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• W2002098156 abstract "The basement membrane glycoprotein, entactin, has previously been shown to promote cell attachment and chemotaxis. We have constructed a panel of glutathione S-transferase fusion proteins that encompasses the four major structural domains of entactin, G1, G2, E, and G3. These proteins have been synthesized in bacteria and purified by affinity chromatography. The connecting stalk of entactin, E, which contains four cysteine-rich EGF homology repeats and the integrin receptor RGD recognition sequence, has been modified by deletion of the RGD sequence and substituting glutamic acid for aspartic acid. Attachment assays reveal that the RGD sequence is one of the major cell attachment sites in entactin and that this sequence is recognized by the αvβ3 integrin receptor. Analysis of cell attachment on mutant forms of full-length entactin expressed in the baculovirus expression system revealed a second attachment site that was independent of the RGD sequence. This second site was localized to a peptide of 39 amino acid residues in the second globular G2 domain of entactin. This peptide represents a cysteine-rich EGF repeat. Inhibition of cell attachment by anti-integrin receptor antibodies indicates that the second attachment site is recognized by a member of the β1 family of integrin receptors, possibly α3β1. The basement membrane glycoprotein, entactin, has previously been shown to promote cell attachment and chemotaxis. We have constructed a panel of glutathione S-transferase fusion proteins that encompasses the four major structural domains of entactin, G1, G2, E, and G3. These proteins have been synthesized in bacteria and purified by affinity chromatography. The connecting stalk of entactin, E, which contains four cysteine-rich EGF homology repeats and the integrin receptor RGD recognition sequence, has been modified by deletion of the RGD sequence and substituting glutamic acid for aspartic acid. Attachment assays reveal that the RGD sequence is one of the major cell attachment sites in entactin and that this sequence is recognized by the αvβ3 integrin receptor. Analysis of cell attachment on mutant forms of full-length entactin expressed in the baculovirus expression system revealed a second attachment site that was independent of the RGD sequence. This second site was localized to a peptide of 39 amino acid residues in the second globular G2 domain of entactin. This peptide represents a cysteine-rich EGF repeat. Inhibition of cell attachment by anti-integrin receptor antibodies indicates that the second attachment site is recognized by a member of the β1 family of integrin receptors, possibly α3β1. INTRODUCTIONThe basement membrane is a sheet-like specialized extracellular matrix that is closely associated with epithelial, endothelial, fat, muscle, nerve, and other cells. It is composed of an interacting complex of extracellular molecules that includes collagen IV(1Martin G.R. Timpl R. Kuhn K. Adv. Protein Chem. 1988; 39: 1-50Crossref PubMed Scopus (114) Google Scholar), isoforms of laminin(2Engel J. Biochemistry. 1992; 31: 10643-10651Crossref PubMed Scopus (187) Google Scholar), entactin (nidogen)(3Chung A.E. Dong L.-J. Wu C. Durkin M.E. Kidney Int. 1993; 43: 13-19Abstract Full Text PDF PubMed Scopus (32) Google Scholar), proteoglycans(4Ruoslahti E. Ann. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (548) Google Scholar), fibronectin(5Yamada K.M. Curr. Opin. Cell Biol. 1989; 1: 956-963Crossref PubMed Scopus (97) Google Scholar), tenascin(6Chiquet-Ehrismann R. Mackie E.J. Pearson C.A. Sakakura T. Cell. 1986; 47: 131-139Abstract Full Text PDF PubMed Scopus (766) Google Scholar), and epiligrin(7Carter W.G. Ryan M.C. Gahr P.J. Cell. 1991; 65: 599-610Abstract Full Text PDF PubMed Scopus (668) Google Scholar). The composition of the basement membrane varies with tissue type and undergoes changes during embryonic development as well as in pathological states. Modulation of cell behavior is mediated through the integrin receptors that recognize specific sequences in the constituent molecules of the basement membrane(8Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). These molecules are often recognized by multiple receptors that bind to distinct cognate peptide sequences(9Ruoslahti E. Annu. Rev. Biochem. 1988; 57: 375-413Crossref PubMed Scopus (1051) Google Scholar, 10Mecham R.P. FASEB J. 1991; 5: 2538-2546Crossref PubMed Scopus (205) Google Scholar, 11Yamada K.M. J. Biol. Chem. 1991; 266: 12809-12812Abstract Full Text PDF PubMed Google Scholar). However, in the majority of cases, the specific recognition sequences have not been identified.In this communication we have examined the cell-attachment sites of entactin and the integrin receptors that recognize these sites. Entactin (12Carlin B. Jaffe R. Bender B. Chung A.E. J. Biol. Chem. 1981; 256: 5209-5214Abstract Full Text PDF PubMed Google Scholar) is a 150-kDa sulfated glycoprotein that has previously been shown to promote the attachment of cells(13Chakravarti S. Tam M.F. Chung A.E. J. Biol. Chem. 1990; 265: 10597-10603Abstract Full Text PDF PubMed Google Scholar), chemotaxis of neutrophils(14Senior R.M. Gresham H.D. Griffin G.L. Brown E.J. Chung A.E. J. Clin. Invest. 1992; 90: 2251-2257Crossref PubMed Scopus (106) Google Scholar), and the outgrowth of trophoblasts(15Yelian F.D. Edgeworth N.A. Dong L.-J. Chung A.E. Armant D.R. J. Cell Biol. 1993; 121: 923-929Crossref PubMed Scopus (79) Google Scholar). The molecule consists of three globular domains and a rigid stalk that together generate an asymmetric dumbbell-like structure(16Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Kreig T. Engel J. Chu M.-L. EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (378) Google Scholar). The globular domains (G1 and G2), connected by a thread-like structure and located at the N-terminal portion of the molecule, are connected by the stalk (E), consisting of four EGF1( 1The abbreviations used are: EGFepidermal growth factorMMTmouse mammary tumorPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferasemAbmonoclonal antibody. )-like cysteine-rich homology repeats and a thyroglobulin-like cysteine-rich repeat, to the C-terminal globule (G3)(17Durkin M.E. Chakravarti S. Bartos B.B. Liu S.-H. Friedman R.L. Chung A.E. J. Cell Biol. 1988; 107: 2749-2756Crossref PubMed Scopus (107) Google Scholar). The first EGF repeat in the connecting stalk contains an RGD peptide sequence that has long been established as a ligand for multiple integrin receptors. The RGD sequence in entactin has been implicated in cell binding(13Chakravarti S. Tam M.F. Chung A.E. J. Biol. Chem. 1990; 265: 10597-10603Abstract Full Text PDF PubMed Google Scholar), chemotaxis(14Senior R.M. Gresham H.D. Griffin G.L. Brown E.J. Chung A.E. J. Clin. Invest. 1992; 90: 2251-2257Crossref PubMed Scopus (106) Google Scholar), and outgrowth of trophoblasts(15Yelian F.D. Edgeworth N.A. Dong L.-J. Chung A.E. Armant D.R. J. Cell Biol. 1993; 121: 923-929Crossref PubMed Scopus (79) Google Scholar), The chemotactic response of neutrophils has been shown to be mediated through the leukocyte response integrin and involves the integrin-associated protein(14Senior R.M. Gresham H.D. Griffin G.L. Brown E.J. Chung A.E. J. Clin. Invest. 1992; 90: 2251-2257Crossref PubMed Scopus (106) Google Scholar). However, the cognate integrin for the RGD sequence in other cell types has not been identified. The existence of a second cell recognition site was suggested by Dedhar et al.(18Dedhar S. Jewell K. Rojiani M. Gray V. J. Biol. Chem. 1992; 267: 18908-18914Abstract Full Text PDF PubMed Google Scholar), who reported that entactin was recognized by the integrin receptor α3β1. This integrin receptor, however, does not interact with the RGD sequence, and the location of its binding site on entactin was not determined. We describe in this communication the identification of a potential second cell-binding site on entactin and putative receptors for both the RGD sequence and the newly identified binding site using human melanoma M21 and mouse mammary tumor (MMT) cells.MATERIALS AND METHODSMutagenesisThe RGD sequence of entactin is located at amino acid residues 672-674(17Durkin M.E. Chakravarti S. Bartos B.B. Liu S.-H. Friedman R.L. Chung A.E. J. Cell Biol. 1988; 107: 2749-2756Crossref PubMed Scopus (107) Google Scholar). Two mutations were made within these residues. In the entactinRGE mutant, the single amino acid change was made at residue 674 by replacing aspartic acid with glutamic acid (Asp → Glu). The mutagenic oligonucleotide was 5′-AGT CTG CCC CTC TCC TCG GAA-3′. The entactin▵RGE mutant was made with the oligonucleotide 5′-ATC ATA GCA AGT CTG CCC GAA GCC GAT GGA GCA TTC-3′ in which the nine nucleotides 5′-GTC TCC TCG-3′ complementary to nucleotides 2109-2117 (17Durkin M.E. Chakravarti S. Bartos B.B. Liu S.-H. Friedman R.L. Chung A.E. J. Cell Biol. 1988; 107: 2749-2756Crossref PubMed Scopus (107) Google Scholar) encoding the the RGD tripeptide were omitted, resulting in deletion of the RGD sequence in this clone. The site-directed mutagenesis was performed as described previously(19Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4544) Google Scholar). The Bluescript cDNA clone E663 containing nucleotides 1661-3520 of the entactin sequence was transformed into a dut-ung- Escherichia coli strain CJ236 (a gift from Dr. Duncan Groebe, University of Pittsburgh, Pittsburgh, PA). The transformed CJ236 bacteria were grown in the presence of excess uracil in order to generate a uracil containing single-stranded DNA template. The exponentially growing bacteria were infected with the helper phage M13KO7 (Bio-Rad) at multiplicity of infection 20 to package the single-stranded phagemid DNA. After 1 h of infection, kanamycin was added to the culture medium, and incubation continued overnight. The phage particles were collected by polyethylene glycol/NaCl precipitation(20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, New York1991: 8.0.3-8.2.5Google Scholar). In order to evaluate the level of the uracil incorporation into the single-stranded DNA, the titer of the phage preparation was determined on both CJ236 (dut- -) and JM109 (dut+ ung+) by following previously described procedures(21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar). A plaque ratio greater than or equal to 105-106 indicated adequate incorporation of uracil into the DNA templates(21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar). The mutagenic oligonucleotides were first phosphorylated using polynucleotide kinase (Life Technologies, Inc.) and then annealed to the single-stranded templates over a period of 30 min. For the second strand synthesis, the oligonucleotide annealed to the template was extended with the Klenow fragment of DNA polymerase (Life Technologies, Inc.), and the ends were ligated using T4 DNA ligase. The resultant heteroduplex was subsequently transformed into JM109 (dut+ ung+), and the mutants were screened by DNA sequence analysis using the dideoxy chain termination method(22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52361) Google Scholar).Construction of Recombinant Baculovirus Transfer VectorThe mutant forms of E663 (pE663-M) were sequentially restricted with SphI and HindIII to yield a 3.8-kilobase pair fragment that included the Bluescript vector sequences and a part of the entactin sequence that contained the mutation. This fragment was isolated by electrophoresis on an agarose gel. An SphI-SacI fragment was removed from a previously described full-length cDNA clone of entactin (pEN) (23Tsao T. Hsieh J.-C. Durkin M.E. Wu C. Chakravarti S. Dong L.-J. Lewis M. Chung A.E. J. Biol. Chem. 1990; 265: 5188-5191Abstract Full Text PDF PubMed Google Scholar) and ligated to the 3.8-kilobase pair mutant fragment to generate a new vector (pE663-M-1). An NcoI-NcoI fragment of this new vector, which contained the mutated sequences, was isolated. This fragment was then used to replace the corresponding fragment in the recombinant baculovirus transfer vector pAcEn(23Tsao T. Hsieh J.-C. Durkin M.E. Wu C. Chakravarti S. Dong L.-J. Lewis M. Chung A.E. J. Biol. Chem. 1990; 265: 5188-5191Abstract Full Text PDF PubMed Google Scholar), thereby generating pAcEn/RGD-M clones of pAcEn with modifications in the RGD sequence. Mutations were confirmed by DNA sequencing as the final step for transfer vector construction.Expression of Mutant Entactin by Baculovirus Expression SystemSf9, Spodoptera frugiperda cells (2.5 X 106 were co-transfected with 12 μg of a 1:5 mixture of baculovirus DNA and the pAcEn/RGD-M. The procedures were as described by Tsao et al.(23Tsao T. Hsieh J.-C. Durkin M.E. Wu C. Chakravarti S. Dong L.-J. Lewis M. Chung A.E. J. Biol. Chem. 1990; 265: 5188-5191Abstract Full Text PDF PubMed Google Scholar), except that for the transfection, lipofectin was used instead of calcium phosphate(24Groebe D.R. Chung A.E. Ho C. Nucleic Acids Res. 1990; 18: 4033Crossref PubMed Scopus (46) Google Scholar). After 7 days of culture, the culture media were collected and screened for the recombinant virus. Briefly, Sf9 cells were seeded in 96-well microtiter plates, and serially diluted virus was added to the wells. After 7 days of culture, the media were saved, and cells were lysed and blotted onto nitrocellulose membrane using a dot-blot apparatus(24Groebe D.R. Chung A.E. Ho C. Nucleic Acids Res. 1990; 18: 4033Crossref PubMed Scopus (46) Google Scholar). The blots were then hybridized to a [32P]cDNA entactin probe. The media from positive clones were diluted and used for the next round of screening.Purification of Entactin and Its Mutant Forms from Sf9 CellsSf9 monolayers were infected with culture medium containing recombinant virus and grown for 3 days. Cells were then harvested and rinsed briefly in PBS containing protease inhibitors. Cell pellets were resuspended in 0.25% CHAPS in PBS, vortexed, and incubated on ice for 1 h. The mixture was centrifuged, and the resulting pellet resuspended by vortexing in 6 M urea, 50 mM Tris (pH 7.4), 2 mM EDTA, and 2 mM dithiothreitol plus protease inhibitors. Entactin was extracted from the mixture by stirring overnight at 4°C. The insoluble materials were removed by centrifigation, and the supernatant solution was collected yielding partially purified entactin that was used for some experiment as indicated in the legends. Purified entactin was obtained from the partially purified sample by gel electrophoresis, as described previously(23Tsao T. Hsieh J.-C. Durkin M.E. Wu C. Chakravarti S. Dong L.-J. Lewis M. Chung A.E. J. Biol. Chem. 1990; 265: 5188-5191Abstract Full Text PDF PubMed Google Scholar). Fractions containing the purified entactin were pooled and dialyzed against 1 M urea in 50 mM Tris (pH 7.4) with 2 mM dithiothreitol. The purity of entactin and its mutant forms was confirmed by SDS-PAGE and Western blotting. Each of the three forms of entactin yielded a single Coomassie Blue stained protein band when analyzed by SDS-PAGE. The bands had the expected molecular weights and were stained by anti-entactin antiserum (data not shown).Expression and Purification of Wild-type and Mutant E Domains of Entactin as GST Fusion ProteinsA slightly modified form of the E domain containing residues 639-893 instead of residues 642-889 of entactin is here referred to as the wild-type E domain. The wild-type E domain, in which the RGD site remain unmodified, and two mutant forms with modifications within the RGD sequence were obtained as GST fusion proteins. These fusion proteins are designated GST-E, GST-ERGE, and GST-E▵RGE, which contain, respectively, the wild-type E domain, the E domain in which the RGD sequence was mutated to RGE, and the E domain in which the RGD sequence was deleted. This was accomplished by polymerase chain reaction with the specific oligonucleotide primers 5′-CAGGATCCAGAATCCATGCTACATT-3′ and 5′-CAGAATTCACTGTACTCAGACACGG-3′. cDNA clones pAcEn and pAcEn/RGD-M served as templates. The polymerase chain reaction products were gel-purified and digested with BamHI and EcoRI. The resulting DNA fragments were subcloned into the pGEX3X GST expression vector (25Hsieh J.-C. Wu C. Chung A.E. Biochem. Biophys. Res. Commun. 1994; 199: 1509-1517Crossref PubMed Scopus (15) Google Scholar) at the BamHI and EcoRI polylinker sites. The E. coli host strain DH5α, containing the three different GST-E constructions, were grown at 37°C to an A600 of 0.6-0.8. The expression of proteins was induced with isopropyl-1-thio-β-D-galactopyranoside (0.4 mM) at 37°C for 7 h. The bacteria were then harvested by centrifugation at 4°C and lysed on ice by incubation with lysozyme (1 mg/ml lysozyme buffer consisting of 25 mM Tris (pH 8.0) and 10 mM EDTA) for 30 min in the presence of 1 mM phenylmethylsulfonyl fluoride. The resulting cell debris was further disrupted by a brief sonication on ice. A small volume of ice-cold extraction buffer (10 mM Tris (pH 8.0)) containing 1 mM phenylmethylsulfonyl fluoride was added to the cell lysate, which was subsequently vortexed at 4°C for 5 min and centrifuged at 15,000 rpm in a SW28 rotor for 30 min at 4°C. The supernatant solution was collected and filtered through a 0.45-μm pore size syringe filter unit. The filtrate was then mixed with glutathione-conjugated Sepharose 4B (Pharmacia Biotech Inc.) and incubated at 4°C, with gentle rocking, for 1 h. The beads were then collected by centrifugation and washed 5 times, each with 10 bed volumes of ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride. Proteins bound to the beads were eluted with 10 mM reduced glutathione (Sigma). The resulting eluate was then dialyzed against PBS at 4°C overnight to remove free glutathione. After the protein concentration was determined by the absorbance at 280 nm, the samples were aliquotted and stored at −70°C. The fusion proteins produced were analyzed by SDS-PAGE and Western blot. The three forms of the GST-E fusion proteins displayed, under reducing conditions, single Coomassie-stained bands with the expected molecular weight of 53,000 and were recognized by a polyclonal antibody against entactin (data not shown).Construction and Expression of the Domains of EntactinThe expression and isolation of GST-fusion proteins containing the G1, G2, and G3 domains of entactin were as described previously(25Hsieh J.-C. Wu C. Chung A.E. Biochem. Biophys. Res. Commun. 1994; 199: 1509-1517Crossref PubMed Scopus (15) Google Scholar). These are here designated GST-G1, GST-G2, and GST-G3 to indicate that the domains are fused to GST.Expression of Subdomains of Entactin GST-G2In order to define more narrowly the peptide sequence responsible for the adhesive activity in the G2 domain of entactin, three subfragments of the G2 domain, GST-G2a, GST-G2b, and GST-G2ab were obtained as GST fusion proteins. GST-G2a contained amino residues 301-357 of entactin, which represents a short connecting peptide between G1 and the first EGF repeat of entactin. GST-G2b contained amino acid residues 358-396, a 39-amino acid peptide forming the first EGF repeat. GST-G2ab contained the amino acid residues 301-396. cDNA sequences encoding the above subfragments of the G2 domain were subcloned into the pGEX3X vector by polymerase chain reaction using specific oligonucleotide primers. For clone G2a, the subcloning primers were 5′-CAGGATCCTCTCTCCTGGCTATGAG-3′ (a) and 5′CAGAATTCTGGGAACCT-GTGTTGTA-3′ (b); for G2b, they were 5′-CAGGATCCAGACTTGTGCCAACAAT-3′ (c) and 5′-CAGAATTCACGCACTGTCTGCCATT-3′ (d). Primers a and d were used to generate clone G2ab. The template for the subcloning was pEn(23Tsao T. Hsieh J.-C. Durkin M.E. Wu C. Chakravarti S. Dong L.-J. Lewis M. Chung A.E. J. Biol. Chem. 1990; 265: 5188-5191Abstract Full Text PDF PubMed Google Scholar). Isolation of the polymerase chain reaction products and insertion into the expression vector as well as protein synthesis and purification were as described in a previous section. The DNA sequences of the three constructions were verified by DNA sequencing. The proteins produced were analyzed by SDS-PAGE and Western blotting with a polyclonal antiserum to entactin (data not shown).Cell Adhesion Assay on Polyvinylidine Difluoride MembraneTo ensure that no major contaminating adhesive activities were present in the partially purified recombinant full-length entactin samples, a cell adhesion assay was performed on Immobilon membranes containing the partially purified entactin samples. Briefly, protein samples including unmodified entactin and mutant forms expressed in the baculovirus expression system, which were partially purified, were reduced and denatured in SDS sample buffer and run on a 7.5% SDS-polyacrylamide gel. The resulting protein bands were subsequently blotted onto a sheet of Immobilon membrane(26Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar). The membrane was rinsed in PBS to remove SDS and to allow the proteins to renature. Nonspecific binding sites were blocked by overnight incubation at 4°C in a solution of 3% bovine serum albumin. This membrane was then used as the substrate for cell adhesion. M21 cells (a gift from Dr. David Cheresh, Scripps Research Institute, La Jolla, CA) at 90% confluence were detached by a brief treatment with 5 mM EDTA in PBS. Cells, 5 X 105 in 15 ml of Dulbecco's modified Eagle's medium, were incubated with the membrane at 37°C for 1 h and then washed 5 times, each for 5 min with gentle agitation, with PBS prewarmed to 37°C. Attached cells were fixed with 3% paraformaldehyde in PBS for 5 min. The membrane was washed in PBS and stained with 1% Amido Black. After destaining in 90% methanol, 2% acetic acid, the membrane was air-dried and examined for the pattern of adherent cells under a stereo microscope.Cell Adhesion Assay in Petri DishesIn order to compare the adhesive activities of the various forms and domains of entactin, the concentrations of the recombinant proteins were adjusted to the same molarity as determined from their sizes and optical densities at 280 nm. The proteins were coated onto 35-mm Petri dishes (Fisher) as described previously(13Chakravarti S. Tam M.F. Chung A.E. J. Biol. Chem. 1990; 265: 10597-10603Abstract Full Text PDF PubMed Google Scholar). Briefly, the Petri dishes were precoated with a thin layer of nitrocellulose. Proteins were diluted to the desired concentrations and loaded onto the dishes in a volume of 200 μl. The protein solutions were spread manually over the entire surface of the dishes and allowed to dry at room temperature. The dishes were washed with PBS 3 times and blocked in a 3% bovine serum albumin/PBS solution at 4°C overnight.Cells at 70% confluence were metabolically labeled with [3H]thymidine 24 h before the experiment. Labeled cells at approximately 90% confluence were detached either by 5 mM EDTA (M21 cells) or by trypsinizing (MMT cells, ATCC, Rockville, MD). Approximately 2 X 105 cells were loaded into each dish and then incubated at 37°C for 1-2 h. Unattached cells were removed by rinsing 3-5 times with PBS. Those remaining attached were collected by trypsinizing and quantitated by determination of radioactivity.Anti-integrin AntibodiesAnti-integrin antibodies were the generous gifts of Dr. David Cheresh (anti-αvβ3) and Dr. Elizabeth Wayner (anti-α3, anti-β1, anti-αvβ5, and αvβ3).Inhibition of Cell Attachment by Anti-integrin AntibodiesThe effects of a panel of anti-integrin antibodies on the attachment of cells to entactin and its domains were determined. 3H-Labeled cells were incubated with different mAbs at certain dilutions in Dulbecco's modified Eagle's medium medium at 4°C for 1 h. The treated cells together with the medium containing the mAbs were transfered to protein-coated dishes for further incubation at 37°C to determine the effects of the antibodies on cell attachment, measured as described above.RESULTSAttachment of Human Melanoma M21 Cells to Recombinant Entactin and Its RGD-modified FormsMutant forms of recombinant entactin were tested for their potency in promoting the attachment of human melanoma M21 cells. In these initial qualitative experiments, M21 cells were allowed to bind directly to the proteins separated by SDS-PAGE and blotted unto an Immobilon membrane. Deletion of the RGD sequence (entactin▵RGE) or replacing the aspartate residue with glutamate (entactinRGE) resulted in a marked decrease in cell attachment as shown in the lower panel of Fig. 1(lanes 2 and 3) when compared with the unmodified entactin (lane 1). The corresponding Coomassie-stained bands in the upper panel indicate that a comparable amount of each protein was used for the cell attachment assay. These results indicated that the RGD sequence was a major contributor to the cell attachment activity of entactin. However, the residual attachment to the modified forms of entactin suggested additional attachment sites.Assessment of the Binding of M21 Cells to RGD-modified EntactinThe attachment of M21 cells to entactin and its modified forms was quantitatively assayed. In these experiments the three forms of entactin were purified by preparative electrophoresis and loaded on nitrocellulose-coated dishes at varying concentrations. The results shown in Fig. 2A clearly demonstrate that deletion or modification of the RGD sequence resulted in approximately 50% reduction of cell attachment activity, confirming the earlier results that additional non-RGD-dependent binding site(s) were retained in entactin. Further examination of the cells attached to entactin and its mutant forms revealed that the morphology of the cells was altered by the nature of the substrate (Fig. 3). Cells attached to the unmodified entactin had a typical flattened spindle or polygonal shape (panel A); however, those cells attached to entactinRGE (panel B) or entactin▵RGE (panel C) were rounded but firmly adherent. The results indicate that the attachment sites mediate different cell responses, engagement of the RGD sequence modifies the cytoskeleton to yield a flattened morphology, while the other site(s) elicits a different response.Figure 2:Mutations in the RGD site of entactin do not completely abolish the cell attachment function of the molecule. Purified entactin, entactinRGE, and entactin▵RGE were used to coat 35-mm Petri dishes at concentrations ranging from 0 to 40 μg/dish. M21 cells were allowed to attach to the precoated dishes in serum-free Dulbecco's modified Eagle's medium supplemented with 0.1 mM MnCl2 for 2 h at 37°C. The data (panel A) are expressed as a percent of cells added to each dish that remain attached, and represent means of triplicates from a single experiment. This experiment, however, was done 3 times independently with two different preparations of proteins. The results from each individual experiment were consistent. The gel electrophoretic pattern of entactin (lanes 1 and 4), entactinRGE (lanes 2 and 5), and entactin▵RGE (lanes 3 and 6) in a 7.5% SDS-PAGE are shown in panel B. Lanes 1-3 contain partially purified samples, and lanes 4-6 contain the purified samples. The purification procedures are described in detail under “Materials and Methods.” As shown in the figure, entactin and its modified forms had similar electrophoretic mobility with a molecular mass of approximately 150 kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:The RGD sequence in entactin mediates cell spreading. Phase contrast micrographs of M21 cells attached to entactin (A), entactinRGE (B), entactin▵RGE (C) are shown. M21 cells were allowed to attach to the proteins under the same conditions as in Fig. 2. At the end of incubation, dishes were washed, and cells that remain attached were fixed in 3% paraformaldehyde in PBS and photographed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Localization of the Cell Attachment Sites in EntactinThe cell attachment domains and sites in entactin were further examined by construction of GST fusion proteins containing each of the major domains, G1, G2, E, and G3 of entactin.The E Domain of EntactinThe E domain of entactin, amino acid residues 639-893, contains cysteine-rich EGF repeats 2-5. RGD, a major cell binding sequence in entactin, is located in repeat 2. GST-fusion proteins containing the E domain (GST-E), the E domain in which the RGD sequence was changed by site directed mutagenesis to RGE (GST-ERGE), and the E domain in which the RGD sequence was deleted (GST-E▵RGE) were synthesized in E. coli and purified by affinity chromatography. These fusion proteins were used to determine if the E domain contained cell attachment sites in addition to the RGD sequence. The data presented in Fig. 4 show that GST-E supported cell attachment; however, modification or deletion of the RGD sequence in the E domain in this fusion protein totally abolished the promotion of M21 cell attachment. The level of attachment to these altered fusion proteins was similar to that of the bovine serum albumin and GST controls and was independent of the assay time allowed for cell attachment (panel A) or concentration of f" @default.
• W2002098156 created "2016-06-24" @default.
• W2002098156 creator A5030295784 @default.
• W2002098156 creator A5072721843 @default.
• W2002098156 creator A5083119433 @default.
• W2002098156 date "1995-06-01" @default.
• W2002098156 modified "2023-10-06" @default.
• W2002098156 title "Two Distinct Cell Attachment Sites in Entactin Are Revealed by Amino Acid Substitutions and Deletion of the RGD Sequence in the Cysteine-rich Epidermal Growth Factor Repeat 2" @default.
• W2002098156 cites W1507411141 @default.
• W2002098156 cites W1508378505 @default.
• W2002098156 cites W1531738684 @default.
• W2002098156 cites W1549719435 @default.
• W2002098156 cites W1561391029 @default.
• W2002098156 cites W1569256916 @default.
• W2002098156 cites W1588115842 @default.
• W2002098156 cites W1661683749 @default.
• W2002098156 cites W1964943252 @default.
• W2002098156 cites W1988297300 @default.
• W2002098156 cites W1999021938 @default.
• W2002098156 cites W2005165360 @default.
• W2002098156 cites W2029231027 @default.
• W2002098156 cites W2032523214 @default.
• W2002098156 cites W2036931150 @default.
• W2002098156 cites W2049720130 @default.
• W2002098156 cites W2053007934 @default.
• W2002098156 cites W2053349622 @default.
• W2002098156 cites W2054307558 @default.
• W2002098156 cites W2075965099 @default.
• W2002098156 cites W2101108802 @default.
• W2002098156 cites W2138270253 @default.
• W2002098156 cites W2147504580 @default.
• W2002098156 cites W2161680930 @default.
• W2002098156 cites W2166227856 @default.
• W2002098156 cites W2212959237 @default.
• W2002098156 cites W2888511473 @default.
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