The Basement Membrane Glycoprotein Entactin Promotes Cell Attachment and Binds Calcium Ions*

Mouse entactin derived from the extracellular matrix of M1536-B3 cells and from insect cells infected with a recombinant virus containing entactin sequences were shown to promote the attachment of mouse mammary tumor, human melanoma, and other cells. The cell attachment was inhibited by antibodies against mouse entactin but not by anti-fibronectin or anti-laminin antibodies. On a weight basis entactin was as effective as laminin in promoting the attachment of mouse mammary tumor cells. The attachment of cells to entactin was in part mediated by the integrin recognition RGD peptide sequence. This was demonstrated by the cell attachment properties of peptides derived from entactin which contained this sequence. Furthermore, the peptide RGDS could inhibit the attachment of mouse mammary tumor cells to entactin to approximately 60% of control. It is suggested that additional cell recognition sequences may be present in entactin. The direct binding of calcium ions to entactin was observed. It is probable that the binding sites reside in peptide sequences located toward the NH2 terminus region of entactin. This conclusion was supported by the demonstration that synthetic peptides, containing potential calcium binding sequences derived from entactin, bound calcium. In addition, a recombinant peptide containing the amino-terminal 330 amino acids of entactin also bound calcium ions. The significance of these properties of entactin is discussed.

The structure and biological functions of the basement membrane have been reviewed in detail in the past several years (1, 2). The basement membrane is a continuous sheet of extracellular matrix that underlies and makes intimate contacts with epithelial and endothelial cells, as well as with muscle, fat, and neural tissues. Information to the cells is transferred through these contacts via membrane receptors specific for one or more of its molecular components, which include type IV collagen (3), laminin (4, 5), entactin (6-8), and heparan sulfate proteoglycans (9-11). The organization, properties, and interactions of the molecular constituents are compatible with the biological function of a particular basement membrane. Rapid and significant advances have been made in understanding basement membrane function by * This research was supported by Grants CA 21246 and GM 25690 from the National Institutes of Health and by the National Science Council, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC. Section 1734 solely to indicate this fact.
Yl To whom correspondence should be addressed.  analysis of the biological properties of each component, and in particular studies on laminin have been revealing and rewarding (12)(13)(14)(15)(16)(17). On the other hand, very little is known about the functions of entactin and its role in the assembly and behavior of basement membranes. Entactin, a 150-kDa sulfated glycoprotein, was first discovered in the extracellular matrix synthesized by the mouse cell line M1536-B3 (18). A similar protein was shown to be present in mouse Reichert's membrane (19). Several years later a degraded form obtained from Englebreth-Holm-Swarm (EHS)' tumors was mistakenly identified as a new basement membrane protein and hence named nidogen (20). The primary structures of mouse (21, 22) and human (23) entactin have been determined by a combination of amino acid and cDNA sequencing. The 85% sequence identity between the mouse and human molecules and the virtual identity of the partial rat sequence (24) with that of the corresponding segment from mouse indicate that the molecular structure is highly conserved. Entactin forms a tight stoichiometric complex with laminin, and images from rotary-shadowed specimens have revealed that the dumbbellshaped entactin binds via one of its globular termini to a short arm of the cruciform laminin in the proximity of the intersection of the arms. It has been suggested that this binding occurs through the carboxyl end of entactin (25,43).
The predicted secondary structure of entactin which is consistent with the physical image obtained by electron microscopy has several additional interesting features. The NH,terminal 639 amino acid residues which are predicted to fold into a 70-kDa globular domain are separated from the car-boxy1 terminus globular domain by a cysteine-rich region which provides the rodlike connection (21). Most of the cysteines in the rodlike domain are organized into an EGFlike repeat pattern (-40 residues long). The amino terminus segment of the molecule (residues 15-26 and 250-261) and two of the EGF-like repeat units contain potential calcium binding sequences (21). The central domain also contains one copy of the Arg-Gly-Asp (residues 672-674) integrin recognition sequence in one of the several EGF-like cysteine-rich homology repeats.
The biological function of entactin has been an enigma. Several lines of evidence suggest, however, that it is important in the organization and normal function of basement membranes. Indirect immunostaining of tissues with antisera raised against entactin derived either from M1536-B3 or EHS tumor extracellular matrix has shown that the molecule appears early in embryogenesis and persists through adulthood (26)(27)(28)(29) In this paper the biological properties of entactin have been investigated with both an insect cell-derived recombinant molecule and that derived from mouse cells. Particular emphasis was placed on the cell and calcium binding potentials of the molecule. In addition, synthetic peptides from entactinderived sequences have been shown to mimic these biological properties and to suggest the assignment of distinct functional domains in the molecule. was not due to contamination by small amounts of laminin, samples of entactin purified (Fig. lA) as described above were immunostained with polyclonal anti-laminin antiserum. The results shown in Fig. 1B demonstrate that this was indeed the case. The anti-laminin antiserum failed to detect any laminin in the entactin preparation (Fig. lB, lane 5). Most importantly, the anti-entactin antiserum did not show any crossreactivity with laminin (Fig. lB, lanes 6 and 7). This antientactin antiserum was used in all the cell adhesion assays described in the following paragraphs. The recombinant entactin isolated from infected Sf9 cells, as expected, was also free of laminin as shown in Fig. 1C (lane 8). Lanes 5-7 and lanes 8 and 9 were immunostained with anti-entactin and anti-laminin antisera, respectively. Lane I, molecular mass standards: 8-galactosydase (116 kDa), phosphorylase b (97.4 kDa), bovine albumin (66 kDa); lanes 2, 7, and 9, M1536-B3 ECM; lanes 3 and 5, SKI cell extract; lanes 4, 6, and 8, partially purified entactin from 14-6/Sf9 cell extract.

Inhibition
supported the attachment of MMT and M21 cells (not shown here). In further quantitative studies with the MMT cell line it was shown to promote cell attachment in a dose-and timedependent manner (Fig. 2, A and B). In these experiments the data were corrected for the background attachment to dishes coated with bovine serum albumin alone. This background was generally in the range of 2-10% of the maximum of the matrix-stimulated attachment. The inhibition of cell attachment to M1536-B3 ECM by antisera to either laminin or entactin (Fig. 3) suggested that both molecules were involved. It is noteworthy that the polyclonal antibodies to either the A or B chains of laminin inhibited cell attachment in contrast to the monoclonal antibodies Lam I (37) and Lam V (38). Two separate preparations of anti-entactin antibodies inhibited cell attachment by approximately 40%. To resolve the contribution of each component to cell attachment, experiments were carried out with purified laminin and entactin preparations.
Attachment of MMT Cells to Laminin and Entactin-As shown in Fig. 4, either laminin or entactin separately enhanced the attachment of MMT cells in a dose-dependent manner. These results demonstrate that at similar quantities, with this particular cell line, the two molecules were equally effective. A comparison with the intact matrix (data not shown) indicated that it was an order of magnitude more effective in promoting cell adhesion than either laminin or entactin. The specificity of cell attachment was further supported by the effects of anti-entactin antiserum on attachment to entactin of either MMT (Fig. 5)  nectin antiserum decreased cell attachment.
The attachment of MMT cells was inhibited to greater than 60% by antientactin antibodies, and the attachment of M21 cells was completely inhibited.
In some instances cell attachment was increased in the presence of anti-laminin or anti-fibronectin antisera. In another approach, direct cell attachment assays were performed with transfer blots of SDS-polyacrylamide gels containing ECM (not shown here) or 14-6/Sf9 cell extracts. Fig. 6 shows the attachment of MMT cells to the recombinant entactin band from 14-6/Sf9 cell extracts (lanes 6 and 7). In the same assay uninfected Sf9 cell extracts did not promote cell attachment (lane 5). These results clearly support the cell attachment potential of entactin.

The RGD Sequence in Entactin Supports Cell Attachment-
The amino acid sequence of entactin contains the integrin recognition RGD sequence. It is known that the cell attachment properties of this peptide sequence depend on its context within a protein. In order to determine if the RGD sequence of entactin was responsible for its cell adhesive properties, synthetic peptides containing this and flanking amino acids derived from the sequence of entactin were analyzed for their cell attachment properties.
Both peptides El and E5 were able to enhance cell attachment (Fig. 7, A and B); the longer peptide E5 was 2-3 times more effective on a molar basis (Fig.  7B). The functional cell attachment sequence in these peptides appeared to be RGD since at a concentration of 20 rg/ ml and above the peptide RGDS could completely inhibit cell attachment (Fig. 7C). Our experiments further show that cell attachment to intact entactin was partially mediated by RGD where 60% inhibition of attachment was observed at concentrations of RGDS above 30 pg/ml (Fig. 8). Cell attachment could not be completely blocked by the higher concentrations of peptide which suggests that other sequences may be involved. Phase contrast micrographs of MMT cells attached to peptides El and E5 and intact entactin are shown in Fig.  9.
Calcium Binding to Entuctin-The primary structure of entactin has revealed the presence of several potential calcium binding sequences, of which two reside in the amino-terminal globular domain. As shown in Fig. lOA, peptide E3, representing the first putative Ca*+ binding sequence, binds 45Ca2+, and somewhat unexpectedly, the two RGD-containing pep-1 23 4 56 7 FIG. 6. Attachment of MMT cells to transblots of M1536-B3 ECM and 14-6/Sf6 cell extracts. MMT cells were incubated with transfer blots df 14-6/W cell extracts resolved by SDS-polyacrylamide gel electrophoresis for 90 min at 37 "C as described in the text. Lanes 1 and 2 were stained with Amido Black. Lanes 3 and 4 were immunostained with the polyclonal anti-entactin antiserum. Lanes 5-7 were incubated with cells. Lanes 1, 3, and 5, uninfected St9 cell extract; lanes 2, 4, and 6, 14-6/W cell extract; lane 7, recombinant entactin band excised from 14-6/W cell extract resolved by SDS-polyacrylamide gel electrophoresis and re-electrophoresed. ['H]Thymidine-labeled MMT cells were plated on Petri dishes coated with the synthetic peptides El (SIGFRGDGQTC) as shown in A or E5 (CYIG-THGCDSNAACRPGPGTQFTCECSIGFRGDGQT) as shown in B. Attachment levels are presented as a percent of average radioactivity added per plate. Each data point represents the mean *SD. of eight observations from 4-day experiments for El and four observations from 2-day experiments for E5. Effects of the RGDS peptide on cell attachment to El and E5 are shown in C. MMT cells were added to Petri dishes coated with 1 ml of 10 @g/ml El or E5. Increasing amounts of the RGDS peptide dissolved in DME medium were added to the Petri dishes to final concentrations in the range of 20-200 rg/ ml, and cell adhesion was assayed as before. The results represent the mean (*SD.) of four observations. Amino acid sequences of the RGD-containing synthetic peptides El and E5 are described in the legend to Fig. 8. Cell attachment assay was performed as for Fig. 2. The results of MMT cells plated on Petri dishes coated with BSA (A), 20 pg/plate El (B) or E5 (C), and 15 pg/plate recombinant entactin (D) are shown here. Rar represents 100 pm.
It is noteworthy that the laminin B subunits but not the A subunits bound "Va"' (Fig. lOB, lane 4). The recombinant baculovirus 2-2 which produces a peptide of molecular mass of -34,500 kDa corresponding to the first 300 amino acids of entactin bound '%a*' (Fig. 1OC). This fragment included both putative calcium binding sequences from the NHa-terminal globular domain of entactin.

DISCUSSION
The recent publication of the primary structure of entactin (21) has enabled us to examine more closely two of its biological properties. The presence of the integrin recognition RGD sequence in a cysteine-rich EGF precursor homology repeat and potential calcium binding sequences in the amino-terminal segment of the molecule provided clues to potential functions of the molecule. In addition, the availability of a recombinant form of entactin, free of laminin, in substantial amounts allowed us to test for the presence of these properties.
The first suggestion that entactin could enhance cell attachment was provided by the partial inhibition of the attachment of mouse mammary tumor cells to the extracellular matrix of the endodermal cell line M1536-B3 by antibodies against entactin. The matrix is composed predominantly of a complex of laminin and entactin. The partial inhibition of cell attachment could have been the result of direct blocking of access to cell membrane receptors for entactin or by indirect steric effects on attachment to other components of the matrix. It was necessary, therefore, to examine the direct attachment of cells to entactin. It was found that entactin derived from either the matrix, representing the natural species, or from the recombinant baculovirus-infected insect cells by itself promoted cell adhesion. The specificity was supported by the observation that cell attachment was inhibited by antientactin antiserum but not by antibodies against either fibronectin or laminin. The direct attachment of cells to entactin in transfer blots of M1536-B3 ECM and 14-6/Sf9 cell extract resolved by SDS-polyacrylamide gel electrophoresis provided additional evidence supporting the cell adhesive potential of entactin. Although MMT cells were studied most extensively, other cells such as human melanoma and ra.s-transformed 3T3 cells could utilize entactin as a substrate for attachment.  Fig. 7B) were applied on nitrocellulose filter paper. The dried filters were incubated with ""CaCly as described under "Materials and Methods," and bound ""CaCL was detected bv autoradioeraohv. i?. M1536-B3 ECM and 14-6/SF-9 cell extracts resolved by SDS-pilyacrylamide gel electrophoresis and transferred to Immobilon filters were assayed for the binding of "Ca" as in A. Lanes l-3 were stained with 0.1% Amido Black, and lanes 4 and 5 were incubated with "Car+. Lane 1, high molecular mass standards; lanes 2 and 5, 14-6/SF-9 cell extract; l&es 3 and 4, ECM. C. bindine of ""CaC1.l bv 14-6/SF-9 and 2-2/SF-9 cell extracts. Lanes l-3 werestained with b.l% kmido Black, and lanes 4-6 were incubated with "Ca". Lane 1, molecular mass standards; lanes 2 and 6, 2-2/SF-9 cell extracts; arrow, recombinant protein corresponding to NHs-terminal 300 amino acids of entactin; lanes 3 and 5, 14-6/SF-9 cell extract; lane 4, wild-type baculovirus-infected SF-9 cell extract. This property of entactin may have interesting implications. Several functional peptide sequences have been identified on the short arms of laminin; these include both cell binding and sites of interaction with collagen type IV (39-41) and heparin or heparan sulfate proteoglycan (42). Since entactin has been shown to bind to one of the short arms of laminin (43), its synthesis and interaction with laminin during development could profoundly influence cell-matrix interactions and consequently gene expression, cell motility, and organogenesis.
The expression of the gene for entactin appears not to be coordinated with that of the laminin chains during mouse embryonic development which suggests that its expression may be temporally linked to the dynamic requirements of the changing embryonic cell population for different types of substrata. The short arm of laminin that is derived from the A chain contains the integrin recognition RGD sequence (16) while the other arms contain sequences that recognize other cell surface receptors (40,44). It is not unlikely that the juxtaposition of two integrin recognition sequences in the laminin-entactin complex could allow them to act in a synergistic manner for cell attachment.
Our results have shown that the RGD sequence in entactin can indeed serve as a recognition signal for cell attachment. Not only can the peptide RGDS inhibit cell attachment to entactin, but entactin sequences which include RGD can support cell attachment. The results further suggest that entactin is recognized by one of the members of the integrin receptor family; however, the nature of this putative receptor must be explored in detail. The observation that RGDS was unable to completely block cell attachment to entactin raises the possibility that entactin may contain additional cell recognition sequences.
The calcium binding properties of entactin were demonstrated with the intact molecule as well as peptide sequences derived from its NH2 terminus region. Previous work has shown that the laminin-entactin complex undergoes polymerization in response to calcium ions (45, 46). There is some evidence which suggests that the globular domain of entactin at the carboxyl end of the molecule binds to laminin (25,43), thus leaving the NH, terminus region free to interact with other molecules. Furthermore, the isolation of the extracellular matrix from M1536-B3 cells in intact form requires the presence of calcium ions, and the extraction of soluble forms of matrix from EHS tumors is facilitated by EDTA (4, 43). These observations taken together clearly suggest that calcium ions may play a key role in the configuration of the extracellular matrix. The free calcium binding amino terminus segment of entactin in the laminin-entactin complex could form a special type of bridge between adjacent complexes. Extracellularly, calcium ion concentrations could regulate such entactin-mediated cross-linking of preformed complexes. Additionally, this region could provide binding sites in the extracellular matrix for other calcium binding molecules or even serve as a reservoir for calcium ions in tissues. It is well documented that entactin is exquisitely sensitive to proteases and that the protease-sensitive sites reside both in the carboxyl-and amino-terminal segments of the molecule (8, 47). The proposed bridging function of entactin in the extracellular matrix would provide a protease-sensitive site for breaching the basement membrane in such processes as metastasis and tissue reorganization.
It is to be noted that a characteristic feature of metastatic cells is their potential to synthesize metal ion-dependent proteases (48). Interestingly, the RGD sequence containing peptides also displayed calcium binding activities in our assays. The entactin RGD sequence is located in one of the EGF-type repeats. Some of the proteins of the coagulation and complement pathways containing such repeat structures have been shown to bind Ca'+, and this is believed to be associated with the presence of @-hydroxylated aspartate and asparagine residues in the EGF-like domains (49). The RGD-containing EGFlike repeat of entactin does not contain the proposed consensus sequence for P-hydroxylation Ca'+ binding. Furthermore, whether this segment or any of the EGF-like repeats actually bind Ca'+ must be demonstrated.
However, an earlier study on the ECM protein thrombospondin reported the binding of calcium by its RGD sequences (50). Whether the RGD sequence of entactin indeed binds calcium with possible effects on its cell binding property remains to be studied. A calciumrelated overall conformational transition of the RGD-containing rodlike domain could affect the cell binding property of entactin. Alternatively, binding of calcium at the RGD site could be involved in stabilizing the RGD site in a cell surface receptor compatible conformation as suggested for thrombos-
The results described represent an attempt to define the 24. biological functions of entactin. The cell attachment and calcium binding functions provide the basis for further exploration of specific cell surface receptors and the role of calcium 25. in the organization and function of basement membranes. 26.