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PubMed1990

Peptide analogs to a fibronectin receptor inhibit attachment of Staphylococcus aureus to fibronectin-containing substrates.

Binding of cells of Staphylococcus aureus to fibronectin has been proposed as a mechanism of bacterial adhesion to host tissues. In this study, we have attempted to define the role of a recently identified fibronectin receptor in the adhesion of staphylococcal cells to fibronectin-containing substrates by using different receptor analogs as potential inhibitors of bacterial adherence. The results showed that synthetic peptides D1, D2, and D3, corresponding to variations of a repeated unit in the fibronectin-binding domain of the receptor, and ZZ-FR, a chimeric protein containing the fibronectin-binding domain of the receptor with the D1, D2, and D3 sequences, inhibited the attachment of staphylococcal cells to microtiter wells coated with intact fibronectin or with the 29-kilodalton amino-terminal fragment of fibronectin. The chimeric protein ZZ-FR also partially inhibited the adherence of staphylococci to human plasma clots formed in vitro but had no effect on bacterial adhesion to clots formed from fibronectin-depleted plasma. These data confirm previous reports suggesting that fibronectin may serve as a substrate for adhesion of staphylococcal cells and indicate that bacterial adhesion is mediated by the identified fibronectin receptor. Furthermore, analogs to the fibronectin receptor can be used to inhibit the adhesion of bacterial cells to these model substrates, and these analogs may be of clinical use.

Animals↗
PubMed1988

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.

Cell Line↗
PubMed1984

Characterization of fibronectin from fetal human plasma in comparison with adult plasma fibronectin.

Fibronectin was purified from fetal human plasma and characterized in comparison with fibronectin from adult human plasma. Fetal plasma fibronectin had an amino-acid composition, immunological properties, and cell-attachment-promoting activity similar to those of adult plasma fibronectin. However, fetal plasma fibronectin was shown to have a distinct carbohydrate composition which is characterized by the presence of fucose. To ascertain the differences in the carbohydrate moiety, 14C-labeled glycopeptides were prepared and sequentially analyzed with columns of immobilized concanavalin A and lentil lectin. Glycopeptides from fetal plasma fibronectin contained a population of glycopeptides which bound to both lectin gels. Almost all of the glycopeptides in this population lost their ability to bind to lentil lectin upon fucosidase digestion, indicating that fetal plasma fibronectin possesses a substantial amount of fucosylated biantennary glycans. In contrast, glycopeptides from adult plasma fibronectin practically lacked such glycopeptides. Another difference observed was that fetal plasma fibronectin had a larger amount of concanavalin A-unbound glycopeptides than did adult plasma fibronectin. These results indicate the presence of age-related variation in glycosylation of human plasma fibronectin.

Adult↗
PubMed1996

Formation of sodium dodecyl sulfate-stable fibronectin multimers. Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin.

Fibronectin exists in a soluble form in body fluids and as a fibrillar component of the extracellular matrix. Matrix fibronectin associates as large complexes in SDS unless a reducing reagent is also present. This observation suggests that complex formation is due to interprotomeric disulfides that form by thiol-disulfide exchange. To localize the presumptive new disulfides, we labeled protomeric fibronectin by the chloramine-T method or with 125I-Bolton-Hunter reagent, incorporated 125I-fibronectin into the matrix of cultured fibroblasts, and subjected matrix fibronectin to acid or cyanogen bromide digestion. When cyanogen bromide digests of matrix 125I-fibronectin and protomeric 125I-fibronectin labeled with Bolton-Hunter reagent were analyzed by two-dimensional polyacrylamide gel electrophoresis in SDS, with the first dimension being nonreducing and the second reducing, we were not able to identify any fragments of matrix fibronectin that migrated as high molecular weight complexes in the first dimension. Limited acid digestion of matrix 125I-fibronectin also dissociated the majority of the high molecular weight complexes. Since we could account for all of the parts of fibronectin that contain cysteine or cystine, we conclude that matrix fibronectin is not stabilized by interprotomeric disulfides. We propose, instead, that stabilization is mediated by noncovalent protein-protein interactions that are sensitive to reduction, cyanogen bromide digestion, or limited acid digestion.

Acids↗
PubMed1999

Characterization of the fibronectin binding motif for a unique mycobacterial fibronectin attachment protein, FAP.

Studies were performed to define the fibronectin binding motif of the previously identified Mycobacterium avium fibronectin attachment protein (FAP-A). Using synthetic peptides of a previously identified fibronectin binding region (amino acids 269-292), the minimal binding sequence was determined to be 12 amino acids, 269-280 (FAP-A-(269-280)). Synthetic peptides were prepared in which each amino acid in the 269-280 sequence was substituted with Ala. Assessment of the effect of Ala substitution on fibronectin binding showed that the presence of Ala at amino acids 273-276 (RWFV) completely abrogated fibronectin binding activity. Furthermore, the ability to inhibit the attachment of viable Mycobacterium bovis BCG to fibronectin was abrogated by Ala substitution at the RWFV sites. To validate the function of RWFV, further studies were performed with recombinant FAP-A in which single Ala mutations were generated for the RWFV sites and as controls at amino acids 269 and 280. Mutant FAP-A containing single Ala substitutions at the RWFV sites (amino acids 273, 274, 275, or 276) showed significant abrogation of fibronectin binding function. Recombinant FAP-A with Ala substitutions at either 269 or 280 showed wild type activity. When the four essential amino acids (RWFV) were either substituted en bloc with Ala or were all deleted, complete loss of fibronectin binding function was observed. Control recombinant proteins with en bloc Ala substitutions or deletions at four positions outside the fibronectin binding region (amino acids 255-257) retained functional activity. These data show that the RWFV sequence is necessary for fibronectin binding function of FAP-A. Furthermore, the data suggest that mycobacterial FAP proteins, all of which share the RWFV binding motif, constitute a family of highly homologous proteins that bind fibronectin in a unique manner.

Adhesins, Bacterial↗
PubMed2002

Chinese hamster ovary cell motility to fibronectin is modulated by the second extracellular loop of CD9. Identification of a putative fibronectin binding site.

CD9, a member of the tetraspanin family of proteins, is characterized by four transmembrane domains and two extracellular loops. Surface expression of CD9 on Chinese hamster ovary (CHO) cells dramatically enhances spreading and motility on fibronectin. To elucidate the mechanistic basis of CD9-fibronectin interaction, binding to fibronectin was investigated using purified and recombinant forms of CD9. The affinity of fibronectin for CD9 in enzyme-linked immunosorbent assay was 81 +/- 25 nm. The binding of fibronectin to immobilized CD9 was enhanced by Ca(2+) ions. Protein binding and peptide competition studies demonstrated that peptide 6 derived from CD9 extracellular loop 2 (amino acids 168-192) contained part of the fibronectin-binding domain. Additionally, enhanced adhesion of CD9-CHO-B2 cells to fibronectin was significantly reduced by peptide 6. CD9-CHO cells had a 5-fold increase in motility to fibronectin as compared with mock-transfected controls, an effect that correlated with CD9 cell surface density. Truncation of CD9 extracellular loop 2 and peptide 6 caused inhibition of CD9-CHO cell motility to fibronectin. Deletion of CD9 extracellular loop 1 had no significant effect on CHO cell motility. These findings demonstrate a critical role for CD9 extracellular loop 2 in cell motility to fibronectin and clarify the mechanism by which CD9-fibronectin interaction modulates cell adhesion and motility.

Amino Acid Sequence↗
PubMed1993

Fibronectin binding site in type I collagen regulates fibronectin fibril formation.

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.

Amino Acid Sequence↗
PubMed1979

Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix.

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.

Animals↗
PubMed1994

Expression of fibronectin and a fibronectin-binding molecule during preimplantation development in the mouse.

Using an indirect immunofluorescent technique, expression of cell surface fibronectin and a cell surface fibronectin-binding molecule was studied during mouse embryo preimplantation development. We also studied the expression of fibronectin on immunosurgically isolated inner cell masses (ICMs) and regenerated mouse blastocysts. Fibronectin and the fibronectin-binding molecule were not detected at the morula stage. From the early to late blastocyst stage, fibronectin expression increased on the trophectoderm. Expression of the fibronectin-binding molecule was found only in the polar trophectoderm region of the early blastocyst, then in the polar and mural trophectoderm regions of the middle blastocyst. In the late blastocyst stage, this fibronectin-binding molecule was only present in the mural trophectoderm. Fibronectin expression by ICMs of early blastocysts was more intense than that of late blastocysts. After 24 h of culture, 10% of ICMs isolated from early blastocysts regenerated a trophectoderm which stained intensively for fibronectin in the mural and polar trophectoderm regions. After 48 h of culture, regenerated blastocyst-like structures closely resembled the normally obtained late blastocysts and stained for fibronectin in the mural and polar trophectoderm regions. The significance of the results is discussed in relation to mouse embryo development, trophectoderm formation and blastocyst implantation.

Amino Acid Sequence↗
PubMed1985

Plasma fibronectin contributes to fibronectin in tissues.

Fibronectin is produced by several cell types, with hepatocytes currently recognized as the main source of plasma fibronectin. Mesenchymal cells produce fibronectin in vitro and probably do so even in vivo. But it is not clear if these cells represent the sole source of tissue fibronectin. In the present study, using direct and indirect immunofluorescence, fibronectin was found to occur ubiquitously in connective tissue in the rat. When 125I-labelled fibronectin was intravenously injected in rat, there was significant uptake of fibronectin in the tissues. The half-time for removal of labelled fibronectin in circulation was c. 15 hours. The authors conclude that flux of fibronectin occurs from plasma into the tissues and that in normal circumstances plasma fibronectin contributes to fibronectin in tissues.

Animals↗
PubMed1994

Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin.

Cultured fibroblasts express binding sites for the amino-terminal region of fibronectin on their cell surface that mediate the assembly of soluble fibronectin into disulfide-stabilized fibrils. These binding sites have been termed matrix assembly sites and have been studied in binding assays using a 125I-labeled 70-kDa fragment derived from the amino terminus of fibronectin. In an attempt to isolate the protein(s) responsible for binding the 70-kDa fragment, cell surface proteins were cleaved from fibroblast monolayers by mild trypsinization. Trypsinization of monolayers generated a series of fibronectin fragments that bound the 125I-labeled 70-kDa fragment by ligand blot assay and affinity chromatography. All of the fibronectin fragments that bound the 70-kDa fragment contained the III-1 module. In solid phase binding assays, the 125I-labeled 70-kDa fragment bound preferentially to reduced fibronectin as compared with unreduced fibronectin fragments. Binding of the 125I-labeled 70-kDa fragment to reduced fibronectin was inhibited by a monoclonal antibody directed against the III-1 domain. Isolated III-1, however, did not bind the 125I-labeled 70-kDa fragment when adsorbed to plastic tissue culture wells. Heat denaturation of III-1 prior to adsorption conferred 70-kDa fragment binding properties on the isolated module. The 125I-labeled 70-kDa fragment did not bind to heat-denatured III-2, suggesting that 70-kDa fragment binding was a property of the III-1 module and not a general characteristic of all type III modules. The binding of 125I-labeled 70-kDa fragment to III-1 was of high affinity (KD = 1.8 x 10(-8) M). These results indicate that a binding site for the 70-kDa amino terminus of fibronectin is contained within a cryptic site found in the first type III module of fibronectin. Unfolding of the III-1 module on the cell surface may control matrix assembly site expression and represent an important step in the initiation of cell-dependent fibronectin polymerization.

Base Sequence↗
PubMed1989

A neuroectoderm-associated ganglioside participates in fibronectin receptor-mediated adhesion of germinal cells to fibronectin.

Previous studies with a rat neural cell line have shown that the D1.1 ganglioside, an O-acetylated derivative of GD3, is involved in cellular adhesion to fibronectin. In vivo, D1.1 is present on germinal cells of the developing rat central nervous system, but not on postmitotic cells that migrate away from the germinal zones. These observations suggest that D1.1 could participate in adhesive interactions with germinal zones and that the loss of D1.1 could be involved in the decision to being migration. In support of this hypothesis, immunofluorescence histochemistry shows that both fibronectin and fibronectin receptor are colocalized with D1.1 in the ventricular zones of the embryonic rat brain and in the external granule cell layer of the postnatal cerebellum. Dishes coated with monoclonal antibody against D1.1 were used to isolate D1.1-positive germinal cells from Embryonic Day 14 cerebrum and from Postnatal Day 6 cerebellum. These cells are able to adhere to fibronectin-coated dishes by a mechanism that is inhibitable by a synthetic hexapeptide containing the arg-gly-asp cell recognition sequence of fibronectin. Adhesion is also partially inhibited by antibody against fibronectin receptor and is slowed by anti-D1.1 antibody, implicating both the receptor and the ganglioside in the adhesion process. During 3 days in culture these D1.1-positive, fibronectin receptor-positive cells exhibit a neuronal phenotype, as judged by morphology and staining with tetanus toxin. This further confirms the neuroepithelial origin of the cells. The cells do not synthesize detectable amounts of fibronectin, thus leaving unidentified the source of the fibronectin seen in the germinal zones in tissue sections. Immunoprecipitation experiments show that the fibronectin receptors present on these cells are heterodimers. Under nonreducing conditions, the immunoprecipitates contain an alpha-subunit of 150-160 kDa and a beta-subunit of 115-125 kDa.

Age Factors↗
PubMed1988

Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin.

We have used a rat neural cell line, B65, to investigate the relative contributions of gangliosides and glycoprotein receptors in adhesion to fibronectin. Monoclonal antibodies against two neuroectoderm-associated gangliosides, D1.1 and GD3, inhibit the rate of B65 attachment to fibronectin, suggesting that these gangliosides are involved in the adhesion process. Adhesion to fibronectin is not affected by a third monoclonal antibody against a separate, unidentified cell-surface component of B65 cells. Furthermore, B65 cells lacking D1.1 adhere to fibronectin at a slower rate than B65 cells that express D1.1. The involvement of glycoprotein receptors in adhesion is demonstrated by the ability of antibodies against human fibronectin receptor to inhibit B65 attachment to fibronectin. In addition, adhesion is blocked by a hexapeptide containing the Arg-Gly-Asp fibronectin sequence which is necessary for binding to the receptor. Trypsin treatment of B65 cells in the absence of divalent cations results in proteolysis of the fibronectin receptor with an accompanying loss of ability of the cells to attach to fibronectin. D1.1 and GD3 expression is not affected by this trypsinization, indicating that the gangliosides alone are incapable of mediating attachment. The glycoprotein receptors must be primarily responsible for adhesion to fibronectin with the gangliosides playing a secondary role as enhancers or modulators.

Animals↗
PubMed1994

Monoclonal antibody FDC-6 exhibits binding to human plasma fibronectin: a caveat for cervicovaginal oncofetal fibronectin testing?

OBJECTIVE: Monoclonal antibody FDC-6 has been used clinically to measure cervicovaginal fibronectin and to predict patients at risk for preterm labor. Because bleeding is often a complicating factor during pregnancy, the aim of this study was to determine whether FDC-6 reactive fibronectin is present in normal human plasma. STUDY DESIGN: Quantitative Western immunoblots and a commercially available enzyme-linked immunosorbent assay were used to measure FDC-6 binding to nonpregnant human and bovine plasma fibronectin. FDC-6 binding to whole sera was also assessed, as was the effect of enzymatic deglycosylation of human plasma fibronectin. RESULTS: Both assays demonstrated measurable FDC-6-reactive fibronectin in nonpregnant human blood, with plasma concentrations ranging from 3 to 12 micrograms/ml (1% to 4% of total plasma fibronectin). FDC-6 had no detectable binding activity to bovine plasma fibronectin. De-O-glycosylated human plasma fibronectin had markedly reduced binding activity for FDC-6. CONCLUSIONS: FDC-6 binds specifically to a clinically significant percentage of circulating O-glycosylated human plasma fibronectin isoforms that are not associated with pregnancy. This caveat should be considered when pregnant patients are evaluated for the presence of FDC-6 reactive oncofetal fibronectin in the cervix or vagina.

Animals↗
PubMed1997

N-terminal type I modules required for fibronectin binding to fibroblasts and to fibronectin's III1 module.

Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fiftBiomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706U.S.A. Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conh as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

Animals↗
PubMed1986

On the origin of species specificity of fibronectin. Immunological identification of a species-specific domain of human fibronectin.

We have investigated whether species-specific epitopes of human fibronectin are localized at a specific domain of fibronectin using rabbit polyclonal antibodies. Tryptic fragments of human fibronectin were tested for reactivity with anti-human fibronectin antibody, which had been previously absorbed with other animal fibronectin to establish species specificity. Human-specific epitopes were found to be present on 75,000, 65,000, and 42,000 dalton fragments. The 42,000-dalton fragment shares almost all the epitopes with the 75,000 and 65,000 dalton fragments. It does not promote BHK cell spreading, whereas the 75,000 and 65,000 dalton fragments do. The amino acid sequence from the amino terminus of the 42,000-dalton fragment is Asp/Gly-Gln/Val-?-Ile-Val-, which is almost identical to the sequence Asp-Gln-Cys-Ile-Val- located in the carboxyl terminal 1/3 of the collagen-binding domain of human fibronectin (Kornblihtt et al. (1985) EMBO J. 4, 1755-1759). These results suggest that human fibronectin bears human-specific epitopes mainly on the amino-terminal half of domain 4 (Hayashi & Yamada (1983) J. Biol. Chem. 258, 3332-3340) located between the collagen and cell binding domains almost at the center of the fibronectin polypeptide. The domain specific for human fibronectin may be a general species-specific domain of animal fibronectins.

Amino Acid Sequence↗
PubMed2004

Proliferating keratinocytes are putative sources of the psoriasis susceptibility-related EDA+ (extra domain A of fibronectin) oncofetal fibronectin.

The extra domain A of fibronectin (EDA+ oncofetal isoform of fibronectin was recently reported to be overexpressed in psoriatic uninvolved epidermis. It has been proposed that the abnormal presence of EDA+ oncofetal protein at the dermal-epidermal junction in the uninvolved skin may provide the "psoriatic" environment in which keratinocytes are in a preactivated state with regard to mitogenic signals (e.g., T cell lymphokines). To determine the possible sources of cellular fibronectin in the non-lesional psoriatic skin, we aimed to investigate whether keratinocytes could produce the EDA+ oncofetal form of fibronectin. RT-PCR studies revealed that both cultured normal keratinocytes and HaCaT cells express the EDA+ splice variant of fibronectin mRNA, and in HaCaT cells the EDA+/EDA- transcript ratio was elevated compared with normal keratinocytes. Cultured keratinocytes and HaCaT cells showed intracytoplasmic staining with an EDA+ fibronectin-specific antibody and among the positively stained cells many showed mitosis. Using RT-PCR, western blot analysis, and flow cytometry, we showed that in synchronized HaCaT cells the amount of both total fibronectin and its EDA+ isoform change with the proliferation/differentiation state of HaCaT cells and peak in highly proliferating cells. We show that in short-term ex vivo cultures, a small population of EDA+ fibronectin containing cell population appear among psoriatic uninvolved, but not normal epidermal cells. We also demonstrate that cell attachment has a strong influence on the expression of both total and EDA+ fibronectin. Our results suggest that proliferating keratinocytes could be the sources of the psoriasis susceptibility-related EDA+ oncofetal fibronectin in the epidermis.

Alternative Splicing↗
PubMed1985

Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains.

Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domain eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps; hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.

Chemical Phenomena↗