FAS1 Domain Research Articles

Identification and characterization of transforming growth factor β induced gene (TGFBIG) from Branchiostoma belcheri: Insights into evolution of TGFBI family

The transforming growth factor β induced gene (TGFBIG) encodes a protein (TGFBI) which plays important roles in many biological processes. However, no TGFBIG homolog has been reported in B. belcheri. Here, we identified a TGFBI-like gene from B. belcheri and extensively studied the evolutionary history of TGFBI family. We found that the amphioxus genome contains a TGFBIG homolog designated as AmphiTGFBI which encodes a protein with 5 Fas1 domains. The TGFBIGs were present in a common ancestor with Amphimedon queenslandica. We also demonstrated expression patterns of AmphiTGFBI in five amphioxus tissues. Interestingly, the gene structures and conserved motifs of invertebrate TGFBIGs were found to present regular changes in the evolution. Positive selection and Fas1 domain loss might cause the regular changes of gene structures and conserved motifs in invertebrate TGFBIGs during evolution. Together, our findings provided an insight into the evolution of the TGFBI family.

A Matrix Metalloproteinase 1–Cleavable Composite Peptide Derived From Transforming Growth Factor β–Inducible Gene h3 Potently Inhibits Collagen‐Induced Arthritis

Transforming growth factor β-inducible gene h3 (βIG-H3), which is abundantly expressed in rheumatoid synovium, and matrix metalloproteinases (MMP) play important roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the therapeutic efficacy of βIG-H3-derived peptides using MMP-1-dependent target tissue delivery in chronic inflammatory arthritis. Peptides developed from βIG-H3 derivatives, including the second and fourth YH peptides, the fourth fas-1 domain, the fourth fas-1 domain truncated for H1 and H2 sequences (dhfas-1), and an MMP-1- cleavable composite peptide (MFK24), were cloned. We confirmed the specificity of MFK24 cleavage by immunoblot analysis after treatment with different proteases. The YH18 peptide in the fourth fas-1 domain of βIG-H3 was weakly effective in suppressing arthritis severity in mice with collagen-induced arthritis (CIA). Treatment with higher-dose dhfas-1 (30 mg/kg) showed remarkable efficacy, whereas treatment with a lower dose (10 mg/kg) resulted in only partial improvement. MFK24, a composite peptide consisting of dhfas-1 and RGD peptide linked by MMP-1 substrate, was cleaved specifically by MMP-1. The adhesion and migration of NIH3T3 cells mediated by βIG-H3 were inhibited by MFK24 at a low concentration. MFK24 suppressed the adhesion of NIH3T3 cells more efficiently compared with murine dhfas-1 (MFK00) or RGD, either alone or in combination. The therapeutic efficacy of MFK24 in mice with CIA was remarkably enhanced, with consistently reduced expression of inflammatory mediators within joint tissue. This proof-of-concept study showed that an MMP-cleavable composite peptide, based on βIG-H3 derivatives, had markedly improved therapeutic efficacy in chronic inflammatory arthritis, implicating a new expandable strategy for enhancement of the efficacy of 2 different active molecules in RA.

Multiple FAS1 domains and the RGD motif of TGFBI act cooperatively to bind αvβ3 integrin, leading to anti-angiogenic and anti-tumor effects

TGFBI, a transforming growth factor β-induced extracellular matrix protein, circulates at a level of ~300ng/ml in humans and modulates several integrin-mediated cellular functions. The protein contains an N-terminal EMI domain, four consecutive FAS1 domains, and the RGD motif. Each FAS1 domain and the RGD motif have been known to interact with avb3 integrin. Here, we found that the binding affinity (Kd) of TGFBI for αvβ3 integrin was approximately 3.8×10−8M, a value ~2300-fold higher than that of a single FAS1 domain, and demonstrated that this greater affinity was due to the cooperative action of the four FAS1 domains and the RGD motif. Moreover, TGFBI exhibited more potent anti-angiogenic and anti-tumorigenic activities, even at a 100-fold lower molar dose than the reported effective dose of the FAS1 domain. Finally, our data showed that TGFBI specifically targeted the tumor vasculature and accumulated at the tumor site. Collectively, our results support the theory that TGFBI acts as a potent endogenous anti-tumor and anti-angiogenic molecule by targeting αvβ3 integrin, and highlights the importance of physiological circulating TGFBI levels in inhibiting tumor growth.

AB0161 A MMP1-cleavable composite peptide derived from BIG-H3 potently inhibits chronic inflammatory arthritis

BackgroundTransforming growth factor β-inducible gene-h3 (βig-h3), an extracellular matrix protein, is composed of four homologous fas-1 domains and an RGD motif. We previously demonstrated that βig-h3 was abundantly expressed in...

Periostin-binding DNA Aptamer Inhibits Breast Cancer Growth and Metastasis

Periostin is an extracellular matrix (ECM) protein that is overexpressed in a variety of human cancers, and its functions appear to be linked to tumor growth, metastasis, and angiogenesis. Recent clinical evidence suggests that aberrant periostin expression is correlated with poor outcome in patients with breast cancer. To identify novel tools to regulate the functional role of periostin, we generated benzyl-d(U)TP-modified DNA aptamers that were directed against human periostin (PNDAs) and characterized their functional roles in breast cancer progression. PNDA-3 selectively bound to the FAS-1 domain of periostin with nanomolar affinity and disrupted the interaction between periostin and its cell surface receptors, αvβ3 and αvβ5 integrins. PNDA-3 markedly antagonized the periostin-induced adhesion, migration, and invasion of breast cancer cells and blocked the activation of various components of the αvβ3 and αvβ5 integrin signal transduction pathways. In a 4T1 orthotopic mouse model, PNDA-3 administration significantly reduced primary tumor growth and distant metastasis. Thus, our results demonstrated that periostin-integrin signaling regulates breast cancer progression at multiple levels in tumor cells and the tumor microenvironment. DNA aptamers targeting periostin may potentially be used to inhibit breast cancer progression.

A C-terminal fragment BIGH3 protein with an RGDRGD motif inhibits corneal neovascularization in vitro and in vivo

An Arg–Gly–Asp (RGD) motif in the fourth FAS1 domain of the human BIGH3 (transforming growth factor-β1-inducible gene-h3) protein has been reported to play an important role in mediating tumor angiogenesis. The aim of this study was to investigate the inhibitory effect of a modified C-terminal fragment BIGH3 protein with an RGDRGD motif on corneal neovascularization in vitro and in vivo. Recombinant C-terminal fragment BIGH3 protein with wild-type sequence and modified C-terminal fragment BIGH3 protein containing an RGDRGD motif were successfully expressed and purified. We demonstrated that both proteins significantly inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) adhesion, migration, and tube formation and induced cell apoptosis but failed to inhibit HUVEC proliferation. We determined that the mechanism underlying this activity was an interaction between BIGH3 and αvβ3 integrin, which blocked the phosphorylation of PI3K/Akt and ERK signaling pathways. The inhibitory effects of wild-type and modified C-terminal fragment BIGH3 proteins on angiogenesis were confirmed by a rabbit corneal neovascularization assay. More importantly, we provided evidence that the modified C-terminal fragment BIGH3 protein with an RGDRGD motif inhibited angiogenic activity far more effectively than did wild-type C-terminal fragment BIGH3. Collectively, our data show that a C-terminal fragment BIGH3 protein containing an RGDRGD motif might be promising as an effective drug in treating corneal neovascularization.

Structure and function of a bacterial Fasciclin I Domain Protein elucidates function of related cell adhesion proteins such as TGFBIp and periostin

Fasciclin I (FAS1) domains have important roles in cell adhesion, which are not understood despite many structural and functional studies. Examples of FAS1 domain proteins include TGFBIp (βig-h3) and periostin, which function in angiogenesis and development of cornea and bone, and are also highly expressed in cancer tissues. Here we report the structure of a single-domain bacterial fasciclin I protein, Fdp, in the free-living photosynthetic bacterium Rhodobacter sphaeroides, and show that it confers cell adhesion properties in vivo. A binding site is identified which includes the most highly conserved region and is adjacent to the N-terminus. By mapping this onto eukaryotic homologues, which all contain tandem FAS1 domains, it is concluded that the interaction site is normally buried in the dimer interface. This explains why corneal dystrophy mutations are concentrated in the C-terminal domain of TGFBIp and suggests new therapeutic approaches.

Expression, purification and characterization of fourth FAS1 domain of TGFβIp-associated corneal dystrophic mutants

Corneal dystrophies (CDs) are a group of inherited bilateral disorders affecting the corneal tissue of the eye. Most of these CDs in the stromal layer of the cornea have been associated with mutations found on the TGFBI gene that codes for a 683-amino acid transforming growth factor induced protein (TGFβIp). These mutations have been found to induce atypical aggregation and progressive accumulation of protein aggregates in the cornea that leads to loss of corneal transparency and hence blindness. At present, 65 distinct pathogenic mutations have been identified in TGFBI that are associated with different clinical phenotypes. More than 80% of these missense mutations occur in the 4th FAS (fasciclin-like) 1 domain. Current treatment includes surgical intervention, which often involves high recurrence rates. Hence, it is imperative to examine the properties of the TGFβIp and the pathogenic mutant proteins to understand the pathology of the disease mechanism and to develop potent therapeutics. Here, we report the recombinant expression, purification, characterization and the effect of four clinically significant pathogenic TGFβIp mutants – R555W, H572R, A620D, and H626R on the biophysical properties of the wild type (WT) 4th FAS1 domain of TGFβIp. While a higher proportion of the R555W, H572R and H626R mutants of the 4th FAS1 domains remained stable, the A620D mutant largely existed as inclusion bodies in native state and aggregates under physiological conditions. These mutants present a unique platform to examine protein aggregation-prone diseases wherein single amino-acid mutations present distinct pathogenic phenotypes. Though pathogenically and phenotypically diverse, these mutants do not exhibit variations in secondary structure and stability, except for the A620D mutant, when examined by CD and UV spectroscopy.

Therapeutic Effect of a Recombinant βig-h3 Fragment-RGD Peptide for Chronic Inflammatory Arthritis

Objective. βig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that βig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with αvβ 3 integrin. We designed a recombinant βig-h3 protein consisting of a fas-1 domain and RGD motif and evaluated the therapeutic efficacy in RA. Methods. Inhibitory effect of adhesion and migration of NIH3T3 cell line was evaluated in 96 well microtiter and transwell plates coated with βig-h3. Clinical arthritis index was evaluated after treating CIA mice with MFK12. Immunohistochemical staining in synovial tissues were performed. Expression of transcripts and proteins of inflammatory mediators were analyzed by semi-quantitative RT-PCR and immunoblotting. Results. Recombinant protein consisted of 4 th fas-1 domain truncated for H1 and H2 sequences and RGD peptide (MFK12), had M.W. of 10.4kDa. βig-h3 mediated adhesion and migration of NIH3T3 cell line were significantly inhibited in a dose-dependent manner. Arthritis severity and incidence were efficiently reduced when CIA mice were treated with MFK12 at 30 mg/kg/day compared with the control. Immunohistochemical staining of joint tissues in MFK12 treated mice exhibited reduced angiogenesis. In treated mice, expression of transcripts regarding inflammatory mediators was markedly suppressed and immunoblotting of ICAM-1 and RANKL from whole extract of hind paws also showed a significant reduction. Conclusion. This study shows that MFK12 is effective in treating RA, although further study is warranted to improve the therapeutic efficacy.

Proteomic Identification of Betaig-h3 as a Lysophosphatidic Acid-Induced Secreted Protein of Human Mesenchymal Stem Cells: Paracrine Activation of A549 Lung Adenocarcinoma Cells by Betaig-h3

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene

Different types of granular corneal dystrophy (GCD) and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571–R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571–R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo.

Expression of periostin during Xenopus laevis embryogenesis

Periostin (postn) is a secreted, extracellular matrix protein containing an EMI domain as well as four fasciclin I-like (Fas1) domains. Postn protein functions in cell adhesion, cell mobility, cell proliferation and gene regulation. Earlier studies have shown that postn is involved in different developmental processes such as somitogenesis, cardiogenesis and bone formation. Intriguingly, postn seems to be a very good candidate to establish novel therapies against cancer and chronic heart defects. Here we describe for the first time the spatio-temporal expression profile of postn during early development of Xenopus laevis. By semi-quantitative RT-PCR approaches, we demonstrate that postn is maternally expressed. Zygotic expression starts during early gastrulation and increases until stage 40. Whole mount in situ hybridization experiments revealed that postn is detectable in somites, the sensory layer of the epidermis, the roof plate, the notochord, the heart, migrating neural crest cells, cranial ganglia and forming cranial cartilage structures. Our results implicate a role of postn during Xenopus embryogenesis and represent a good starting point for future functional analyses.

Identification of a novel cell binding site of periostin involved in tumour growth

IntroductionPeriostin (PN), a member of the fasciclin family of proteins, is a TGF-β-induced extracellular matrix protein involved in cell survival, angiogenesis, invasion and metastasis. It is considered a potent angiogenic factor and a marker of tumour progression in many types of human cancer. Many different kinds of cells bind to PN by means of the integrins αvβ3 and αvβ5, but the periostin epitope recognised by these integrins is not formally demonstrated. The aim of our study was to identify which domain of PN could be involved in cell adhesion and its potential role in tumour growth. MethodsWe generated the monoclonal antibody OC-20 (mAb OC-20) by hybridoma technology. Different PN recombinant fragments were used to characterise the periostin epitope recognised by the mAb OC-20 and to localise a new cell binding site of the protein. A murine model of human melanoma was used in the preclinical in vivo experiments. ResultsWe formally demonstrate that the periostin epitope recognised by OC-20 is a new binding site for the integrins αvβ3 and αvβ5, localised in the second FAS1 domain (FAS1–2) of the protein. Moreover the in vivo use of this antibody significantly inhibits tumour growth and angiogenesis. ConclusionOur results show that the FAS1–2 domain of PN plays a role in tumour progression. Moreover this novel antibody may likewise prove to be very useful in clarifying the role of PN in angiogenesis and may contribute to the design of novel anti-angiogenesis drugs.

SAXS Models of TGFBIp Reveal a Trimeric Structure and Show That the Overall Shape Is Not Affected by the Arg124His Mutation

Human transforming growth factor β induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple “beads-on-a-string” arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.

Comparative study on the cellular activities of osteoblast-like cells and new bone formation of anorganic bone mineral coated with tetra-cell adhesion molecules and synthetic cell binding peptide

PurposeWe have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg-Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of βig-h3. Therefore, the purpose of this study was to evaluate the cellular activity of osteoblast-like cells and the new bone formation on ABM coated with T-CAM, while comparing the results with those of synthetic cell binding peptide (PepGen P-15).MethodsTo analyze the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, andto analyze gene expression, northernblot was performed. Mineral nodule formations were evaluated using alizarin red stain. The new bone formations of each group were evaluated using histologic observation and histomorphometrc analysis.ResultsExpression of alkaline phosphatase mRNA was similar in all groups on days 10 and 20. The highest expression of osteopontin mRNA was observed in the group cultured with ABM/P-15, followed by those with ABM/T-CAM and ABM on days 20 and 30. Little difference was seen in the level of expression of collagen type I mRNA on the ABM, ABM/T-CAM, and ABM/P-15 cultured on day 20. There were similar growth and proliferation patterns for the ABM/T-CAM and ABM/P-15. The halo of red stain consistent with Ca2+ deposition was wider and denser around ABM/T-CAM and ABM/P-15 particles than around the ABM particles. The ABM/T-CAM group seemed to have bone forming bioactivity similar to that of ABM/P-15. A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups.ConclusionsABM/T-CAM, which seemed to have bone forming bioactivity similar to ABM/P-15, was considered to serve as effective tissue-engineered bone graft material.

Expression, Purification and Characterization of Soluble Recombinant Periostin Protein Produced by Escherichia coli

Periostin is a matricellular protein participating in the tissue remodelling of damaged cardiac tissue after acute myocardial infarction and of the periodontal ligament in mice. However, further studies on the periostin protein have been limited by the intrinsic difficulty of purifying this protein produced in Escherichia coli due to its insolubility. Here, we demonstrate the expression of recombinant periostin protein with high solubility and monodispersity in E. coli. Periostin is composed of an amino-terminal EMI domain, a tandem repeat of 4 fas1 domains (RD1-4), and a carboxyl-terminal region (CTR). We expressed the RD4-CTR region tagged with GST at amino-terminal and 6x Histidine at carboxyl-terminal end in E. coli. The recombinant protein was purified by using GSH-Sepharose and nickel chelation affinity chromatography, followed by gel filtration chromatography. The RD4-CTR protein exhibited high solubility and monodispersity. On average, 9.1 mg of purified RD4-CTR was routinely obtained from 1 L of culture media. Furthermore, the RD4-CTR was biochemically active, because it bound to the RD1-4, the same as intact periostin protein that had been purified from mammalian cells. Our results should enable us to produce the periostin recombinant protein in large quantities and facilitate future studies on functional and structural analyses of periostin.

Stabilin-2 mediates homophilic cell–cell interactions via its FAS1 domains

Stabilin-2 was recently shown to mediate a heterophilic interaction with integrin alphaMbeta2 via its FAS1 domain. Here, we demonstrate that stabilin-2 also mediates homophilic cell–cell interactions. L cells expressing stabilin-2 mediate a significant level of cell aggregation, and this aggregation is significantly inhibited by anti-stabilin-2 antibody. Stabilin-2-mediated aggregation is mediated by homophilic interactions and enhanced in the presence of Ca 2+ and Mg 2+. Interestingly, exogenous addition of FAS1 domains but not EGF-like domains enhances stabilin-2-mediated cell aggregation, suggesting that exogenous FAS1 domains may form polymeric structure with FAS1 domains of stabilin-2. Together, these data show the participation of stabilin-2 in homophilic cell adhesion and role of FAS1 domains.

First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.

Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9(th) and 10(th) type III domains of fibronectin, and 4(th) FAS1 domain of betaig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9(th) and 10(th) type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4(th) FAS1 domain of betaig-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4(th) FAS1 domain of betaig-h3 or 9(th) and 10(th) type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.

Crystallization and preliminary crystallographic analysis of the fourth FAS1 domain of human BigH3

The protein BigH3 is a cell-adhesion molecule induced by transforming growth factor-beta (TGF-beta). It consists of four homologous repeat domains known as FAS1 domains; mutations in these domains have been linked to corneal dystrophy. The fourth FAS1 domain was expressed in Escherichia coli B834 (DE3) (a methionine auxotroph) and purified by DEAE anion-exchange and gel-filtration chromatography. The FAS1 domain was crystallized using the vapour-diffusion method. A SAD diffraction data set was collected to a resolution of 2.5 A at 100 K. The crystal belonged to space group P6(1) or P6(5) and had two molecules per asymmetric unit, with unit-cell parameters a = b = 62.93, c = 143.27 A, alpha = beta = 90.0, gamma = 120.0 degrees.