Identification of Motifs for Cell Adhesion within the Repeated Domains of Transforming Growth Factor-β-induced Gene,βig-h3
2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês
10.1074/jbc.m002752200
ISSN1083-351X
AutoresJung‐Eun Kim, Song Ja Kim, Byung-Heon Lee, Rang‐Woon Park, Ki-San Kim, In-San Kim,
Tópico(s)NF-κB Signaling Pathways
Resumoβig-h3 is a transforming growth factor-β-inducible cell adhesion molecule that has four characteristic homologous repeated domains. We made recombinant βig-h3 proteins, which were highly active in mediating human corneal epithelial (HCE) cell adhesion and spreading. The 2nd and the 4th repeated domains were sufficient to mediate HCE cell adhesion. A sequence analysis showed that aspartic acid (Asp) and isoleucine (Ile) of the 2nd and the 4th domains are highly conserved in many fasciclin 1 homologous (fas-1) domains. Substitution mutational study identified these two amino acids are essential for cell adhesion. Synthetic peptides containing Asp and Ile, NKDIL and EPDIM derived from the 2nd and the 4th domains, respectively, almost completely blocked cell adhesion mediated by not only wild type βig-h3 but also each of the 2nd and the 4th domains. These peptides alone were fully active in mediating cell adhesion. In addition, we demonstrated the functional receptor for βig-h3 is α3β1integrin. These results, therefore, establish the essential motifs within the 2nd and the 4th domains of βig-h3, which interact with α3β1 integrin to mediate HCE cell adhesion to βig-h3 and suggest that other proteins containing Asp-Ile in their fas-1 domains could possibly function as cell adhesion molecules. βig-h3 is a transforming growth factor-β-inducible cell adhesion molecule that has four characteristic homologous repeated domains. We made recombinant βig-h3 proteins, which were highly active in mediating human corneal epithelial (HCE) cell adhesion and spreading. The 2nd and the 4th repeated domains were sufficient to mediate HCE cell adhesion. A sequence analysis showed that aspartic acid (Asp) and isoleucine (Ile) of the 2nd and the 4th domains are highly conserved in many fasciclin 1 homologous (fas-1) domains. Substitution mutational study identified these two amino acids are essential for cell adhesion. Synthetic peptides containing Asp and Ile, NKDIL and EPDIM derived from the 2nd and the 4th domains, respectively, almost completely blocked cell adhesion mediated by not only wild type βig-h3 but also each of the 2nd and the 4th domains. These peptides alone were fully active in mediating cell adhesion. In addition, we demonstrated the functional receptor for βig-h3 is α3β1integrin. These results, therefore, establish the essential motifs within the 2nd and the 4th domains of βig-h3, which interact with α3β1 integrin to mediate HCE cell adhesion to βig-h3 and suggest that other proteins containing Asp-Ile in their fas-1 domains could possibly function as cell adhesion molecules. fasciclin 1 homologous domain human corneal epithelium plasma fibronectin bovine serum albumin Hepes-buffered saline polyacrylamide gel electrophoresis wild type phosphate-buffered saline βig-h3 is an extracellular matrix protein that can be induced by transforming growth factor-β in several cell types, including human melanoma cells, mammary epithelial cells, keratinocytes, and lung fibroblasts (1Skonier J. Benenett K. Rothwell V. Kosowski S. Plowman G. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (261) Google Scholar). Several studies suggest βig-h3 is involved in cell growth (1Skonier J. Benenett K. Rothwell V. Kosowski S. Plowman G. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (261) Google Scholar), cell differentiation (2Dieudonne S.C. Kerr K.M. Xu T. Sommer B. DeRubeis A.R. Kuznetsov S.A. Kim I.-S. Robey P.G. Young M.F. J. Cell. Biochem. 1999; 76: 231-243Crossref PubMed Scopus (61) Google Scholar, 3Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (111) Google Scholar), wound healing (4Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. & Visual Sci. 1997; 38: 893-900PubMed Google Scholar), and cell adhesion (5LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavele R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (202) Google Scholar, 6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar), although the underlying mechanisms for these effects are still unclear. In addition, some βig-h3 missense mutations were identified in families affected with human autosomal dominant corneal dystrophies (7Munier F.L. Korvatska E. Djemai S. LePaslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Crossref PubMed Scopus (533) Google Scholar). Yet the exact role of mutant βig-h3 proteins in developing corneal dystrophies is unidentified. βig-h3 contains an RGD motif and four internal repeated domains, which have highly conserved sequences found in some secretory and membrane proteins of several species including mammals, insects, sea urchins, plants, yeast, and bacteria (8Kawamoto T. Noshiro M. Shen M. Nakamasu K. Hashimoto K. Kawashima-Ohya Y. Gotoh O. Kato Y. Biochim. Biophys. Acta. 1998; 1395: 288-292Crossref PubMed Scopus (105) Google Scholar). These proteins include periostin, fasciclin I, sea urchin HLC-2, alga Algal-CAM, and mycobacterium MPB70. The homologous domain (designated fas-1)1 of these proteins is 110–140 amino acids long and is characterized by two highly conserved stretches of about 10 amino acids (H1 and H2). Some proteins including βig-h3, periostin, and fasciclin I have four sets of fas-1, HLC-2 has two sets, and MPB70 contains only one set. Although their biological functions are poorly characterized, some of them have been reported to function as cell adhesion molecules. βig-h3, periostin, and fasciclin I have been reported to mediate attachment of fibroblasts (5LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavele R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (202) Google Scholar), osteoblasts (9Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. J. Bone Miner. Res. 1999; 14: 1239-1249Crossref PubMed Scopus (824) Google Scholar), and neuronal cells (10Wang W.-C. Zinn K. Bjorkaman P.J. J. Biol. Chem. 1993; 268: 1448-1455Abstract Full Text PDF PubMed Google Scholar), respectively. Algal-CAM is also known to be a cell adhesion molecule in embryos of the alga Volvox (11Huber O. Sumper M. EMBO J. 1994; 13: 4212-4222Crossref PubMed Scopus (120) Google Scholar). According to a recent report (6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar), βig-h3 enhanced the spreading of fibroblasts via integrin α1β1, and its RGD motif was not necessary for mediating cell spreading. They also reported that the conserved H1 and H2 peptides were not sufficient to affect βig-h3-mediated cell adhesion. This suggests that the essential amino acids for the cell adhesion activity of βig-h3 may exist elsewhere rather than H1 and H2 regions. A computer search based on homologies not only among the repeated fas-1 domains of βig-h3 but also among fas-1 domains of other proteins revealed that there are a few highly conserved amino acids in addition to H1 and H2. Particularly, we focused on aspartic acid near H2 because aspartic acid is known to be an essential amino acid for interaction with several integrins (12Ebel J.A. Kuhn K. Eble J.A. Molecular Biology Intelligence Unit: Integrin-Ligand Interaction. Springer-Verlag, Heidelberg, Germany1997: 1-40Google Scholar). Based on the above findings, we hypothesized that a highly conserved sequence including aspartic acid may support βig-h3-mediated cell adhesion. To prove our hypothesis, we have generated wild type and several mutant recombinant βig-h3 proteins. By using these proteins, we found that βig-h3 mediates human corneal epithelial (HCE) cell adhesion through α3β1integrin and that each of the 2nd fas-1 domain and the 4th fas-1 domain of βig-h3 is sufficient to mediate cell adhesion via α3β1 integrin. Then, we demonstrated that two conserved amino acids, Asp and Ile, near H2 in both repeated domains are essential and that synthetic peptides including Asp and Ile from the 2nd and the 4th domains are sufficient to mediate cell adhesion via α3β1 integrin. These results, therefore, establish the essential amino acid residues within the fas-1 domains of βig-h3, which interact with α3β1 integrin to support βig-h3-mediated HCE cell adhesion. Expression plasmids for recombinant βig-h3 proteins, βigh3-wild type (WT), and βigh3-ΔRGD were described in the previous report (3Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (111) Google Scholar) where βigh3-WT and βigh3-ΔRGD were named Hisβ-c and Hisβ-d, respectively. Each fragment of βig-h3 cDNA, encoding amino acids 129–241, 237–377, 368–506, and 498–637, was generated by polymerase chain reaction and cloned into the EcoRV and XhoI sites of pET-29b, named βigh3 D-I, D-II, D-III, and D-IV, respectively. Substitution mutants of βigh3 D-IV were generated by a two-step polymerase chain reaction as described previously (13Kim I.-S. Sinha S. deCrombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 4003-4013Crossref PubMed Scopus (129) Google Scholar). The DNA sequences of all mutants were verified. Recombinant βig-h3 proteins were induced and purified as described previously (3Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (111) Google Scholar). HCE cells were cultured in Dulbecco's modified Eagle's medium with Nutrient Mixture F-12 (Life Technologies, Inc.) supplemented with 15% fetal bovine serum, gentamicin (40 μg/ml), 5 μg/ml insulin, 0.1 μg/ml cholera toxin, and 10 ng/ml human epidermal growth factor at 37 °C in 5% CO2. The human erythroleukemic cell line, K562, was grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. The cell adhesion assay was performed as described previously (14Landegren U. J. Immunol. Methods. 1984; 67: 379-388Crossref PubMed Scopus (628) Google Scholar). Briefly, 96-well microculture plates (Falcon, Becton-Dickinson, Mountain View, CA) were incubated with recombinant βig-h3 proteins or other extracellular matrix proteins at 37 °C for 1 h and then blocked with PBS containing 0.2% BSA for 1 h at 37 °C. The coated extracellular matrix proteins used were as follows: human plasma vitronectin (Promega), purified human plasma fibronectin (pFN), chicken collagen types I and II (Chemicon International Inc., Temecula, CA), bovine collagen types IV and VI (Chemicon), mouse laminin (Chemicon), and bovine serum albumin (BSA) (Sigma). Cells were trypsinized and suspended in the culture media at a density of 2 × 105 cells/ml, and 0.1 ml of the cell suspension was then added to each well of the plates. Cell attachment was analyzed as follows. After incubation for 1 h at 37 °C, unattached cells were removed by rinsing twice with PBS. Attached cells were incubated for 1 h at 37 °C in 50 mm citrate buffer, pH 5.0, containing 3.75 mm p-nitrophenyl-N-acetyl β-d-glucosaminide (hexosaminidase substrate) and 0.25% Triton X-100. Enzyme activity was blocked by the addition of 50 mm glycine buffer, pH 10.4, containing 5 mmEDTA, and the absorbance was measured at 405 nm in a Multiscan MCC/340 microplate reader (Titertek Instruments, Inc., Huntsville, AL). To determine cell area, 4×104 cells were applied to substrates in 48-well culture plates. The attached cells were fixed with 8% glutaraldehyde (Sigma) and then stained with 0.25% Crystal Violet (Sigma) in 20% methanol (w/v). Cell area was measured using Image-Pro plus software (Media Cybernetics, Silver Spring, MD). Experiments were repeated in triplicate with 200 or 300 measurements per site for each experiment. Data are reported as the mean area at specific time points ± S.E. Various reagents and synthetic peptides were examined for their ability to prevent cells from adhering to the prepared substrata. Synthetic peptides were synthesized on an automated multiple peptide synthesizer (PE/ABD 433, PE Corp., Norwalk, CT) using standard solid phase procedures. Peptides were purified by reverse phase high performance liquid chromatography. Cell adhesion assay was done as described above. To analyze the divalent cation sensitivity of βig-h3-mediated adhesion, cells were suspended at 2 × 105 cells/ml in Hepes-buffered saline (HBS), 150 mm NaCl, 25 mm Hepes, pH 7.4, and incubated at 37 °C for 30 min. They were then washed twice in HBS and resuspended in the same buffer. Aliquots of cells (50 μl) were then added to the microculture plate wells and incubated with 50-μl aliquots of HBS containing twice the final concentration of divalent cations (MnCl2, MgCl2, or CaCl2) for 30 min at 37 °C in a humidified atmosphere of 5% CO2. They were then plated on ligand-coated dishes to perform the adhesion assays, as described above. To identify the receptor for βig-h3, monoclonal antibodies to different types of integrins (Chemicon) were preincubated individually with HCE in 0.05 ml of incubation solution (2 × 105 cells/ml) at 37 °C for 30 min. The preincubated cells were transferred onto plates precoated with βig-h3 proteins and then incubated further for 1 h at 37 °C. Attached cells were then quantified as described above. For flow cytometry analysis, cells at confluence were detached by gentle treatment with 0.25% trypsin, 0.05% EDTA in PBS, washed, and incubated with the antibodies for 1 h at 4 °C. Cells were then incubated with 10 μg/ml affinity purified fluorescein-labeled secondary antibodies for 1 h at 4 °C and analyzed on the flow cytometer FACSCalibur system (Becton Dickinson, San Jose, CA) equipped with a 5-watt argon laser at 488 nm. HCE cells or α3-transfected HCE cells were solubilized in 200 mm n-octyl β-d-glucopyranoside, 1 mm phenylmethylsulfonyl fluoride, 100 mmTris-HCl, pH 7.4. Immunoprecipitations were carried out by overnight incubation at 4 °C of the immunoadsorbents (antibodies adsorbed onto protein A-Sepharose (Amersham Pharmacia Biotech) with samples of cell lysates. Precipitated proteins were separated on 10% SDS-polyacrylamide gel. After separation of precipitated proteins by SDS-PAGE and transfer to a nitrocellulose membrane (Schleicher & Schuell), blots were incubated for 2 h with either anti-α3 or anti-βig-h3 polyclonal antibodies, then detected using horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Sigma), followed by enhanced chemiluminescence (ECL) system (NEN Life Science Products). Anti-integrin monoclonal antibodies utilized were α1 (Fb12), α3 (ASC-1), α4 (P1H4), α5β1 (HA6), α6 (CLB701), αv (P3G8), αvβ3 (LM609), αvβ5 (P1F6), and β1 (12G10) from Chemicon. Polyclonal rabbit anti-α3 (AB1948) antiserum and affinity purified fluorescein-labeled secondary antibodies were also purchased from Chemicon. Polyclonal anti-βig-h3 antiserum against recombinant βig-h3 protein was generated in rabbit and described previously (15Lee E.H. Seomun Y. Hwang K.-H. Kim J.-E. Kim I.-S. Kim J.H. Joo C.-K. Invest. Ophthalmol. & Visual Sci. 2000; 41: 1840-1845PubMed Google Scholar). Full-length cDNA for the human α3subunit (clone 3.10) was purchased from the American Type Culture Collection. A fragment (3.47 kilobases) containing the entire cDNA excised by digestion with XbaI/SalI was cloned into the EcoRV site of the mammalian expression vector pcDNA 3.1+ (Invitrogen, Carlsbad, CA). The α3integrin expression plasmid DNA (1 μg) was transfected into HCE cells using the LipofectAMINE (Life Technologies, Inc.). For cell adhesion assay, we used two recombinant βig-h3 proteins that have been described previously (3Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (111) Google Scholar). We changed the nomenclature from Hisβ-c and Hisβ-d as used in the previous paper (3Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (111) Google Scholar) to βigh3-WT and βigh3-ΔRGD, respectively (Fig.1 A). βig-h3 was previously demonstrated to support the adhesion and spreading of fibroblasts (5LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavele R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (202) Google Scholar,6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar), and the RGD motif was proposed not to be necessary for such activity (6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar). The numbers and surface areas of HCE cells that adhered to βigh3-WT were clearly greater than those attached to albumin and were comparable to those of cells that adhered to fibronectin (Fig.1 B). The cell adhesion and spreading activities of βig-h3 were concentration-dependent (Fig. 1, C andD). Similar results were also obtained with Chinese hamster ovary cells (data not shown). As expected, βigh3-ΔRGD lacking the RGD motif was almost equally effective at supporting cell adhesion and spreading (Fig. 2, A andB). These results confirm that βig-h3 supports cell adhesion and spreading independent of the RGD motif.Figure 2βig-h3-mediated cell adhesion and spreading are independent of the RGD motif. Plastic culture dishes were coated with 10 μg/ml of each protein, i.e.BSA, plasma FN, βigh3-WT, or βigh3-ΔRGD, and were incubated for 1 h at 37 °C. Cells were rinsed, fixed, and stained with crystal violet. Adhesion (A) and spreading (B) of HCE cells were quantified as described under “Experimental Procedures.” The values are the means ± S.D. of triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify the nature of the cell surface receptor for βig-h3, several reagents were used. Cell adhesion to βig-h3 was significantly inhibited by βig-h3 itself, RGD peptide, and EDTA, and it was partially inhibited by fibronectin and EGTA but not inhibited by RGE peptide. Cell adhesion to fibronectin was also significantly inhibited by fibronectin itself, RGD peptide, and EDTA and partially inhibited by βig-h3 and EGTA but not by RGE peptide (Fig. 3 A). Then we examined the effects of Mn2+, Mg2+, and Ca2+ on βig-h3-mediated cell adhesion. Cell adhesion to βig-h3 was strongly promoted by Mn2+, and to a lesser extent by Mg2+, but only marginally by Ca2+(Fig. 3 B). These results suggest that the cell surface receptor for βig-h3 could be one of the RGD-dependent integrins, which require divalent cations for interaction with ligands. To identify the βig-h3 receptor, the effects of function-blocking monoclonal antibodies to integrin subunits were examined on the adhesion of HCE cells to the surface coated with βig-h3. Adhesion to the βig-h3-coated surface was specifically inhibited by antibody to α3 subunit but not by antibodies to other α subunits (Fig. 3 C). Because the integrin α3 subunit is known to couple with the integrin β1 subunit, anti-β1 antibody was also expected to inhibit cell adhesion to βig-h3. As expected, anti-β1 antibody significantly blocked cell adhesion (Fig. 3 C). To determine whether βig-h3 interacts with α3 integrin, we carried out co-immunoprecipitation assays. Immunoblotting with anti-βig-h3 antiserum showed that βig-h3 was detected in immunoprecipitates formed by anti-α3 integrin antibody (Fig. 3 D,lanes 2–4). Conversely, α3 integrin was also detected in immunoprecipitates formed by anti-βig-h3 antiserum (Fig.3 D, lanes 6–8). α3 integrin was detected in all four cell lysates (Fig. 3 D, lanes 1–4) among which α3 overexpressed cell lysate showed the highest amount of α3 integrin in immune complex. Because the basal expression level of βig-h3 in cell lysate is very low, it was barely detected in immune complex precipitated by anti-βig-h3 antiserum (Fig. 3 D, lane 5). However, when we added recombinant βig-h3 protein to the culture medium (Fig. 3 D, lanes 6 and8) or to cell lysate (Fig. 3 D, lane 7), it was detected in immunoprecipitates. Nothing was detected in immunoprecipitates with nonimmune rabbit serum (data not shown). To determine which integrins are expressed in HCE cell, we performed fluorescence-activated cell sorter analysis. Fig.4 shows that all the integrins tested were detected on the HCE cell surface although their expression levels varied. The expression level of α3 integrin was relatively high, whereas that of α5β1 was low. All the others had similar expression levels. Taken together, these results suggest integrin α3β1 is a specific functional receptor for βig-h3 in HCE cells. In an attempt to identify essential amino acid residues conferring cell adhesion activity of βig-h3, we first tested whether each repeated domain is capable of mediating cell adhesion. We made four recombinant proteins corresponding to each repeated domain (Fig. 5, A andB) and tested their cell adhesion activities. We found that the 2nd fas-1 domain and the 4th fas-1 domain were equally active compared with the wild type βig-h3, whereas the 1st fas-1 domain was moderately active and the 3rd fas-1 domain was very weakly active (Fig.5 C). Both 2nd fas-1 and 4th fas-1 domain-mediated cell adhesions were almost blocked by antibodies to α3 and β1 integrin subunits (Fig. 5 D) suggesting that both 2nd fas-1 and 4th fas-1 domains have essential amino acid residues for interacting with integrin. These results also support that neither H1 nor H2 is mediating cell adhesion activity of βig-h3 because the 1st and the 3rd domains are not active in cell adhesion although they have H1 or H2. To identify cell adhesion motifs within the 2nd and the 4th domains, we performed a computer search using Prodom Release 99.2 based on homologies not only among the repeated fas-1 domains of βig-h3 but also among fas-1 domains of other proteins. In many fas-1 domains including the 2nd and the 4th fas-1 domains of βig-h3, two amino acid residues, aspartic acid and isoleucine near H2, are highly conserved, whereas in some cases including the 1st fas-1 domain of βig-h3, only aspartic acid is conserved (Fig. 6). Although not shown here, some fas-1 domains such as the 3rd fas-1 domain of βig-h3 do not have aspartic acid near H2. In order to examine the sequence containing aspartic acid that is needed for cell adhesion, we generated mutated 4th fas-1 proteins of βig-h3 where each 616 proline, 617 aspartic acid, and 618 isoleucine was replaced with serine, alanine, and serine, respectively (Fig. 7,A and B). D617A (βigh3 d-IV-PaI) and I618S (βigh3 D-IV-PDs) mutations significantly blocked cell adhesion whereas P616S (βigh3 D-IV-sDI) mutation did not affect cell adhesion. Consequently, three amino acids mutation, P616S/D617A/I618S (βig-h3 D-IV-sas) also blocked cell adhesion (Fig. 7 C). These results support our hypothesis that the 617 aspartic acid is essential for cell adhesion and indicate that the 618 isoleucine is also important for cell adhesion.Figure 7Effect of substitution mutations of βig-h3-domain IV on HCE cell adhesion and spreading. A, diagram of substitution mutant proteins of βigh3 D-IV. Four substitution mutations were made. Thelowercase letters indicate substituted amino acids.B, purified recombinant proteins were subjected to 15% polyacrylamide gel electrophoresis. 1st lane, βigh3 D-IV;2nd lane, βigh3 D-IV-PaI; 3rd lane, βigh3 D-IV-sDI; 4th lane, βigh3 D-IV-PDs; 5th lane, βigh3 D-IV-sas. Molecular mass standards are indicated in kDa on theleft of the gel. C, adhesion of HCE cells to mutants of βig-h3-domain IV. HCE cells were seeded onto 96-well microculture plates coated with 10 μg/ml of each protein and incubated for 1 h at 37 °C. Cell attachment was quantified as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm further aspartic acid and isoleucine are essential for cell adhesion, four synthetic peptides were generated. As shown in Fig.8 A, the first three peptides, KADHH (amino acids 219–223), NKDIL (amino acids 354–358), and EPDIM (amino acids 615–619), correspond to each conserved sequence of the 1st, the 2nd, and the 4th fas-1 domains of βig-h3, respectively. The last one, DEMPI is from the 4th fas-1 but is scrambled and used as a control peptide. We tested whether these four peptides could inhibit βig-h3-mediated cell adhesion. As shown in Fig. 8 B, NKDIL from the 2nd fas-1 and EPDIM from the 4th fas-1 were capable of blocking cell adhesion to βigh3-WT, whereas KADHH from the 1st fas-1 weakly inhibited cell adhesion, and control peptide DEMPI did not affect cell adhesion. Similar results were obtained when we used the 2nd fas-1 and the 4th fas-1 proteins as cell substrata (Fig.8 C). Then we tested whether each peptide itself is capable of mediating cell adhesion. Several different concentrations of each peptide were used as cell substrata and tested for cell adhesion activity. As shown in Fig. 9 A, NKDIL and EPDIM were capable of mediating cell adhesion in a dose-dependent manner. KADHH was also capable of mediating cell adhesion in a dose-dependent manner, but the activities were relatively weak. The control peptide was not active in cell adhesion. In the next experiment, we examined whether peptide-mediated cell adhesion was also mediated via α3β1 integrin. Fig. 9 B showed that cell adhesion to NKDIL or EPDIL was blocked by antibodies to α3 and β1 integrin subunits. These results suggest that the conserved aspartic acid and isoleucine in the 2nd fas-1 and the 4th fas-1 domains are essential for βig-h3-mediated cell adhesion through α3β1 integrin. To examine whether inhibition of cell adhesion by these two peptides was specific to βig-h3, we tested the effects of peptides on cell adhesion to other adhesion molecules. As is shown in Fig.10 A, NKDIL and EPDIM efficiently blocked cell adhesion not only to βig-h3 but also to laminin, whereas they moderately inhibited cell adhesion to fibronectin and did not affect cell adhesion at all to collagen type I, type II, and vitronectin. These inhibitory effects were dose-dependent (Fig. 10 B). These results suggest that NKDIL and EPDIM specifically compete with α3β1 integrin-interacting molecules.Figure 9Adhesion of HCE cells onto surfaces coated with synthetic peptides. A, HCE cell adhesion to synthetic βig-h3 peptides. 96-Well microculture plates were coated with the indicated concentrations of each synthetic peptide and incubated with HCE cells at 37 °C for 1 h. After incubation, cells attached to the substrates were quantified by hexosaminidase assay as described under “Experimental Procedures.” B, α3β1 integrin mediates HCE cell adhesion to EPDIM peptide and NKDIL peptide. HCE cells were preincubated with function-blocking monoclonal antibodies to integrin subunits at a concentration of 5 μg/ml for 30 min at 37 °C and then seeded to the precoated wells with 100 μm EPDIM or 100 μm NKDIL. After 1 h incubation, cells attached to the substrates were quantified by hexosaminidase assay as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Effect of synthetic peptides on HCE cell adhesion to several extracellular matrix proteins. A, inhibition of HCE cell adhesion by synthetic peptides onto surfaces coated with matrix proteins. HCE cells were preincubated in medium in the absence or presence of 100 μm each synthetic βig-h3 peptide and seeded onto surfaces coated with 10 μg/ml of each protein, i.e. βigh3-WT, FN, Col I, Col II, LM, or VN. After 1 h of incubation, cells attached to the substrates were quantified by hexosaminidase assay as described under “Experimental Procedures.” B, dose-dependent inhibition of HCE cell adhesion to surfaces coated with 10 μ g/ml βigh3-WT, FN, orLM was measured in the presence of the indicated concentrations of EPDIM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although βig-h3 has been considered to promote cell adhesion and spreading, the interacting cell receptor and the specific motifs of βig-h3 for cell adhesion have not been characterized. In this report, we identified that the functional receptor for βig-h3 is α3β1 integrin and the sequences, NKDIL of the 2nd and EPDIM of the 4th fas-1 domains, are active sites and sufficient to induce cell adhesion through α3β1 integrin. In addition, aspartic acid and isoleucine turned out to be the essential amino acid residues of these motifs. βig-h3 was first identified by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-β (16Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (512) Google Scholar). Its cell adhesion activity was first reported with human dermal fibroblasts (5LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavele R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (202) Google Scholar) and then with chondrocytes, peritoneal fibroblasts, and human MRC5 fibroblasts (6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar). Because βig-h3 has an RGD motif at the carboxyl terminus, βig-h3 was thought to mediate cell adhesion through its RGD motif. Ohnoet al. (6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar), however, reported that the RGD motif at the carboxyl terminus of βig-h3 was not necessary for enhancing the spreading of chondrocytes. This result was predictable because the RGD motif was not present in the mature βig-h3 protein as a result of carboxyl-terminal processing, and the mature form was able to inhibit cell adhesion when it was added to the culture medium (1Skonier J. Benenett K. Rothwell V. Kosowski S. Plowman G. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (261) Google Scholar). Our result also supports the RGD motif is not necessary for mediating cell adhesion activity of βig-h3. Several efforts were made to identify a cell surface receptor for βig-h3. The fact that βig-h3-mediated cell adhesion was blocked by an RGD peptide and EDTA suggests that the surface receptor for βig-h3 could be one of RGD-dependent integrins of which activity requires divalent cations such as Mn2+ and Mg2+. The result of assay using function-blocking antibodies to α1, α2, α3, α4, α5, α6, αv, and β1 integrins suggests that the specific integrin interacting with βig-h3 is α3β1 integrin, which has been known to belong to the RGD- and divalent cation-dependent integrins (12Ebel J.A. Kuhn K. Eble J.A. Molecular Biology Intelligence Unit: Integrin-Ligand Interaction. Springer-Verlag, Heidelberg, Germany1997: 1-40Google Scholar). Indeed, we showed that α3 integrin was co-immunoprecipitated by anti-βig-h3 antiserum, and conversely, βig-h3 was also co-immunoprecipitated by anti-α3 integrin antibody indicating that α3β1 integrin is a specific functional receptor for βig-h3 in HCE cells. fas-1 domain is found in several proteins including βig-h3, periostin, fasciclin, HLC-2, and algal-CAM, all of which are known as cell adhesion molecules, but they have different numbers of fas-1 domain (8Kawamoto T. Noshiro M. Shen M. Nakamasu K. Hashimoto K. Kawashima-Ohya Y. Gotoh O. Kato Y. Biochim. Biophys. Acta. 1998; 1395: 288-292Crossref PubMed Scopus (105) Google Scholar). It suggests that βig-h3 may not require all four fas-1 domains to mediate cell adhesion and even a single fas-1 domain could mediate cell adhesion. This hypothesis was proved by demonstrating that each of the 2nd and the 4th fas-1 domains was sufficient for cell adhesion activity. However, why were the 1st and the 3rd fas-1 domains not active in mediating cell adhesion? Complete lack of cell adhesion activity of the 3rd fas-1 domain may be due to its less homology with other three domains. Particularly, the region around H2 homologous sequence of the 3rd fas-1 domain is not well conserved. In contrast, although the 1st fas-1 domain is relatively highly homologous with the 2nd fas-1 and the 4th fas-1 domains, it is not as active as the 2nd and the 4th domains in mediating cell adhesion. These findings suggest that cell adhesion motifs in the 2nd and the 4th domains might be lacking or altered in the 1st domain and the 3rd domain. To answer this question, we first analyzed sequences of each domain of βig-h3 and several other fas-1 domains looking for conserved amino acids rather than H1 and H2. In particular, we focused on aspartic acid, which is known to be essential for interacting with integrins. A sequence analysis uncovered that aspartic acid and isoleucine near H2 are highly conserved in many fas-1 domains. It is noteworthy that these two amino acids are not found in the 3rd domain and isoleucine is replaced by histidine in the 1st domain. In fact, mutation of either aspartic acid or isoleucine almost completely blocked the 4th fas-1 domain-mediated cell adhesion. In addition, synthetic peptides, NKDIL and EPDIM from the 2nd and 4th domains, respectively, were efficiently able to block cell adhesion mediated not only by each domain but also by wild type βig-h3, whereas the synthetic peptide KADHH derived from the 1st domain was less efficient to block cell adhesion. These results indicate that both aspartic acid and isoleucine are required for cell adhesion and also give us an answer why the 1st domain is weakly active and the 3rd domain is not active in mediating cell adhesion. Synthetic peptides, NKDIL and EPDIM, themselves were found to be good substrates for cell adhesion. Like wild type βig-h3, cell adhesion mediated by the 2nd domain, the 4th domain, and two synthetic peptides were also specifically blocked by antibodies to α3 and β1 subunits. This implies that βig-h3 interacts with α3β1 integrin via two major motifs, where one resides in the 2nd fas-1 domain and the other resides in the 4th fas-1 domain. This finding is different from what Ohno et al. (6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar) reported. They reported that the cell surface receptor for βig-h3 was α1β1 integrin in MRC5 fibroblasts. This discrepancy may not be due to simple differences in surface integrin profiles because we found that both HCE cells and MRC5 cells have not only α3 integrin but also α1integrin on their cell surfaces. Actually, MRC5 has more α3 integrin than α1 integrin (data not shown). Our experiments with MRC5 fibroblasts revealed that their adhesion to βig-h3 was incompletely blocked by antibody to α1 integrin but, interestingly, was almost completely blocked by αv integrin antibody which Ohno et al. (6Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashim-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (102) Google Scholar) have not tried in their experiment (data not shown). Furthermore, all mutant forms of the 4th fas-1 domain, which failed to mediate corneal epithelial cell adhesion in our experiments, still retain cell adhesion activities for MRC5 fibroblasts (data not shown). These results strongly suggest that βig-h3 can mediate cell adhesion through different integrins depending on cell types, and that its interacting domains could be different. A number of studies have defined multiple ligands for α3β1 integrin, including laminin (17Delwel G.O. de Melker A.A. Hogervorst F. Jaspars L.H. Fles D.L. Kuikman I. Lindblom A. Paulsson M. Timpl R. Sonnenberg A. Mol. Biol. Cell. 1994; 5: 203-215Crossref PubMed Scopus (195) Google Scholar), certain types of collagen (18Elices M.J. Urry L.A. Hemler M.E. J. Cell Biol. 1991; 112: 169-181Crossref PubMed Scopus (385) Google Scholar), fibronectin (18Elices M.J. Urry L.A. Hemler M.E. J. Cell Biol. 1991; 112: 169-181Crossref PubMed Scopus (385) Google Scholar), and nidogen (19Dedhar S. Jewell K. Rojiani M. Gray V. J. Biol. Chem. 1992; 267: 18908-18914Abstract Full Text PDF PubMed Google Scholar). Although there are some conflicting reports that some of these proteins do not support α3β1-mediated cell adhesion (19Dedhar S. Jewell K. Rojiani M. Gray V. J. Biol. Chem. 1992; 267: 18908-18914Abstract Full Text PDF PubMed Google Scholar, 20Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar) despite apparent P1B5 blocking effects on some of them, α3β1 integrin is considered to respond to a broad spectrum of extracellular ligands (21DiPersio C.M. Shah S. Hynes R.O. J. Cell Sci. 1995; 108: 2321-2336Crossref PubMed Google Scholar). There seems to be no conserved binding motif for α3β1 integrin because no apparent sequence homology is observed among active peptides from thrombospondin (22Krutzsch H.C. Choe B.J. Sipes J.M. Guo N. Roberts D.D. J. Biol. Chem. 1999; 274: 24080-24086Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), laminin (23Gehlsen K.R. Sriramara P. Furcht L.T. Skubitz A.P.N. J. Cell Biol. 1992; 117: 449-459Crossref PubMed Scopus (109) Google Scholar), and type IV collagen (24Miles A.J. Knutson J.R. Skubitz A.P.N. Furcht L.T. McCarthy J.B. Fields G.B. J. Biol. Chem. 1995; 270: 29047-29050Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which have been suggested to interact with α3β1. Our peptides from βig-h3 also do not share any sequence homology with the active peptides mentioned above. However, it is interesting to note that the α3β1-interacting peptide corresponding to α1(IV)-(531–543) has two Asp residues, both of which are important for cell adhesion activity and that the first Asp is flanked by Leu (24Miles A.J. Knutson J.R. Skubitz A.P.N. Furcht L.T. McCarthy J.B. Fields G.B. J. Biol. Chem. 1995; 270: 29047-29050Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Miles A.J. Skubitz A.P.N. Furcht L.T. Fields G.B. J. Biol. Chem. 1994; 269: 30939-30945Abstract Full Text PDF PubMed Google Scholar). This Asp-Leu residue is reminiscent of our peptides, NKDIL and EPDIM, where Ile replaces Leu. Both Ile and Leu are hydrophobic and have bulky side chains which, together with Asp, are known to be important for interacting with integrins (12Ebel J.A. Kuhn K. Eble J.A. Molecular Biology Intelligence Unit: Integrin-Ligand Interaction. Springer-Verlag, Heidelberg, Germany1997: 1-40Google Scholar). Further investigations, however, are required to identify and prove α3β1 integrin-interacting motifs more precisely from several ligands. Transfection of βig-h3 expression plasmids into Chinese hamster ovary cells led to marked decreases in cell growth and the ability of these cells to form tumors in nude mice (1Skonier J. Benenett K. Rothwell V. Kosowski S. Plowman G. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (261) Google Scholar). We previously reported βig-h3 could inhibit osteoblast differentiation (3Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (111) Google Scholar), and its expression is down-regulated in bone marrow stem cells treated with dexamethasone (2Dieudonne S.C. Kerr K.M. Xu T. Sommer B. DeRubeis A.R. Kuznetsov S.A. Kim I.-S. Robey P.G. Young M.F. J. Cell. Biochem. 1999; 76: 231-243Crossref PubMed Scopus (61) Google Scholar). Other studies imply a role for βig-h3 in wound healing in corneal and vascular tissues (4Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. & Visual Sci. 1997; 38: 893-900PubMed Google Scholar, 26O'Brien E.R. Bennett K.L. Garvin M.R. Zdric T.W. Hinohara T. Simpson J.B. Kimura T. Nobuyoshi M. Mizgala H. Purchio A. Schwartz S.M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 576-584Crossref PubMed Scopus (94) Google Scholar). A role for disturbed morphogenesis has been assigned to βig-h3 missense mutations in families affected with human autosomal dominant corneal dystrophies (7Munier F.L. Korvatska E. Djemai S. LePaslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Crossref PubMed Scopus (533) Google Scholar). Given that βig-h3 is highly induced by transforming growth factor-β in several cells and that it functions as a cell adhesion molecule, together with all above reports, we suggest that βig-h3 may play an important role in the regulation of morphogenesis and the maintenance of several tissues in normal and pathological conditions. In conclusion, we have demonstrated that two motifs, NKDIL and EPDIM within the 2nd and the 4th fas-1 domains of βig-h3, are sufficient to mediate human corneal epithelial cell adhesion through α3β1 integrin. We have also identified that two amino acid residues, Asp and Ile, in the context of motifs are essential for cell adhesion activity. These results, therefore, establish the mechanism of βig-h3-mediated cell adhesion and suggest that other proteins containing Asp-Ile near H2 in their fas-1 domains could function as cell adhesion molecules. We thank Drs. Kaoru Araki-Sasaki (Kinki Central Hospital, Japan) and Masatsugu Nakamura (Santen Pharmaceutical Co., Japan) for the use of human corneal epithelial cells and Drs. Marian F. Young and Kenneth M. Yamada (NIDCR, National Institutes of Health) for critical review of the manuscript.
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