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• W2040372150 abstract "Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093–3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin α1I, α2I, and α10I domains and cell spreading assays with α1β1- and α2β1-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by α5β1 and αVβ1integrins, and it could be blocked by RGD peptides. The collagen α-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three α-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration. Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093–3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin α1I, α2I, and α10I domains and cell spreading assays with α1β1- and α2β1-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by α5β1 and αVβ1integrins, and it could be blocked by RGD peptides. The collagen α-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three α-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration. Chinese hamster ovary phosphate-buffered saline polymerase chain reaction glutathione S-transferase maltose-binding protein The collagens are a family of extracellular matrix proteins (1Prockop D. Kivirikko K. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1378) Google Scholar). Among the 19 different collagen subtypes that have been identified, types XIII and XVII are the only transmembrane proteins (2Hägg P.O. Rehn M. Huhtala P. Väisänen T. Tamminen M. Pihlajaniemi T. J. Biol. Chem. 1998; 273: 15590-15597Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 3Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Type XVII collagen (BP180) is hemidesmosomal transmembrane protein that is mutated in junctional epidermolysis bullosa and is targeted by autoantibodies in blistering skin diseases (4Borradori L. Sonnenberg A. J. Invest. Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 5Bruckner-Tuderman L. Royce P. Steinmann B. Connective Tissue and Its Heritable Disorders: Molecular, Genetic and Medical Aspects. Wiley-Liss Inc., New York2001Google Scholar, 6Schumann H. Baetge J. Tasanen K. Wojnarowska F. Schäcke H. Zillikens D. Bruckner-Tuderman L. Am. J. Pathol. 2000; 156: 685-695Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It occurs in two forms: as a full-length transmembrane protein and as a distinct, soluble ectodomain that is shed from the cell surface by a furin-mediated, proteolytic process (7Schäcke H. Schumann H. Hammami-Hauasli N. Raghunath M. Bruckner- Tuderman L. J. Biol. Chem. 1998; 273: 25937-25943Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8Hirako Y. Usukura J. Uematsu J. Hashimoto T. Kitajima Y. Owaribe K. J. Biol. Chem. 1998; 273: 9711-9717Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This collagen possesses intracellular and transmembrane domains that are of 560 and 23 amino acids in length, respectively. In addition, it has an extracellular domain of 914 amino acids that contains multiple noncollagenous interruptions, dividing it into 15 collagenous subdomains (3Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). The longest of these subdomains, COL15, consists of 242 amino acids (residues 567–808 of collagen XVII). Thus, it is much larger than any of the other collagenous domains, which vary from 14 to 45 residues in length (3Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Most collagen subtypes can be recognized by a group of integrin-type cell adhesion receptors. Although all collagen-receptor integrins share the common β1 integrin subunit, they have unique α subunits resulting in four heterodimers: α1β1, α2β1, α10β1, and α11β1 (9Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar, 10Takada Y. Hemler M.E. J. Cell Biol. 1989; 109: 397-407Crossref PubMed Scopus (252) Google Scholar, 11Camper L. Hellman U. Lundgren-Åkerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 12Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Unlike many other integrins, such as the fibronectin receptor α5β1 and the αV class of integrins, the collagen receptors do not recognize the putative binding motif, arginine-glycine-aspartic acid (RGD). In contrast, they require the triple helical structure of native collagen. Different from other matrix receptor integrins, the collagen receptors have an independently folding protein structure called the I domain (inserted domain) (9Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar, 10Takada Y. Hemler M.E. J. Cell Biol. 1989; 109: 397-407Crossref PubMed Scopus (252) Google Scholar, 11Camper L. Hellman U. Lundgren-Åkerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 12Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). This 200-amino acid structure is located between the second and the third NH2-terminal repeated sequences in the ectodomain of an α subunit. The I domain adopts a structure called the “Rossman fold” configuration in which several β sheets are surrounded by α helices and support a divalent metal-binding site, referred to as the metal ion-dependent adhesion site (13Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (805) Google Scholar). Both the α1I and α2I domains are known to be essential for the primary recognition of the collagens (14Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 15Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar). The published experiments have revealed interesting differences in the recognition of different collagens by α1β1and α2β1 integrins (16Kern A. Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar, 17Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). All collagens tested so far have been recognized by at least one of these two receptors (16Kern A. Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar, 17Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 18Saelman E.U. Nieuwenhuis H.K. Hese K.M. de Groot P.G. Heijnen H.F. Sage E.H. Williams S. McKeown L. Gralnick H.R. Sixma J.J. Blood. 1994; 83: 1244-1250Crossref PubMed Google Scholar, 19Tuckwell D.S. Reid K.B. Barnes M.J. Humphries M.J. Eur. J. Biochem. 1996; 241: 732-739Crossref PubMed Scopus (52) Google Scholar). Still, the cellular receptors for many collagen subtypes remain unidentified. Here, we show that the largest collagenous domain, COL15, in type XVII collagen (20Tasanen K. Eble J.A. Aumailley M. Schumann H. Baetge J Tu H. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2000; 275: 3093-3099Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) is an exception to the established trend. However, human HaCaT keratinocytes can use their RGD-dependent integrins to spread on COL15 and COL15 as a migration substrate. Although COL15 does not contain an RGD motif, it has numerous, highly related KGD sequences, and, as previous studies have shown, snake venom poisons can bind integrins through this sequence (21Scarborough R.M. Rose J.W. Hsu M.A. Phillips D.R. Fried V.A. Campbell A.M. Nannizzi L. Charo I.F. J. Biol. Chem. 1991; 266: 9359-9362Abstract Full Text PDF PubMed Google Scholar, 22Oshikawa K. Terada S.,. J. Biochem. (Tokyo). 1999; 125: 31-35Crossref PubMed Scopus (48) Google Scholar). Human kidney 293-EBNA cells (Invitrogen, Groningen, the Netherlands) constitutively expressing EBNA-1 protein from Epstein-Barr virus were grown in Dulbecco's modified Eagle's/Nutrient mix F-12 medium (Life Technologies, Inc.) containing 10% fetal calf serum (Life Technologies, Inc.) and 0.35 µg/ml G418 (Invitrogen). One million cells/10-cm culture dish were transfected with 25 µg of an expression vector, pCEP-Col15, coding for the amino acids 567–807 of human collagen XVII (for details see Ref. 20Tasanen K. Eble J.A. Aumailley M. Schumann H. Baetge J Tu H. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2000; 275: 3093-3099Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) using the calcium phosphate method. Following a selection with 0.5 µg/ml puromycin (Sigma), the transfected cells were grown to confluency, washed twice with phosphate-buffered saline, and switched to the same serum-free medium containing 50 µg/ml ascorbic acid (Fluka, Deisenhofen, Germany). To maintain sufficient levels of ascorbic acid in the medium, 10 µl of freshly made ascorbic acid stock solution (5 mg/ml) per ml of medium was added every 24 h. The media were collected every 48 h, centrifuged shortly to remove cellular debris, and transferred to ice, and protease inhibitors were added immediately to a final concentration of 1 mm Pefablock (Merck) and 1 mmN-ethylmaleimide (Sigma). The recombinant COL15 was dialyzed against 50 mm Tris, pH 8.6, and contaminating protein was removed by separation on a DEAE-cellulose column (Whatman). COL15 did not bind to DEAE-cellulose, but a significant amount of contaminating protein was removed with this purification step (20Tasanen K. Eble J.A. Aumailley M. Schumann H. Baetge J Tu H. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2000; 275: 3093-3099Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For spreading and binding experiments, COL15 was concentrated using 60% ammonium sulfate precipitation followed by a dialysis against 0.05 m Tris-HCl, pH 8.6, at +4 °C, to remove ammonium sulfate. CHO1 cells obtained from the American Type Culture Collection (Manassas, VA) were used as hosts for transfection and expression of integrin α1 or α2 subunits (17Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Integrin α1 cDNA (9Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar) was a gift from Dr. E. Marcantonio (Columbia University, New York, NY), and α2 cDNA (10Takada Y. Hemler M.E. J. Cell Biol. 1989; 109: 397-407Crossref PubMed Scopus (252) Google Scholar) was a gift from Dr. M. Hemler (Dana-Farber Cancer Research Center, Boston, MA). CHO cells were grown in α-minimum essential medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum, 2 mm glutamine, 100 IU/ml penicillin-G, and 100 µg/ml streptomycin. 20 µg of expression plasmid consisting of α2 cDNA in pAW (23Ohashi P.S. Mak T.W. van den Elsen P. Yanagi Y. Yoshikai Y. Calman A.F. Terhorst C. Stobo J.D. Weiss A. Nature. 1985; 316: 606-609Crossref PubMed Scopus (198) Google Scholar) was used to transfect cells by electroporation (0.3 kV, 960 microfarad, 0.4-cm cuvette in RPMI plus 1 mm sodium pyruvate, 2 mml-glutamine, without serum). Integrin α1cDNA in pLEN (20 µg; Ref. 9Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar) was cotransfected with 1 µg of pAWneo2. Similarly, 20 µg α2/pAW was used. Transfected cells were plated and allowed to recover for 1 day in culture medium. G418 (Life Technologies, Inc.) was added to the medium at a concentration of 1 mg/ml. G418-resistant clones were selected for 1–2 weeks, isolated, and analyzed for their expression of α1 or α2 integrin. The cell surface expression levels of the integrins were checked using anti-integrin antibodies (12F1 for α2 integrin, a gift from Dr. V. Woods, UCSD; SR-84 for α1 integrin, a gift from Dr. W. Rettig, Boehringer Ingelheim) and flow cytometry (17Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). 96-well microtiter plates were precoated by exposure (12 h, 4 °C) to 0.1 ml of PBS containing purified collagens or either native or heat-denatured COL15. COL15 was denatured for 20 min at 56 °C. Residual protein sites on all wells were blocked for 1 h at 37 °C with 0.1% heat-inactivated bovine serum albumin in PBS. Three cell lines were used: a mixture of CHO clones expressing the α1β1 integrin (CHO-α1β1), monoclonal CHO cells expressing the α2β1 integrin (CHO-α2β1), and an immortalized human keratinocyte cell line (HaCaT) that was obtained from Dr. N. E. Fusenic (Deutsches Krebsforschungszentrum, Heidelberg, Germany). Semiconfluent cell cultures were detached with 0.01% trypsin and 0.02% EDTA. Trypsin activity was inhibited by washing the cells with 0.2% soybean trypsin inhibitor. The cells were suspended in serum-free Dulbecco's modified Eagle's medium or serum-free α-minimum essential medium containing 0.1 mg/ml cycloheximide to avoid endogenous matrix synthesis. The cells were added to microtiter plates (104 cells/well) and allowed to attach and spread for 120 min. The role of specific integrin subunits in cell adhesion to COL15 was tested by adding to cell suspensions, function-blocking antibodies (2 µg/ml) against either the β1 (Chemicon), αV (American Type Culture Collection; L230) α2 (Serotec), α3 (Chemicon), or α5 (Chemicon) subunits. In inhibition studies, either linear or cyclic synthetic peptides were added to the cell suspensions. Linear peptides were used at concentrations of 0.01, 0.1, or 0.7 mm. The cyclic RGD peptide used to inhibit integrin function was GACRGDCLGA, which contained a covalent bond between the cysteins. A similar RGE peptide (GACRGECLGA) was used as a nonfunctional control. These peptides were produced as described and were used at 500 µm. After incubation, the medium containing nonadhered cells was poured out; no additional washes were performed. The cells were fixed for 30 min with a solution of 30% formaldehyde and 5% sucrose. The cells in at least three parallel fields in each of three wells were analyzed using phase contrast microscopy. The total number of cells attached and the percentage of spread cells were counted. A spread cell was characterized as one having a clearly visible ring of cytoplasm around the nucleus. A 24-well cell culture cluster (Costar) was precoated for 2 h at 37 °C with 0.5 ml of PBS, pH 7.4, containing human fibronectin (5 µg/cm2;Roche Molecular Biochemicals) or either native or heat denaturated COL15 (5 µg/cm2). Semiconfluent HaCaT cell cultures were detached with 0.01% trypsin and 0.02% EDTA. Trypsin activity was inhibited by washing the cells with 0.2% soybean trypsin inhibitor (Sigma). The cells were suspended in serum-free Optimem 1 medium (Life Technologies, Inc.), and 2 × 104 cells/well were transferred into a custom-made stainless steel cylinder with an opening of 2.8 mm in diameter in the center. The cells were allowed to attach to the substrate for 2 h at 37 °C. The cylinders were removed, nonadhered cells were washed away with Optimem, and the adhered cells were then allowed to migrate in Optimem for 2 or 4 days at 37 °C. The cells were fixed with 2% paraformaldehyde, stained with 5% crystal violet, and washed with distilled water. The rate of migration was estimated by measuring the surface area (mm2) covered by the cells by using NIH Image 1.62 software (National Institute of Health). cDNAs encoding for α1I and α2I domains were generated by PCR as described earlier (17Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) using human integrin α1 and α2 cDNAs as templates. Vectors pGEX-4T-3 and pGEX-2T (both Amersham Pharmacia Biotech) were used to generate recombinant glutathione S-transferase (GST) fusion proteins of human α1I and α2I domains, respectively. Competent Escherichia coli BL21 cells were transformed with the plasmids for protein production. LB medium (500 ml; Biokar) containing 100 g/ml ampicillin was inoculated with 50 ml of overnight culture of either BL21/pα1I or BL21/pα2I, and the cultures were grown at 37 °C until theA600 of the suspension reached 1.0–2.0. An inducer, isopropyl-1-thio-β-d-galactopyranoside, was added, and the cells were allowed to grow for an additional 4–6 h before harvesting by centrifugation. Pelleted cells were resuspended in PBS, pH 7.4, and then lysed by sonication followed by addition of Triton X-100 to a final concentration of 10%. After incubation for 30 min, the suspensions were centrifuged, and the supernatants were pooled. Glutathione-Sepharose (Amersham Pharmacia Biotech) was added to the lysate, which was incubated at room temperature for 30 min with gentle agitation. The lysate was then centrifuged, the supernatant was removed, and glutathione-Sepharose with bound fusion protein was transferred into disposable chromatography columns (Bio-Rad). The columns were washed with PBS, and the fusion proteins were eluted using 30 mm glutathione. Native polyacrylamide gel electrophoresis was done for both recombinant proteins. The recombinant α1I domain produced was 227 amino acids in length, corresponding to sequence 123–338, whereas the α2I domain was 223 amino acids long, which corresponded to sequence 124–339. The carboxyl termini of the α1I and α2I domains contained ten and six nonintegrin amino acids, respectively. Recombinant I domains were used as GST fusion proteins for binding experiments. The α10I domain cDNA was generated by reverse transcriptase-PCR from RNA isolated from KHOS-240 cells (human Caucasian osteosarcoma). Total cellular RNA was isolated by using a RNeasy mini kit (Qiagen). Reverse transcriptase-PCR was done using the Gene Amp PCR kit (PerkinElmer Life Sciences). The amplified α10I domain cDNA was connected to a pMAL-c vector (New England BioLabs). The construct was transformed to the E. coli BL21 strain for production. The DNA sequence of the construct was checked with DNA sequencing. The α10I domain was produced as a fusion with maltose-binding protein (MBP) and tested to show specific binding to several fibril-forming collagens as well as to basement membrane type IV collagen in a Mg2+-dependent manner. 2M. Tulla, O. T. Pentikäinen, T. Viitasalo, J. Käpylä, U. Impola, P. Nykvist, L. Nissinen, M. S. Johnson, and J. Heino, submitted. The coating of a 96-well high binding microtiter plate (Nunc) was done by exposure to 0.1 ml of PBS containing 5 µg/cm2 (15 µg/ml) collagens or either native or heat-denaturated COL15 for 12 h at +4 °C. Blank wells were coated with 0.1 ml of Delfia® Diluent II (Wallac). Residual protein absorption sites on all wells were blocked with 0.1 ml of Delfia® Diluent II (Wallac). Recombinant proteins, α1I-GST, α2I-GST, or α10-MBP were added to the coated wells at a concentration of 10 or 15 µg/ml in Delfia® assay buffer and incubated for 1 h at +37 °C. Anti-GST antibody (Amersham Pharmacia Biotech) at a concentration of 63 ng/ml or anti-MBP antibody (New England BioLabs) diluted in 1:1000 was added to wells and incubated for 1 h at +37 °C. Europium-labeled protein-G (Wallac) was then added, and the mixtures were incubated for 1 h at +37 °C. All of the incubations mentioned above were done in the presence of 2 mmMgCl2. Delfia® enhancement solution (Wallac) was added to each well, and the Europium signal was measured by time-resolved fluorometry (Victor2 multilabel counter, Wallac). At least three parallel wells were analyzed. Wild-type CHO cells show endogenous expression of the β1 integrin subunit, but they do not express any collagen-binding α subunits. Here, cDNAs coding for either the integrin α1 or α2 subunit were transfected into CHO cells. The expression of these exogenous integrin subunits in transfected cells was verified by flow cytometry. In our previous studies the cell spreading assay has turned out to be a more sensitive and reproducible method to study integrin function than the cell attachment assay. CHO-α1β1 and CHO-α2β1 cells were allowed to spread for 120 min on microtiter wells that had been precoated with type I or type IV collagen or the largest collagenous domain of type XVII collagen, COL15. The total number of attached and spread cells were counted. Spread cells were described as those with cytoplasm around the nucleus and with either a flattened, circular shape with a string-of-pearls-like plasma membrane structure or a fibroblast-like morphology. Nonspread cells were characterized by either a round or a splinter-like shape and a yellow shimmer when viewed under a phase contrast microscope. After the 120-min incubation, more of the CHO-α1β1 cells on type IV collagen had spread (62 ± 3%) than those on type I (18 ± 3%; Fig.1A). In contrast, CHO-α2β1 cells spread faster on type I collagen; 88 ± 3% had spread versus 75 ± 6% on type IV collagen. These values are consistent with our previous observations (15Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar). Both α1β1- and α2β1-expressing cells attached and spread on COL15, although the spreading was much slower than on either types I or IV collagen. After incubation only 14 ± 6% of CHO-α1β1 cells and 17 ± 6% of CHO-α2β1 cells had spread on COL15. The spreading of vector-transfected CHO cells was at the same level (15 ± 4%), suggesting that COL15 was not recognized by α1β1 or α2β1, but probably by some other cell adhesion receptor expressed on CHO cells. Recombinant human integrin α1I, α2I, and α10I domains were produced as fusion proteins in E. coli and purified. The fusion proteins were characterized using both SDS- and native polyacrylamide gel electrophoresis (not shown). A solid phase assay was used to investigate the binding of αI domains to type I collagen and native and denatured COL15. Because the collagen-binding activity of integrin I domains has been shown to be Mg2+-dependent, the assays were carried out in the presence of 2 mm MgCl2. Binding levels of each αI domain to bovine serum albumin were used as background controls. The fusion proteins of each αI domain were applied to immobilized COL15, and europium-labeled protein G was linked to the bound αI domain via an anti-GST or anti-MBP antibody. The binding was measured using time-resolved fluorescence as the number of counts/s. Integrin α1I (Fig. 1C), α2I (Fig. 1D), and α10I (Fig. 1E) domains bound to the fibril-forming collagen used as a positive control (type I collagen for α1I and α2I domains and type II collagen for α10I domain). The binding of each domain to either native or denatured COL15 was insignificant. Importantly, this suggests that COL15 is one of the very few collagens that cannot be recognized by the collagen receptors or their corresponding αI domains. Human HaCaT keratinocytes were tested in cell spreading assays on both denatured and native COL15. Spreading on the native COL15 fragment at concentrations of 5 and 20 µg/cm2 was 27 ± 6 and 36 ± 5%, respectively (Fig. 2). Although HaCaT cell spreading was slow on native COL15, it became significantly faster after denaturation. The spreading on denatured COL15 at concentrations 5 and 20 µg/cm2 was 31 ± 6 and 53 ± 2%. Because COL15 has a relatively low melting temperature (26 °C; Ref. 20Tasanen K. Eble J.A. Aumailley M. Schumann H. Baetge J Tu H. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2000; 275: 3093-3099Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), it is probable that at room temperature a portion of native collagen is partially denatured and that only the denatured COL15 mediates cell adhesion. The capability of keratinocytes to migrate on COL15 was tested using coated cell culture wells. Migration was measured in serum-free conditions after 2 or 4 days. Most cells plated on native COL15 detached during the first 2 days, and no migration could be measured. Meanwhile, HaCaT cells stayed attached on denatured COL15, and cell migration during the first 4 days was comparable with that on fibronectin (Fig. 3). In 2 days, HaCaT cells migrated 9.3 ± 1.7 mm2 on denatured COL15 domain. Within 4 days, the extents of migration on denatured COL15 and fibronectin were 10.8 ± 1.8 and 10.3 ± 1.5 mm2, respectively. Thus, denatured COL15 forms an excellent matrix for keratinocyte migration. Similarly to the nonactivated basal keratinocytes in skin, the HaCaT cells express the following integrins: α2β1 collagen receptor and laminin receptors α3β1 and α6β4. However, they also have integrins that can usually be seen only in activated skin keratinocytes: fibronectin receptors α5β1, αVβ1, and αVβ6, and vitronectin receptor αVβ5 (24Koivisto L. Larjava K. Hakkinen L. Uitto V.J. Heino J. Larjava H. Cell Adhes. Commun. 1999; 7: 245-257Crossref PubMed Scopus (57) Google Scholar, 25Zambruno G. Marchisio P.C. Marconi A. Vaschieri C. Melchiori A. Giannetti A. Luca M. J. Cell Biol. 1995; 129: 853-865Crossref PubMed Scopus (313) Google Scholar). Here, antibodies against β1 integrin (5 µg/ml) could inhibit 75% of HaCaT cell spreading on denatured COL15 (Fig.4A). This is in accordance with the previous report showing that anti-β1 antibody can block cell adhesion to both native and denatured COL15 (20Tasanen K. Eble J.A. Aumailley M. Schumann H. Baetge J Tu H. Bruckner P. Bruckner-Tuderman L. J. Biol. Chem. 2000; 275: 3093-3099Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, in our experiments the inhibition was not complete. A cyclic RGD peptide (26Koivunen E. Gay D.A. Ruoslahti E. J. Biol. Chem. 1993; 268: 20205-20210Abstract Full Text PDF PubMed Google Scholar), unlike a control peptide with an RGE sequence, could effectively inhibit cell spreading (Fig. 4B). In this experiment, when no synthetic peptide was present, 30 ± 9% of HaCaT cells spread on denatured COL15. In the presence of the RGE peptide, 41 ± 17% of cells spread. When the RGD peptide was present, spreading decreased to only 12 ± 4% of attached cells, which was equivalent to bovine serum albumin background (Fig.4B). Thus, the data indicate that the main cellular receptors for COL15 are the RGD-dependent β1integrins. In HaCaT cells this suggests the involvement of α5β1 and αVβ1. This was further supported by the fact that anti-αVintegrin antibody could partially (about 40%) inhibit HaCaT cell adhesion to COL15 (Fig. 5A) and that specific antibodies against integrin α2 and α3 subunits had no effect (data not shown). The involvement of α5β1" @default.
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