From the
Laminin-5 (kalinin) was purified from spent cell culture media
(SCC25 cells) by affinity chromatography on monoclonal antibody BM165.
The protein was recovered as a mixture of the typical polypeptides of
165-155, 140, and 105 kDa as judged by SDS-polyacrylamide gel
electrophoresis analysis under reducing conditions. The amino acid
composition of purified laminin-5 was in agreement with that compiled
from the recently published cDNA sequences of the
Kalinin is a specific component of basement membranes underlying
stratified epithelia (Rousselle et al., 1991). It was
immunolocalized to the anchoring filaments (Rousselle et al.,
1991), which are supposed to play a major structural role in the
cohesion between epidermal cells, the basal lamina, and the underlying
stroma (Haber et al., 1985). The molecule is synthesized as a
precursor consisting of three chains of 200, 140, and 155 kDa, with two
of them, the 200- and 155-kDa chains, being rapidly processed into 165-
and 105-kDa polypeptides, respectively (Rousselle et al.,
1991; Marinkovich et al., 1992). Sequencing of corresponding
cDNA clones (Kallunki et al., 1992; Vailly et al.,
1994; Gerecke et al., 1994; Ryan et al., 1994)
demonstrated that kalinin represents one more member of the growing
family of laminin molecules, and it was therefore renamed laminin-5
according to the new nomenclature for laminin family members (Burgeson
et al., 1994).
Laminins are heterotrimers formed by
assembly of three genetically distinct chains,
Sequencing of cDNA clones
corresponding to the
We have now addressed the question of whether
laminin-5 cell adhesion activity is dependent or not on a triple
coil-coiled conformation, as is the case for laminin-1 (Deutzmann
et al., 1990). Circular dichroism spectroscopy analysis
provided experimental evidence for the predicted presence of a helical
conformation in laminin-5. Cell adhesion was found to be strictly
dependent on the conformation and was totally abrogated by thermal
denaturation of laminin-5 between 65 and 75 °C.
Using a two-step procedure, laminin-5 was purified by
affinity chromatography from spent culture media of SCC25 cells. The
first chromatography over gelatin-Sepharose (25-ml bed volume) was
sufficient to remove fibronectin from cell culture medium as shown by
SDS-PAGE and immunoblotting analysis of the eluted material
(Fig. 1C). After a second chromatography on
BM165-Sepharose, the eluted material was found to contain only the
typical laminin-5 polypeptides of 165-155, 140, and 105 kDa as
shown by SDS-PAGE under reducing conditions (Fig. 1A).
In addition, none of the laminin-5 was retained on the
gelatin-Sepharose affinity column (Fig. 1B, compare
lanes1 and 2).
Laminin-5 showed a
circular dichroism spectrum with a minimum in negative ellipticity
between 215 and 220 nm that indicated a high
By its localization to anchoring filaments, laminin-5 is
supposed to play major mechanical and biological roles. The former is
based on the fact that there is a lack of or alterations in anchoring
filaments and an absence of immunoreactivity toward monoclonal antibody
GB3 (Verrando et al., 1991), a laminin-5-specific antibody, in
hereditary forms of epidermolysis bullosa junctionalis, a lethal
blistering disease characterized by intracutaneous splits within the
lamina lucida of skin basement membranes. Recently, mutations in the
Cloning and sequencing of
the cDNA corresponding to the
Cell adhesion-promoting activity of
laminin-5 was strictly dependent on the helical conformation, as was
observed for laminin-1 fragment 8 (Deutzmann et al.(1990),
Goodman et al.(1991), and this report), suggesting that all
the cell-binding activity of laminin-5 is restricted to the
carboxyl-terminal domain of the molecule. This is in contrast to
laminin-1, which contains additional cell-binding sites on
amino-terminal portions of the molecule contributing the short arms
(Hall et al., 1990; Goodman et al., 1991). Laminin-5
lacks several of the amino-terminal domains found in laminin-1 that
probably contribute to the cell adhesion activity of laminin-1 short
arms.
The stability and biological properties of basement membranes
might be adapted to specific functions of the tissue that they border
by varying the composition in the isoforms of the basic constitutive
molecules (Engvall et al., 1990; Sanes et al., 1990).
Laminin-5 is located in basement membranes underlying stratified
epithelia such as the epidermis and the esophagus (Rousselle et
al., 1991). These tissues are exposed to high mechanical and
temperature constraints and should therefore be adapted to variations
in these parameters. It is interesting to note that the conformation
and cell adhesion activity of laminin-1, which has been shown to be
expressed in early stages of embryonic development, are labile at lower
temperatures compared with laminin-2/4 or laminin-5, which appear later
in development. The cell-binding site of another cell adhesion molecule
of basement membranes, collagen IV, has a denaturation temperature of
50 °C, which is well above the denaturation temperature of other
collagens located deeper in the dermis (Eble et al., 1993).
Subtle variations in the sequences involved in the ionic interactions
could provide the different basement membranes with specific molecular
structures that are adapted to their polymorphic properties and that
remain to be elucidated.
We gratefully acknowledge Marie-Marguerite Boutillon
and Josiane Pradines-Grillet for expert technical assistance and Alain
Bosch for artwork. We thank Dr. Rupert Timpl for kind gifts of reagents
and for valuable suggestions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-,
-, and
-laminin chains. Moreover, the
content of half-cystine residues in laminin-5 was about two-thirds that
in laminin-1, which confirms the prediction of a smaller number of
epidermal growth factor-like repeats in the amino-terminal portion of
the three chains. The content of coiled-coil
-helices (27%)
determined by CD spectroscopy was comparable to that reported for
laminin-1, which indicates that the long arm portion of laminin-5 is
equivalent to that of other laminin isoforms. The melting temperature
was recorded at 72 °C by CD monitoring of unfolding and refolding
of the coiled-coil structures during thermal denaturation and
renaturation, respectively. The thermal stability of laminin-5 is
therefore significantly higher than that of laminin-1 or
-chain-containing laminins, which suggests higher
ionic interactions between the three polypeptide chains of laminin-5.
Cell adhesion-promoting activity of laminin-5 was found to be strictly
and entirely dependent on the presence of coiled-coil structures. It
decreased gradually after heat denaturation of the protein above 65
°C and was totally abrogated at 75 °C. This is in contrast to
laminin-1, which contains both conformation-dependent and -independent
cell-binding sites on the long and short arm domains, respectively.
,
, and
(Timpl and Brown, 1994). The first member of this family, laminin-1,
was isolated from the Engelbreth-Holm-Swarm tumor transplantable to
mice and was shown to be constituted by an
-chain of
400 kDa and two light chains,
and
,
of
200 kDa assembled into a cross-shaped structure (Timpl, 1989).
The carboxyl-terminal domains of laminin-1 chains, domains I and II,
contain long heptad peptide regions (
600 residues), where specific
ionic interactions are responsible for initiating and folding into a
triple-stranded coiled-coil domain corresponding to the long arm of the
molecule (Paulsson et al., 1985; Hunter et al., 1992;
Beck et al., 1993; Utani et al., 1994). Another
characteristic feature of laminin-1 is that it contains a relatively
high proportion of cysteine residues, with most being clustered in
repeats of
50 amino acids with homology to epidermal growth
factor. The repeats are arranged into rows forming rod-like structures
intercalated with globular domains, with both structures constituting
the short arms of the molecules (Timpl, 1989; Beck et al.,
1990). Different structural domains of laminin-1 are endowed with
mechanical and biological functions including induction of cell
adhesion. A major cell-binding site has been located in a polypeptide
fragment encompassing the carboxyl-terminal half of the laminin-1 long
arm (Aumailley et al., 1987; Goodman et al., 1987)
and has been shown to be strictly dependent on the coiled-coil
conformation (Deutzmann et al., 1990; Sung et al.,
1993). Another cell-binding site is located in the short arms (Hall
et al., 1990) and is maintained after heat denaturation of
laminin-1 (Goodman et al., 1991).
-chain (Ryan et al.,
1994),
-chain (Gerecke et al., 1994), and
-chain (Kallunki et al., 1992; Vailly et
al., 1994) of laminin-5 showed that they all contain the classical
heptad repeats suitable for
-helical folding of their
carboxyl-terminal parts. Laminin-5 sequences differ, however, from
those of laminin-1 by a reduced number of epidermal growth factor-like
repeat rows and globular domains (Kallunki et al., 1992;
Gerecke et al., 1994; Vailly et al., 1994; Ryan
et al., 1994). Due to difficulties in extracting from tissues
the corresponding protein in significant quantities, information on the
chemical and biological characterization of laminin-5 has been scarce.
It is only recently that laminin-5 was purified from cell culture
medium in amounts suitable for studies showing that laminin-5 is
endowed with specific cell adhesion-promoting activity (Rousselle and
Aumailley, 1994).
Cell Cultures
Squamous carcinoma cells (SCC25,
American Type Culture Collection) were grown in 50% Ham's F-12
medium and 50% Dulbecco's modified Eagle's medium (Gibco
BRL, Cergy-Pontoise, France) supplemented with 10% fetal calf serum, 2
mM glutamine, hydrocortisone (0.4 µg/ml), and a mixture of
antibiotics. For laminin-5 purification, spent culture media were
regularly harvested from confluent cultures, clarified, and kept frozen
at -20 °C after the addition of protease inhibitors (5
mM EDTA and 50 µM each phenylmethylsufonyl
fluoride and N-ethylmaleimide). Human fibrosarcoma HT1080
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 2 mM glutamine, a mixture of antibiotics,
and 10% fetal calf serum (Seromed/Biochrom, Polylabo, Strasbourg,
France) and used for cell adhesion assays as described previously
(Aumailley et al., 1987).
Laminin-5 Purification
Spent culture medium (500
ml) was passed sequentially over 25 ml of gelatin-Sepharose (Pharmacia
Fine Chemicals, St.-Quentin, France) and 10 ml of BM165-Sepharose (G1
fraction of monoclonal antibody BM165) (Rousselle et al.,
1991), both equilibrated in phosphate-buffered saline. Material bound
to BM165-Sepharose was eluted using 1 M acetic acid. Aliquots
of peak fractions were analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE)(
)
and immunoblotting. The
fractions containing laminin-5 were pooled, neutralized by dialysis
against phosphate-buffered saline, and kept frozen at -20 °C.
Protein concentration was determined by the micro-bicinchoninic acid
assay (Pierce, Interchim, Montluon, France) or amino acid analysis
after hydrolysis.
Thermal Denaturation
Circular dichroism was used
to demonstrate the -helical structure of laminin-5 and to record
unfolding and refolding of the coiled-coil structure during thermal
denaturation and renaturation, respectively. Laminin-5 (40 µg/ml)
was in phosphate-buffered saline at pH 7.2. Mean residue ellipticities
([
]R) were monitored in the far-UV spectrum
from 195 to 250 nm in a Mark IV autodichrograph (ISA, Jobin Yvon,
Division d'Instruments S. A.) in a 1-mm cell (Hellma,
Mülheim, Federal Republic of Germany). The
-helical content
was calculated according to Greenfield and Fasmann(1969) assuming that
[
]
= -11,800 degrees
cm
dmol
. Thermal transition curves were
monitored from 20 to 90 °C at a fixed wavelength of 220 nm with a
1-mm thermostated cell and a linear temperature gradient of 20 °C/h
using a thermostat (RKS20, Lauda) with an automatic programmer (PM351,
Lauda) as described previously (Eble et al., 1993). The degree
of conversion was calculated as described (Bächinger et
al., 1980).
Cell Adhesion Assays
Multiwell tissue culture
plates (96-well; Costar, Dutscher, France) were coated by overnight
adsorption at 4 °C with serial dilutions (0-40 µg/ml, 100
µl/well) of purified laminin-5, laminin-1-nidogen complex extracted
from a murine Engelbreth-Holm-Swarm tumor, or laminin fragment E8
(Paulsson et al., 1987), the latter being kindly donated by
Dr. R. Timpl. To study the effect of thermal denaturation, the proteins
were heat-denatured for 10 min at the indicated temperature,
immediately cooled, and used for coating the wells. After saturation of
the wells with 1% bovine serum albumin (fraction V, Sigma Chimie,
St.-Quentin-Fallavier, France), cell adhesion assays were performed in
serum-free medium, and the extent of adhesion was determined using a
colorimetric method as detailed previously (Aumailley et al.,
1989). Each assay point was derived from triplicate wells.
Gel Electrophoresis and Immunoblotting
SDS-PAGE
was carried out according to Laemmli(1970) using 5 or 3-5%
gradient acrylamide gels. Samples were analyzed after reduction with 2%
2-mercaptoethanol. Proteins were either stained with Coomassie
Brilliant Blue or transferred to nitrocellulose filters (Bio-Rad
Laboratories, Ivry, France) for Western blotting according standard
procedures. Antigens were detected with monoclonal antibody BM165 or a
polyclonal antiserum raised in rabbits against purified human
fibronectin and visualized with the corresponding anti-mouse or
anti-rabbit secondary antibodies coupled to horseradish peroxidase
(Bio-Rad Laboratories). Low molecular weight markers were from
Pharmacia Fine Chemicals.
Analytical Procedures
For amino acid analysis,
proteins (10 µg) were hydrolyzed (24 h, 110 °C) in 6 M
HCl in the presence of -mercaptoethanol using the PicoTag
workstation (Waters, Millipore Division, St. Quentin-en-Yvelines,
France) according to standard procedures. For determination of cysteine
and half-cystine residues, the samples were reduced with
dithiodiglycolic acid in 0.2 M NaOH and hydrolyzed in the
presence of 6 N HCl/trifluoroacetic acid/phenol (60:30:1; 16
h, 120 °C) as described (Hoogerheide and Campbell, 1992). Amino
acid analysis was performed with a Model 6300 automated analyzer
(Beckman, Gagny, France).
Figure 1:
SDS-PAGE and Western blot analysis of
immunoaffinity-purified laminin-5 from cell culture medium. SCC25 cell
culture medium was chromatographed on gelatin-Sepharose followed by
BM165-Sepharose. Fifty-µl aliquots of the eluted peak fractions (1
ml) were ethanol-precipitated and resolved by SDS-PAGE on a 5%
acrylamide gel under reducing conditions. Protein bands were either
stained with Coomassie Blue (A) or transferred to
nitrocellulose filters for immunoblotting with monoclonal antibody
BM165 (B) or a polyclonal antiserum against human fibronectin
(C). A: lane1, starting material;
lane2, material bound to BM165-Sepharose.
B: lane1, starting material; lane2, material not bound to gelatin-Sepharose; lane3, material not bound to BM165-Sepharose; lane4, material bound to BM165-Sepharose. C:
lane 1, fibronectin; lane2, material bound
to BM165-Sepharose. Migration positions of molecular weight markers are
shown to the left of A. Arrowheads to the right of
A and to the left of B and C indicate the
positions of the polypeptide bands corresponding to laminin-5
(165-155, 140, and 105 kDa) and to
fibronectin.
The amino acid
composition of laminin-5 was determined after hydrolysis of the
purified material and compared with that of the laminin-1-nidogen
complex used as a control. The amino acid content of laminin-5 was very
similar to that of laminin-1 except for the number of half-cystine
residues, which was about two-thirds that of laminin-1 ().
The values were in good agreement with the theoretical composition of
human laminin-5 and of the mouse laminin-1-nidogen complex calculated
from the data obtained by sequencing cDNA clones corresponding to the
different polypeptide chains (Sasaki et al., 1987, 1988;
Sasaki and Yamada, 1987; Mann et al., 1989; Kallunki et
al., 1992; Gerecke et al., 1994; Vailly et al.,
1994; Ryan et al., 1994), which indicated the high purity of
the preparations used for further studies.
-helix content
(Fig. 2). Calculations according to Greenfield and Fasmann(1969)
indicated that the helical conformation accounted for 26.9% in the
native protein. Denaturation of laminin-5 by raising the temperature to
90 °C was followed by a loss of the signal for ellipticity. Partial
refolding was observed after gradually lowering the temperature back to
20 °C (Fig. 2). The melting curve monitored at 220 nm
exhibited a transition temperature at 72 °C (Fig. 3).
Figure 2:
Circular dichroism spectrum of laminin-5.
Laminin-5 was in phosphate-buffered saline, pH 7.2, at a concentration
of 40 µg/ml. Spectrum1, CD spectrum recorded at
20 °C; spectrum2, CD spectrum recorded at 20
°C after denaturation by heating at 80 °C and
renaturation.
Figure 3:
Thermal denaturation profile of laminin-5.
A laminin-5 solution (40 µg/ml) in phosphate-buffered saline, pH
7.2, was heated from 20 to 80 °C with a linear temperature gradient
of 20 °C/h. Profiles were recorded by CD monitoring at 220 nm. The
melting temperature is indicated by a verticaldashedline.
To
investigate the role of the coil-coiled structures in the cell
adhesion-promoting activity of laminin-5, dose-response curves were
constructed with HT1080 cells adhering to coats of the native protein
or of the protein denatured by heating at various temperatures ranging
from 55 to 85 °C with a 5 °C increment (data not shown). The
maximal adhesion obtained in each dose-response curve was then
expressed as a percent of the maximal adhesion recorded for cells
adhering to native laminin-5 (Fig. 4). Maximal cell
adhesion-promoting activity of laminin-5 gradually decreased after
heating the protein at temperatures above 65 °C and was completely
abolished at 75 °C. Similar experiments were performed with the
laminin-1-nidogen complex or with its major cell-binding domain,
laminin-1 fragment E8 (Fig. 4). A strict dependence on the
conformation was observed for cell adhesion to fragment E8, which
confirmed previous results (Deutzmann et al., 1990; Goodman
et al., 1991). However, the cell adhesion-promoting activity
of fragment E8 was destroyed at a lower temperature (between 60 and 65
°C) than that of laminin-5. Parallel analysis of thermal
denaturation-dependent cell adhesion to the laminin-1-nidogen complex
showed that the transition occurred as for fragment 8 between 60 and 65
°C, but that a residual cell adhesion activity about half that of
the unheated protein was maintained above 65 °C.
Figure 4:
Cell adhesion activity of laminin-5, the
laminin-1-nidogen complex, and laminin-1 fragment E8 after heat
denaturation performed at various temperatures. Multiwell plates were
coated with laminin-5 (), the laminin-1-nidogen complex (
),
or laminin-1 fragment E8 (
) at concentrations ranging from 0 to
20 µg/ml prior or after heating at the indicated temperatures.
HT1080 cells were seeded onto triplicate wells, and the extent of
adhesion was determined after 30 min of incubation using a colorimetric
assay. The plateau values observed for dose-response curves at each
temperature were recorded and are expressed as a percent of the
adhesion obtained using unheated coats (25 °C). Each point
represents the average of triplicate wells.
-chain gene (LAMC2) of patients affected with this
disease have been reported (Pulkkinen et al., 1994; Aberdam
et al., 1994), indicating that a structural defect in or an
absence of laminin-5 in the tissue could impair the cohesion between
basal epidermal cells and the underlying basement membrane. Early
reports had suggested that kalinin/laminin-5 could have cell
adhesion-promoting activity (Rousselle et al., 1991; Carter
et al., 1991), and this was further documented by detailed
in vitro analysis with highly purified preparations of the
protein (Rousselle and Aumailley, 1994).
-chain (Ryan et
al., 1994),
-chain (Gerecke et al.,
1994) and
-chain (Kallunki et al., 1992;
Vailly et al., 1994) have been recently completed, allowing
predictions to be made on the structure of laminin-5. We provide here
experimental evidence supporting these predictions. Amino acid analysis
of laminin-5 showed that the half-cystine content was about two-thirds
that of the laminin-1-nidogen complex (4.1% versus 6.1%). This
difference is in good agreement with the data obtained from sequencing
cDNA clones and confirm that a smaller number of cysteine-rich
epidermal growth factor-like motifs should be present in the
amino-terminal ends of the protein. The circular dichroism spectrum of
laminin-5 indicated that it contains a high content of
-helical
structures, in agreement with the presence of heptad repeats in the
sequences of the
-,
-, and
-chains. The
-helix content was calculated
according to Greenfield and Fasmann(1969) assuming that
[
]
= -11,800 degrees
cm
dmol
and was found to be 26.9%, a
value similar to that reported for native laminin-1 (Ott et
al., 1982; Paulsson et al., 1987). Laminin-5 could be
denatured by raising the temperature up to 90 °C and was partially
renatured by lowering the temperature back to 20 °C. Partial
refolding has also been observed for placenta and heart laminin
isoforms (Lindblom et al., 1994). The transition between
folded and unfolded structures occurred at 72 °C, while laminin-1
has a lower melting temperature of 58 °C (Ott et al.,
1982). Intermediate melting temperatures of 65.0 and 64.2 °C were
recorded for bovine heart and human placenta laminins, respectively
(Lindblom et al., 1994). The
-chain of the former was not
precisely identified, but does not seem to be
or
(Lindblom et al., 1994), while the latter
contains the
-chain associated either with
and
or with
and
(Brown et al., 1994). This suggests that ionic
interactions between the
-,
-, and
-chains of laminin-5 are higher than those present in
other laminin isoforms.
Table:
Amino acid composition of laminin-5 and
comparison with laminin-1
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.