interaction of fibulin-2 with laminin-5. Fibulin-2 was also found to interact with laminin-1 (alpha 1beta 1gamma 1) through a region (residues 654-665) of the alpha 1 chain short arm whose sequence is similar to that of the fibulin-2 binding site of the gamma 2 chain. Together these results suggest that fibulin-2 functions to bridge laminin-1 and laminin-5 with other extracellular matrix proteins, providing a linkage between the cell surface and the basement membrane.


INTRODUCTION

At the dermal-epidermal junction, there is stable attachment of epithelia to the underlying stroma through various protein-protein interactions. Electron microscopy and immunohistochemical studies have defined the topographical linkage between hemidesmosomes on the basal surface of epithelium, anchoring filaments, and anchoring fibrils. These structures form an extended network, which surrounds the stromal fibers and inserts into the basement membrane. Hemidesmosomes of basal keratinocytes contain several molecules including BP180, BP230, HD1, and integrin alpha 6beta 4. The anchoring filaments contain laminin-5 (kalinin/nicein, epiligrin) and colocalize with hemidesmosomes at the supra basement membrane. The basement membrane components laminin-1, type IV collagen, and nidogen/entactin and the anchoring fibrils consisting of type VII collagen are located on the dermal side of the basement membrane (1-3).

Mutations in the genes for the components of the dermal-epidermal junction in human patients with skin blister-forming disease have revealed the importance of these protein linkages in maintaining the structural stability of the dermal-epidermal junction. Mutations in BP180 (4), integrin beta 4 (5), laminin-5 (6-9), and type VII collagen (10-12) have been identified. Acquired skin blister-forming diseases have also been shown to be due to autoantibodies to BP180 (13), laminin-5 (14), and type VII collagen (15).

Basement membrane components have been shown to interact with each other and self-assemble to form a supramolecular network. Laminin-1 polymerizes through interactions at the N-terminal short arms of the monomeric molecules to form a hexagonal array of molecules (16). Nidogen is also a crucial molecule required for network formation since it binds several components of basement membrane including laminin-1, type IV collagen, and perlecan (17-19). Fibulin-1 (BM-90) and fibulin-2 were recently identified as a family of extracellular matrix proteins that interact with the laminin-1-nidogen complex, type IV collagen, and fibronectin (20-23). The interaction of fibulins with multiple components of the extracellular matrix suggests that they function as mediators of supramolecular assembly at the basement membrane.

Epithelial cell-specific laminin-5 consists of three chains, alpha 3, beta 3, and gamma 2, and is an adhesive substrate for keratinocytes in vitro (24). Recently, both alpha 6beta 4 and alpha 3beta 1 integrins were identified as cellular receptors for laminin-5 (25-29). Laminin-5 forms a disulfide-bonded complex with laminin-6 (alpha 3beta 1gamma 1) (30), possibly via the N-terminal globular domain VI of the laminin beta 3 chain. The N-terminal globular domain VI of the laminin beta 3 chain may be involved in the complex formation, since there is an uncoupled cysteine residue within this domain (31, 32).

In order to study the interaction between laminin-5 and other extracellular matrix components, proteins in the culture medium of Pam 212 epidermal cells were screened for binding recombinant laminin chains by affinity chromatography. Fibulin-2, which is prominently expressed in skin and heart, was found to bind laminin-5 through the short arm of the gamma 2 chain and we have identified a 9-amino acid sequence in domain IV of the gamma 2 chain critical for this binding. We have also found that fibulin-2 binds to laminin-1 via the N terminus of the alpha 1 chain, a site showing sequence homology to the 9-amino acid sequence of the gamma 2 chain. Together these results suggest that fibulin-2 functions in assembling the laminin network in the basal lamina at the dermal-epidermal junction in bridging laminin-1 and laminin-5 with other matrix proteins.


MATERIALS AND METHODS

Cell Lines, Antibodies, and Reagents

The murine epidermal cell line Pam 212 (33) and the human epidermoid carcinoma cell line A431 were obtained from ATCC (Bethesda, MD). Primary cultures of mouse dermal fibroblasts were isolated from newborn FVB/N mouse. All cells were maintained with 10% fetal bovine serum/Dulbecco's modified Eagle's medium (Life Technologies Inc.). Anti-mouse fibulin-2 antiserum was made by immunizing rabbits with recombinant fibulin-2 (34) kindly provided by Dr. R. Timpl (Max-Planck-Institut für Biochemie, Munich, Germany). Anti-laminin gamma 2 chain antiserum was prepared as described previously (35). Anti-mouse type IV collagen antibody was made with Engelbreth-Holm-Swarm-derived type IV collagen in rabbits and purified using type IV collagen-coupled Sepharose column. Anti-human thrombospondin antibody was a generous gift from Dr. D. D. Roberts (NCI, NIH, Bethesda, MD) (36). Laminin-1 was prepared from the Engelbreth-Holm-Swarm tumor and purified as described previously (37). Bacterial collagenase form III was purchased from Advanced Biotechnologies Inc.

Recombinant Proteins and Synthetic Peptides

cDNAs for murine laminin alpha 2, gamma 2, and beta 3 chains were produced as reported previously (32, 35, 38). cDNA fragments generated by restriction enzymes or polymerase chain reaction were subcloned into either the pGEX-2T or -4T bacterial expression vector (Pharmacia Biotech Inc.). Recombinant proteins were expressed and purified as described previously (39). These recombinant proteins include: gamma 2-r1 (residues 17-306), gamma 2-r2 (residues 129-306), gamma 2-r3 (residues 197-306), gamma 2-r4 (248-306), gamma 2-r5 (residues 197-247) of the gamma 2 chain, beta 3-r1 (residues 18-253) of the beta 3 chain, alpha 2-r1 (residues 23-242) of the alpha 2 chain. All constructs used in these studies were confirmed by automated DNA sequencing (model 370A, Applied Biosystems, Foster City, CA).

Synthetic peptides, gamma 2-pC and gamma 2-pN, were synthesized with a peptide synthesizer (Applied Biosystems, model 431A) by the t-butoxycarbonyl-based solid-phase strategy (40). All other synthetic peptides were manually synthesized by the Fmoc (9-fluorenylmethoxycarbonyl)-based solid-phase strategy and prepared as the C-terminal amide form as described previously (41). All synthetic peptides were purified by reverse phase high performance liquid chromatography. The purity and identity of the synthetic peptides were confirmed by analytical reverse phase high performance liquid chromatography and amino acid analysis. The location and amino acid sequences of the synthetic peptides from the gamma 2 chain are listed in Table I and Fig. 3. Synthetic peptides listed in Fig. 7B include: pN-21, 9 amino acids (residues 199-207) of the gamma 2 chain; palpha 1-654, 12 amino acids (residues 654-665) of the alpha 1 chain; palpha 1-279, 9 amino acids (residues 279-287) of the alpha 1 chain; pbeta 1-461, 9 amino acid (residues 461-469) of the beta 1 chain with two arginine residues added to increase the solubility of the peptide; pgamma 1-587, 9 amino acids (residues 587-595) of the gamma 1 chain with two added arginine residues to increase the solubility of the peptide; and palpha 2-287, 9 amino acids (residues 287-295) of the alpha 2 chain.

Table I.

A list of synthetic peptides from the N-terminus of domain IV of the gamma 2 chain and their activities for competing fibulin-2 binding to gamma 2-r

The activities shown were from the competition assays in Fig 4. ++, active at 200 µg/ml; +, active at 1 mg/ml; -, inactive even at 1 mg/ml. gamma 2-pN corresponds to residues 197-226 of the gamma 2 chain of laminin-5. *pN-18, *pN-19, and *pN-20 are scrambled peptides of pN-4.
Peptide Sequence Activity

 gamma 2-pN HASADFSVHKITSTFSQDVDGWKAVQRNGA ++
pN-1                     GWKAVQRNGA  -
pN-2           ITSTFSQDVDGWKAVQRNGA  -
pN-3 HASADFSVHKITSTF ++
pN-4 HASADFSVHKIT ++
pN-5 HASADFSVHKI ++
pN-6 HASADFSVHK +
pN-7 HASADFSVH  -
pN-8 HASADFSV  -
pN-9 HASADFS  -
pN-10 HASADF  -
pN-11 HASAD  -
pN-12      FSVHKIT  -
pN-13    ADFSVHKIT +
pN-14   SADFSVHKIT ++
pN-15  ASADFSVHKIT ++
pN-16    ADFSVHK  -
pN-17     DFSV  -
*pN-18 IDSKVHATSHAF  -
*pN-19 KHHADASSVTFI  -
*pN-20 VFAADKTHSHIS  -


Fig. 3. Deletion analysis for the active site of fibulin-2 binding. A, a schematic illustration of the laminin gamma 2 chain. Domains V and III contain several epidermal growth factor-like repeats (boxes), and domain IV is a globular region (oval) of the short arm region. Domains II/I indicate alpha -helical-rich long arm region forming a triple-stranded coiled-coil structure with other laminin-5 chains. A set of deletion mutant recombinant proteins, gamma 2-r1 to gamma 2-r5, and synthetic peptides, gamma 2-pN and gamma 2-pC, are shown with the amino acid residue numbers of the murine laminin gamma 2 chain. B, affinity chromatography assays were performed with gamma 2-r1-GST to gamma 2-r5-GST agarose beads using [35S]methionine-labeled Pam 212 conditioned media and followed by fluorography.
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Fig. 7. Identification of the fibulin-2 binding site on laminin-1. A, beads coupled with laminin-1 (left panel) or gamma 2-r1 (right panel) were incubated with Pam 212 cell conditioned media in the presence of increasing amounts of gamma 2-r1-derived synthetic peptide pN-4: lane 1, none; lane 2, 4 µg/ml; lane 3, 10 µg/ml; lane 4, 40 µg/ml; lane 5, 200 µg/ml. The bound fibulin-2 was detected by Western blotting. B, amino acid sequence and activities of a set of synthetic peptides derived from the gamma 2 chain (pN-21), alpha 1 chain (palpha 1-654 and palpha 1-279), beta 1 chain (pbeta 1-461), gamma 1 chain (pgamma 1-587), alpha 2 chain (palpha 2-287) are shown in the lower panel. The rr at the C terminus of pbeta 1-461 and (pgamma 1-587) indicates two arginine residues artificially added to improve the solubility of the peptides. Upper panel, left, the competition assays with laminin-1-coupled beads (lanes 1-6) or gamma 2-r1-coupled beads (lanes 7-9) were performed with 200 mg/ml each of the synthetic peptides depicted above each lane. Upper panel, right, the competition assays with laminin-1-coupled beads using increasing amounts of palpha 1-654: lane 1, none; lane 2, 4 µg/ml; lane 3, 10 µg/ml; lane 4, 100 µg/ml; lane 5, 200 µg/ml. Lane 6, 200 µg/ml pN-21.
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Affinity Chromatography and Competition Assays

The recombinant proteins fused to glutathione S-transferase (GST)1 were bound to glutathione-agarose (Pharmacia) at 0.2 mg/ml. Laminin-1 prepared from Engelbreth-Holm-Swarm tumor was coupled to CNBr-activated Sepharose beads (1 mg/ml) (Pharmacia). Pam 212 cells and murine dermal fibroblasts were labeled with 50 µCi/ml [35S]methionine (ICN, Costa Mesa, CA) in methionine-free Dulbecco's modified Eagle's medium (ICN) for 4 h. The conditioned media were adjusted to 2 mM phenylmethylsulfonyl fluoride and centrifuged at 3,000 revolutions/min for 10 min. These supernatants were precleared with 30% v/v Sepharose CL-4B (Pharmacia) by incubation for 30 min at 4 °C, and these precleared supernatants were stored at -80 °C until use. The conditioned media (500 ml) were incubated with 30 ml of affinity beads for 2 h with rotary shaking at 4 °C. Following three washings with 1 ml of 0.1% Triton X-100, phosphate-buffered saline, 2 mM phenylmethylsulfonyl fluoride, the proteins bound to the affinity beads were extracted with SDS-sample buffer. The samples were boiled with or without 100 mM dithiothreitol, analyzed on 4-12% SDS-PAGE, and treated with Enlightning (DuPont NEN) prior to autoradiograph. For competition assay, the synthetic peptides were added to the mixture during the incubation period.

Western Blotting and Immunoprecipitation Analysis

For Western blotting, the conditioned media were prepared from early confluent Pam 212 cells in 80-cm2 flasks by incubation with 8 ml of Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum for overnight and treated as mentioned above. The proteins bound to the affinity beads were electrophoresed through 4-12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Marlborough, MA). The membrane was blocked with 5% milk, Tris-buffered saline, pH 7.4, 0.1% Tween 20 for 30 min and incubated with antibody either overnight at 4 °C or for 2 h at room temperature. Bound antibody was detected by peroxidase-conjugated anti-rabbit IgG antibody (Pierce) and antibody reactivity followed by ECL (Amersham Life Science). The anti-fibulin-2 antiserum and anti-laminin gamma 2 chain antiserum were diluted at 1:20,000 and at 1:5,000, respectively. For immunoprecipitation, the antibody to the gamma 2 chain was diluted at 1:100. Conditioned media (1 ml) were incubated with the antibody and protein G beads (Pharmacia) for 2 h at 4 °C. After washing three times with 1 ml of 0.1% Triton X-100, phosphate-buffered saline, 2 mM phenylmethylsulfonyl fluoride, the bound proteins were eluted by slow agitation in washing buffer containing 5 mM EDTA for 20 min at room temperature. The eluted proteins were precipitated with 10% trichloroacetic acid for 30 min on ice with 10 mg of bovine serum albumin added as a carrier. After centrifugation at 30,000 × g for 15 min at 4 °C, the pellets were washed with 1 ml of 0.5% trichloroacetic acid once and then washed two times with 1 ml of acetone. These pellets were resuspended in sample buffer and separated by SDS-PAGE.

Sequence Analysis

Protein sequence analysis were performed using a software package from the University of Wisconsin Genetics Computer Group; optimal alignment was provided by the program BESTFIT.


RESULTS

Screening of Extracellular Proteins Bound to the Short Arms of Laminin-5

Since the short arms of laminins have been shown to interact with other basement membrane proteins, we examined whether additional extracellular matrix proteins bind to the short arms of the gamma 2 and beta 3 chains of laminin-5 using recombinant laminin chains. [35S]Methionine-labeled conditioned media from murine epidermal Pam 212 cells were incubated with various recombinant laminin proteins-coupled to agarose beads. After washing, bound proteins were eluted and analyzed on SDS-PAGE. The N terminus of the gamma 2 chain short arm bound a protein with an apparent molecular mass of 195 kDa, while there was no protein binding to either the short arm of the beta 3 chain of laminin-5 or the alpha 2 chain of laminin-2 (Fig. 1A, lanes 1-3). Binding of a 195-kDa molecule to the gamma 2 chain short arm was also observed with the conditioned media from mouse dermal fibroblasts (Fig. 1A, lane 5). Since 5 mM EDTA abolished the binding, this interaction was likely dependent on a divalent cation (Fig. 1A, lane 4). Electrophoresis containing 6 M urea showed a single protein band, strongly suggesting that this 195-kDa band consists of a single molecule (Fig. 1B). Furthermore, the shift of the molecular size from 195 kDa to ~600 kDa under non-reducing conditions suggested that the 195-kDa molecule might form a disulfide-bonded homotrimer (Fig. 1B). Digestion of this 195-kDa protein with collagenase did not cleave this protein, suggesting it does not contain a collagenous domain (Fig. 1C, lanes 3 and 4).


Fig. 1. Affinity chromatography of conditioned media on the recombinant short arm fragments of laminin. [35S]Methionine-labeled conditioned media from murine epidermal Pam 212 cells (A, lanes 1-3, B and C) or from murine newborn dermal fibroblasts (A, lanes 4 and 5) were incubated with recombinant protein-coupled affinity beads. A, bound proteins were analyzed by 4-12% SDS-PAGE under reducing conditions and followed by fluorography. Lane 1, proteins bound to recombinant alpha 2-r1; lane 2, proteins bound to gamma 2-r1; lane 3, proteins bound to beta 3-r1. The protein binding to gamma 2-r1 was analyzed in the presence (lane 4) or the absence of 5 mM EDTA (lane 5). B, proteins bound to gamma 2-r1 were analyzed on 4% SDS-PAGE containing 6 M urea under non-reducing (lane 1) or reducing (lane 2) conditions. Upper arrow, electrophoretic mobility of laminin alpha 1 chain; lower arrow, that of laminin beta 1 and gamma 1 chains. C, proteins bound to gamma 2-r1 (lanes 3 and 4) and immunoprecipitated type IV collagen (lanes 1 and 2) were digested on beads with bacterial collagenase in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4) of 5 mM N-ethylmaleimide. The prestained molecular size markers 199, 120, 87, and 47 kDa were used.
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Fibulin-2 Binding to the Recombinant gamma 2 Chain Short Arm

Judging from its molecular size and its ability to form a homotrimer, we speculated that this non-collagenous 195-kDa extracellular protein might be either fibulin-2 (21) or thrombospondin (36). To test these possibilities, we used antibodies to both fibulin-2 and thrombospondin in Western blotting. The 195-kDa protein bound to the recombinant gamma 2 chain short arm (gamma 2-r1) was recognized by an antibody against mouse fibulin-2 (Fig. 2A, lane 2), but not by an antibody to thrombospondin (Fig. 2B). Furthermore, this protein that reacted with the anti-fibulin-2 antibody formed a trimer under non-reduced conditions (Fig. 2A, lane 3) and exhibited divalent cation-dependent binding to gamma 2-r1 (Fig. 2A, lanes 4 and 5). These data confirm the identity of the 195-kDa protein as fibulin-2.


Fig. 2. Fibulin-2 binding to the recombinant fragment of the gamma 2 chain (gamma 2-r1). The conditioned media from Pam 212 cells were incubated with GST alone (lane 1) or gamma 2-r1-GST (lanes 2-5). EDTA (5 mM) was added to the incubation mixture (lane 4). The bound proteins were separated on 4-12% SDS-PAGE in the presence of 100 mM dithiothreitol except for lane 3. The same amounts of conditioned media were applied on the gel under reducing conditions (lane 6). Western blotting was performed with anti-fibulin-2 antiserum. B, the conditioned media from the human epidermoid cell line A431 were incubated with gamma 2-r1-GST and the bound proteins were analyzed under reducing condition (lane 1). One-tenth the amount of input conditioned media was applied on the next lane (lane 2). Western blotting was performed with anti-thrombospondin antiserum. Fibulin-2 and thrombospondin were detected with ECL chemiluminescence.
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Deletion Analysis of the Fibulin-2 Binding Site of the gamma 2 Chain

In order to identify the binding site for fibulin-2, a series of deletion mutants of gamma 2-r1 were prepared (Fig. 3A). The binding activities were analyzed by affinity chromatography using conditioned media from murine epidermal Pam 212 cells (Fig. 3B). Recombinant gamma 2-r3 containing a deletion of domain V still interacted with fibulin-2 (Fig. 3B, lane 3). The N-terminal 51-amino acid region in domain IV (gamma 2-r5, residues 197-247) was active for binding but the C-terminal 59-amino acid region (gamma 2-r4, residues 248-306) was inactive (Fig. 3C, lanes 4 and 5). These results indicate that the active region for fibulin-2 binding is located within residues 197-247 in domain IV of the gamma 2 chain.

Delineation of the Fibulin-2 Binding Sequence of the gamma 2 Chain

To delineate the fibulin-2 binding sequence of the gamma 2 chain, synthetic peptides derived from the gamma 2 chain were tested for their ability to compete for binding to fibulin-2 (Table I). The synthetic peptide gamma 2-pN (residues 197-226) showed inhibition of fibulin-2 binding to gamma 2-r1 in a dose-dependent manner, while no inhibition was observed with gamma 2-pC (residues 219-248) (Figs. 3A and 4A). Inhibition studies with smaller peptides, pN-1, -2, and -3, demonstrated that only pN-3 was active in competing with fibulin-2 binding to gamma 2-r1 (Fig. 4A, lanes 6-9). The inhibitory activities of synthetic peptides with incremental 1-amino acid deletions (pN-4 to pN-11) from the C terminus of pN-3 were analyzed (Fig. 4, B and C, lanes 1-5). pN-4 (HASADFSVHKIT) and pN-5 (HASADFSVHKI) showed significant inhibition in fibulin-2 binding. pN-6 (HASADFSVHK) was less active than pN-4 and pN-5. The inhibitory activity of pN-6 was also confirmed by increasing the concentration of the peptide from 0.2 mg/ml to 1 mg/ml in the inhibition reaction mixtures (Fig. 4C, lane 13). pN-7 (HASADFSVH) was inactive even at 1 mg/ml (Fig. 4C, lane 12). These results indicate that the 10-amino acid sequence HASADFSVHK (residues 197-206) was necessary for the fibulin-2 binding and that Ile-207 was required for full inhibitory activity. To determine the minimum size for the inhibitory activity, another set of N-terminal deletions (pN-12 to pN-15) were prepared and tested. pN-14 (SADFSVHKIT) and pN-15 (ASADFSVHKIT) were active at a concentration of 0.2 mg/ml (Fig. 4C, lanes 8 and 9). Both pN-12 (FSVHKIT) and pN-13 (ADFSVHKIT) showed no activity at 0.2 mg/ml, whereas only pN-13 was active at 1 mg/ml (Fig. 4C, lanes 6, 7, 10, and 11). These results indicate that HAS (residues 197-199) were not essential, but Ser-199 was required for the full binding activity. Heptapeptide pN-16 (ADFSVHK), containing the core sequence for the activity, however, was not active even at 1 mg/ml (Fig. 4C, lane 14), suggesting that either Ile-207 or Ser-199 was required for activity. Furthermore, none of three scrambled peptides containing the pN-4 residues were active (Fig. 4C, lanes 16-18), indicating that the activity depends on the specific sequence of amino acids and not on the amino acid composition.


Fig. 4. Affinity competition assays using synthetic peptides. Synthetic peptides derived from domain IV of the gamma 2 chain are listed in Fig. 3A and Table I. The peptides were added to the mixture of gamma 2-r1-GST and Pam 212 conditioned media to test whether they competed for fibulin-2 binding to gamma 2-r1-GST. Bound substrates were analyzed on 4-12% SDS-PAGE and detected by either fluorography (A) or Western blotting using anti-fibulin-2 antiserum (B-D). A, peptides depicted above each lane were added at 40 µg/ml (lanes 2 and 4) or 200 µg/ml (lanes 3, 5, and 7-9). B, peptides depicted above each lane were added at 200 µg/ml. C, peptides depicted above each lane were added at 200 µg/ml (lanes 1-9 and 15-18). In lanes 10-14, the synthetic peptides were increased to 1 mg/ml. Molecular markers indicate 199 and 120 kDa. D, synthetic peptides shown in the boxes with alanine substitutions (left panel) or other single amino acid point mutations (right panel) were added at 200 µg/ml.
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Analysis with the truncated peptides described above demonstrated that the 9-amino acid sequence, SADFSVHKI (residues 199-207), of the gamma 2 chain was necessary for fibulin-2 binding. We introduced single amino acid substitutions (Fig. 4D, left panel) in this 9-amino acid peptide (pN-21) to identify residues important for the activity. pN-21A1 and pN-21A5 completely abolished the activity, indicating that Phe-202 and Lys-206 were critical for the activity (Fig. 4D, lanes 3 and 8). The Val-204 and Ile-207 were also important but not as important as Phe-202 and Lys-206, because pN-21A3 and pN-21A6 were more active than pN-21A1 and pN-21A5 (Fig. 4D, lanes 5 and 7). The significant loss of activity in pN-21A6 is consistent with a loss observed with pN-6 containing the deletion of Ile-207 of pN-5 (Fig. 4B). The fact that pN-21A2 and pN-21A4 peptides were active in binding suggests that Ser-203 and His-205 were not essential for binding (Fig. 4D, left panel). The change of Phe-202 to Leu (pN-21L1) completely abolished binding (Fig. 4D, right panel, lanes 2 and 3). Although a leucine substitution at Val-204 (pN-21L2) did not decrease its activity, a threonine substitution at Val-204 (pN-21T) abolished its activity (Fig. 4D, right panel, lanes 4 and 6). Furthermore, a glycine substitution at Asp-201 (pN-21G) did not reduce the activity, indicating that this residue was not essential for activity (Fig. 4D, right panel, lane 5). Taken together, these results demonstrate that the nonapeptide 199-207 was the minimum active region with residues Ser, Ala, Phe, Val, Lys, and Ile, critical for the activity. The importance of Ala-200 was not confirmed by a deletion peptide since the peptide was not soluble after deletion of this residue from pN-13 (Table I).

Fibulin-2 Binds to Native Laminin-5

Binding of fibulin-2 to native laminin-5 was examined by immunoprecipitation assays. The conditioned media from Pam 212 cells were immunoprecipitated with the antibody to the gamma 2 chain. The precipitates were analyzed by Western blotting with the anti-fibulin-2 antibody. The anti-gamma 2 chain antibody immunoprecipitated fibulin-2 (Fig. 5A). This co-immunoprecipitation of fibulin-2 was inhibited by peptide pN-4 (Fig. 5B, upper panel). pN-4 did not affect the amount of the gamma 2 chain immunoprecipitated by the anti-gamma 2 antibody (Fig. 5B, bottom panel). These results suggest that fibulin-2 binds to native laminin-5 via the pN-4 site in domain IV of the gamma 2 chain.


Fig. 5. Fibulin-2 binds to native laminin-5. Pam 212 cell conditioned media were analyzed by immunoprecipitation (I.P.) and subsequently by Western blotting (W.B.). The conditioned media were treated with preimmune rabbit serum (preimmune) or anti-laminin gamma 2 chain antiserum (anti gamma 2). The precipitates were separated on SDS-PAGE followed by Western blotting with anti-fibulin-2 antiserum (anti fib-2). Arrow indicates rabbit IgG. B, immunoprecipitations with an anti-gamma 2 antiserum were performed in the presence of different amounts of the active synthetic peptide pN-4: lane 1, none; lane 2, 50 µg/ml; lane 3, 200 µg/ml; lane 4, 600 µg/ml. Upper panel, eluates with 5 mM EDTA from the immunoprecipitates were reacted by anti-fibulin-2 antiserum after blotting. Lower panel, the remaining proteins on the beads were probed with an anti-laminin gamma 2 chain antiserum after blotting. Double arrows, 155- and 105-kDa laminin gamma 2 chains. Arrow, rabbit IgG.
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Fibulin-2 Binds to Laminin-1

We examined whether fibulin-2 binds to laminin-1 by affinity chromatography. The conditioned media from Pam 212 cell culture were applied on a native laminin-1-coupled Sepharose affinity column. Western blotting of the eluant from this column showed the presence of fibulin-2, indicating that fibulin-2 interacts with laminin-1 (Fig. 6A). Inclusion of 5 mM EDTA to the conditioned media completely eliminated the binding of fibulin-2 to laminin-1, suggesting that this interaction is cation-dependent. Addition of both Ca2+ (8 mM) and Mn2+ (5 mM) to the 5 mM EDTA-containing conditioned media restored binding of the fibulin-2 to laminin-1 (Fig. 6B, upper panel). Mg2+ alone also restored the binding activity, although to a lesser extent. Similar divalent cation dependence was also seen for the binding of fibulin-2 to the recombinant laminin gamma 2 chain (Fig. 6B, bottom panel). These results indicate that the fibulin-2 binding to laminin-1 and laminin-5 requires divalent cations.


Fig. 6. Fibulin-2 binds to native laminin-1. A, laminin-1-coupled Sepharose beads (lanes 1, 2, and 4) or bovine serum albumin-coupled beads (lane 3) were incubated with Pam 212 conditioned media. Lane 5 represents laminin-coupled beads alone without conditioned media. Control, without incubation. Bound proteins were eluted by SDS-sample buffer and analyzed by 4-12% SDS-PAGE under reducing (lanes 2-5) or nonreducing (lane 1) conditions followed by Western blotting with anti-fibulin-2 antiserum. B, laminin-1-coupled (upper panel) or gamma 2-r1-coupled beads (lower panel) and conditioned media were combined and adjusted to either 5 mM EDTA (lanes 2-5) or 0 mM EDTA (lane 1). Three different divalent cations were added to the incubation mixture at 8 mM Ca2+ (lane 3), Mg2+ (lane 4), and Mn2+ (lane 5). The bound proteins were separated on 4-12% SDS-PAGE and analyzed by Western blotting with anti-fibulin-2 antiserum.
[View Larger Version of this Image (31K GIF file)]


A Fibulin-2 Binding Site of Laminin-1

Peptide pN-4 from the gamma 2 chain had a dose-dependent inhibitory activity for the binding of fibulin-2 to laminin-1 (Fig. 7A). These unexpected observations suggest that laminin-1 binds to fibulin-2 at a site similar in pN-4. A protein sequence homology search showed that there are two homologous amino acid stretches in the laminin alpha 1 chain and one region in each of the alpha 2, beta 1, and gamma 2 chains (Fig. 7B, lower panel). The peptides containing these sequences were analyzed for their inhibitory activity in the binding of fibulin-2 to laminin-1 using the affinity column assay. Palpha 1-654 from the laminin alpha 1 chain was active (Fig. 7B, left panel, lane 2) and showed a dose-dependent inhibition (Fig. 7B, right panel, lanes 1-5), whereas the other four peptides, pbeta 1-461, pgamma 2-587, palpha 1-279, and palpha 2-287, were inactive (Fig. 7B). These results indicate that fibulin-2 binds to laminin-1 through the palpha 1-654 sequence (residues 654-665) of the globular domain IVb of the alpha 1 chain short arm. In competition assays, pN-4 and palpha 1-654 showed similar inhibitory activities and blocked binding of fibulin-2 to laminin-1 and to gamma 2-r1 (Fig. 7A). palpha 1-654 completely inhibited the binding of fibulin-2 to gamma 2-r1 at 0.2 mg/ml comparable to pN-4 (Fig. 7B, lanes 7-9). These results suggest that fibulin-2 binds to both laminin-1 and -5 through similar sequence in the alpha 1 and gamma 2 chain.


DISCUSSION

We have demonstrated through a number of independent methods that fibulin-2 binds to laminin-1 and laminin-5 through the alpha 1 and gamma 2 chain short arms, respectively.

The binding of fibulin-2 to laminin-5 appears to be a relatively strong interaction, since the anti-gamma 2 antibody co-immunoprecipitates the complex in the conditioned media. The finding that a synthetic peptide from the gamma 2 chain could inhibit this complex formation suggests that the fibulin-2 binding site of the gamma 2 chain is not cryptic and is active in the native laminin-5 molecule. A heptapeptide sequence within laminin gamma 1 has been delineated for nidogen binding (42-44). As the nonapeptides from the gamma 2 and alpha 1 chains inhibit fibulin-2 binding, it is likely that fibulin-2 interacts with a small region in the laminins of similar size to that identified for nidogen. Since the 10 epidermal growth factor-like repeats of fibulin-2 have a calcium-binding motif (21), this region likely contains a site(s) for laminin binding. Consistent with these data is our finding that fibulin-2 binds to both laminin-1 and -5 and this binding is abolished by the addition of EDTA. Recombinant fibulin-2 has been shown to bind strongly to fibronectin in calcium-dependent manner by solid phase radioligand binding assays (23). It also binds to nidogen, although this interaction is only blocked partially by EDTA. However, little binding of fibulin-2 to laminin-1 was found in the solid phase binding assay system. This discrepancy of fibulin-2 binding to laminin-1 may be due to differences in the two assays.

Amino acid truncation and substitution analysis to delineate the region of the gamma 2 chain responsible for binding to fibulin-2 suggested that residues Ser-199, Phe-202, Val-204, Lys-206, and Ile-207 within the nonapeptide sequence of the gamma 2 chain (pN-21, residues 199-207) were required. Although pbeta 1-461 from the beta 1 chain contains similar residues including Phe, Val, and Ile at the positions similar to pN-21, it was inactive in inhibiting fibulin-2 binding to laminin-1. The inactive peptide pbeta 1-461 also contains Leu at the position corresponding to Lys-206 in pN-21. This is also consistent with the result that Lys-206 was critical for the activity. The active peptide palpha 1-654 possesses Arg at the position of Lys-206 in pN-21, suggesting that a positively charged residue at this position is also involved in an ionic interaction with the binding site of fibulin-2. Moreover, palpha 1-654 with Leu at the position of Ile-207 in pN-21 was active, and an Ala substitution at this position reduced the activity of pN-21, suggesting that a hydrophobic residue Leu or Ile at the position of Ile-207 in pN-21 was preferable for binding activity. Since an Ala-200 in pN-21 is not conserved in palpha 1-654, this alanine residue does not seem to be essential for the activity. Together, it was concluded that consensus critical residues in laminin-1 and -5 for the fibulin-2 binding are F, V, (K/R), and (I/L). The laminin sequences for fibulin-2 binding defined in this report are not present in fibronectin and nidogen. Hence, fibulin-2 may interact with these molecules via different sites (23).

The biological importance of the short arm of the gamma 2 chain of laminin-5 has been revealed by the finding of a gamma 2 chain mutation in a human patient with junctional epidermolysis bullosa (7). This patient has an internal deletion of domains III and IV of the gamma 2 chain short arm, suggesting that the short arm of the gamma 2 chain is critical for the structural stability of the dermal-epidermal junction. Although the deleted region does not correspond exactly to the site for fibulin-2 binding, it is possible that the deletion perturbs the native conformation of domain IV, resulting in the masking or inactivation of the binding site. Since both molecular abnormalities in laminin-5 and autoantibodies specific to laminin-5 cause blister formation in skin, laminin-5 appears to be important for the integrity of skin. Since fibulin-2 binds to laminin-5, it is possible that it plays a critical role in stabilizing or organizing the epithelial basement membrane during development of skin or wound healing. The recent report that expression of fibulin-2 is markedly increased during skin repair supports this hypothesis (45). It will be of interest to examine whether the active peptide from the gamma 2 or alpha 1 chain can block formation of basal lamina in an in vitro reconstitution cell culture system (46). It is also interesting to examine whether blistering could be produced by subcutaneous-injection of the active peptide. Further studies will examine the significance of the fibulin-2 binding to laminins.


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.
Dagger    To whom correspondence should be addressed: Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bldg. 30, Rm. 405, Bethesda, MD 20892.
1    The abbreviations used are: GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

Acknowledgments

We acknowledge the expert technical assistance of Nikki Hayes for DNA sequencing. We thank Rupert Timpl for anti-fibulin-2 antibody. We thank Peter Burbelo, Hynda K. Kleinman, and Sharon Powell for critical reading of the manuscript.


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