(Received for publication, September 3, 1996, and in revised form, October 25, 1996)
From the Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892
Epithelial cell-specific laminin-5, consisting of
three chains, 3,
3, and
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
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
2 chain was defined as a critical site
for fibulin-2 binding. An anti-
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 (
1
1
1) through a region (residues
654-665) of the
1 chain short arm whose sequence is similar to that
of the fibulin-2 binding site of the
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.
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 6
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 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, 3,
3, and
2, and is an adhesive substrate for keratinocytes in
vitro (24). Recently, both
6
4 and
3
1 integrins were
identified as cellular receptors for laminin-5 (25-29). Laminin-5
forms a disulfide-bonded complex with laminin-6 (
3
1
1) (30),
possibly via the N-terminal globular domain VI of the laminin
3
chain. The N-terminal globular domain VI of the laminin
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 2 chain and we have identified a 9-amino acid
sequence in domain IV of the
2 chain critical for this binding. We
have also found that fibulin-2 binds to laminin-1 via the N terminus of
the
1 chain, a site showing sequence homology to the 9-amino acid
sequence of the
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.
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 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.
cDNAs for
murine laminin 2,
2, and
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:
2-r1 (residues
17-306),
2-r2 (residues 129-306),
2-r3 (residues 197-306),
2-r4 (248-306),
2-r5 (residues 197-247) of the
2 chain,
3-r1 (residues 18-253) of the
3 chain,
2-r1 (residues 23-242) of the
2 chain. All constructs used in these studies were
confirmed by automated DNA sequencing (model 370A, Applied Biosystems,
Foster City, CA).
Synthetic peptides, 2-pC and
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
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
2 chain;
p
1-654, 12 amino acids (residues 654-665) of the
1 chain;
p
1-279, 9 amino acids (residues 279-287) of the
1 chain;
p
1-461, 9 amino acid (residues 461-469) of the
1 chain with two
arginine residues added to increase the solubility of the peptide;
p
1-587, 9 amino acids (residues 587-595) of the
1 chain with
two added arginine residues to increase the solubility of the peptide;
and p
2-287, 9 amino acids (residues 287-295) of the
2
chain.
|
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.
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 2 chain antiserum were diluted at 1:20,000 and at
1:5,000, respectively. For immunoprecipitation, the antibody to the
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.
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.
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
2 and
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
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
3 chain
of laminin-5 or the
2 chain of laminin-2 (Fig.
1A, lanes 1-3). Binding of a
195-kDa molecule to the
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).
Fibulin-2 Binding to the Recombinant
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 2 chain short arm (
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
2-r1 (Fig. 2A,
lanes 4 and 5). These data confirm the identity
of the 195-kDa protein as fibulin-2.
Deletion Analysis of the Fibulin-2 Binding Site of the
In order to identify the binding site for fibulin-2, a
series of deletion mutants of 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
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 (
2-r5, residues 197-247) was active for binding but
the C-terminal 59-amino acid region (
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
2 chain.
To delineate the fibulin-2 binding sequence of the 2
chain, synthetic peptides derived from the
2 chain were tested for their ability to compete for binding to fibulin-2 (Table
I). The synthetic peptide
2-pN (residues 197-226)
showed inhibition of fibulin-2 binding to
2-r1 in a
dose-dependent manner, while no inhibition was observed
with
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
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.
Analysis with the truncated peptides described above demonstrated that
the 9-amino acid sequence, SADFSVHKI (residues 199-207), of the 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).
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 2 chain. The precipitates were analyzed by Western
blotting with the anti-fibulin-2 antibody. The anti-
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
2 chain immunoprecipitated by the anti-
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
2 chain.
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 2
chain (Fig. 6B, bottom panel). These results
indicate that the fibulin-2 binding to laminin-1 and laminin-5 requires
divalent cations.
A Fibulin-2 Binding Site of Laminin-1
Peptide pN-4 from the
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
1
chain and one region in each of the
2,
1, and
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. P
1-654 from
the laminin
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, p
1-461, p
2-587,
p
1-279, and p
2-287, were inactive (Fig. 7B). These
results indicate that fibulin-2 binds to laminin-1 through the
p
1-654 sequence (residues 654-665) of the globular domain IVb of
the
1 chain short arm. In competition assays, pN-4 and p
1-654
showed similar inhibitory activities and blocked binding of fibulin-2
to laminin-1 and to
2-r1 (Fig. 7A). p
1-654 completely inhibited the binding of fibulin-2 to
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
1 and
2 chain.
We have demonstrated through a number of independent methods that
fibulin-2 binds to laminin-1 and laminin-5 through the 1 and
2
chain short arms, respectively.
The binding of fibulin-2 to laminin-5 appears to be a relatively strong
interaction, since the anti-2 antibody co-immunoprecipitates the
complex in the conditioned media. The finding that a synthetic peptide
from the
2 chain could inhibit this complex formation suggests that
the fibulin-2 binding site of the
2 chain is not cryptic and is
active in the native laminin-5 molecule. A heptapeptide sequence within
laminin
1 has been delineated for nidogen binding (42-44). As the
nonapeptides from the
2 and
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 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
2 chain (pN-21, residues 199-207) were
required. Although p
1-461 from the
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 p
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
p
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, p
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 p
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 2 chain of
laminin-5 has been revealed by the finding of a
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
2 chain short
arm, suggesting that the short arm of the
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
2 or
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.
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.