©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Requirement for Cell Adhesion to Kalinin (Laminin-5) (*)

Patricia Rousselle (1), Ralph Golbik (2), Michel van der Rest (1), Monique Aumailley (1)(§)

From the (1) Institut de Biologie et Chimie des Protéines, CNRS UPR 412, 7, Passage du Vercors, 69367 Lyon Cedex 07, France and the (2) Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -, -, 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.


INTRODUCTION

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, , , 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).

Sequencing of cDNA clones corresponding to the -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).

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.


MATERIALS AND METHODS

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).


RESULTS

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).


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.

Laminin-5 showed a circular dichroism spectrum with a minimum in negative ellipticity between 215 and 220 nm that indicated a high -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.




DISCUSSION

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 -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).

Cloning and sequencing of the cDNA corresponding to the -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.

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.

  
Table: Amino acid composition of laminin-5 and comparison with laminin-1



FOOTNOTES

*
This work was supported in part by a grant from the Association pour la Recherche sur le Cancer (to P. R.). 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.: 33-72-72-26-35; Fax: 33-72-72-26-02.

The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


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