Cell Attachment Domain Research Articles

Fibronectin mediates adherence of rat alveolar type II epithelial cells via the fibroblastic cell-attachment domain.

The lung alveolar surface is composed of types I and II epithelial cells. Extremely attenuated type I cells cover 90% of the surface and are prone to necrosis during acute lung injury. After denudation of type I cells, the alveolar epithelium is restored by proliferation of type II cells. During reepithelialization in vivo the type II cells have been observed to reorganize on an extracellular matrix that contains fibronectin. We thus sought to determine whether type II cells would adhere to purified fibronectin. Adherence assays of primary rat type II cells were performed in protein-coated bacteriologic microtiter wells for 24 h at 37 degrees C. Concentrations of fibronectin from 1 to 300 micrograms/ml mediated type II cell adherence, 10 micrograms/ml gave maximal adherence, and 4 micrograms/ml gave 50% maximal adherence. Adherence progressively increased from 1 to 72 h. Adherence on fibronectin was at least 50% greater than adherence on laminin, types I and III collagen, or IV collagen. Little or no adherence was observed on bacteriologic plastic or albumin. Spreading on these various substrata paralleled adherence. Adherence to fibronectin, laminin, and fibrinogen was specifically blocked by their respective polyclonal antibodies. Monoclonal antibodies (MoAb) to the fibronectin cell-attachment domain blocked adherence to fibronectin, whereas MoAb to other domains did not. From the data reported here and the previously mentioned in vivo study we propose that fibronectin is an important functional component of the extracellular matrix that supports type II cells during alveolar reepithelialization.

Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast.

NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found. Our results further establish that the ability of a cell to organize fibronectin into an extracellular matrix is dependent on certain gangliosides, but they also indicate that cell adhesion to fibronectin is independent of these gangliosides. We suggest that matrix organization and cell attachment and spreading are based on separate mechanisms and that these functions are associated with different cell surface "receptors."

Evolution of the fibronectin gene. Exon structure of cell attachment domain.

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.

Functional expression in Escherichia coli of cloned reovirus S1 gene encoding the viral cell attachment protein σ1

A cDNA clone encompassing the complete reovirus (serotype 3) Sl gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5′ end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-σ1 antibodies. Like authentic soluble σ1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. 2 2 Abbreviations used: HA, hemagglutination; IPTG, isopropyl-β- d-thiogalactoside; PBS, phosphate-buffered saline [0.14 M NaCl, 2.7 m M KCI, 8 m M Na 2HPO 4, 1.5 m M KH 2PO 4 (pH 7.2)]; PMSF, phenylmethylsulfonylfluoride; IgG, immunoglobulin G; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. Such hemagglutination was inhibited by a monoclonal anti-σl antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.

Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.

The ability of fibronectin to bind cells can be accounted for by the tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine, a sequence which is part of the cell attachment domain of fibronectin and present in at least five other proteins. This tetrapeptide may constitute a cellular recognition determinant common to several proteins.

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin.

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.

Synthetic peptide with cell attachment activity of fibronectin.

Four synthetic peptides that together constitute the cell attachment domain of fibronectin [Pierschbacher, M.D., Ruoslahti, E., Sundelin, J., Lind, P. & Peterson, P. (1982) J. Biol. Chem. 257, 9593-9597] were constructed and tested for their ability to induce cell attachment and spreading. One of these peptides, consisting of the 30 amino acid residues nearest the COOH terminus of the domain, contained all of the cell attachment activity of the whole domain. Under suitable conditions the peptide was approximately as active as intact fibronectin on a molar basis. The activity could be demonstrated by binding the peptide to polystyrene directly, or via albumin, or by coupling it to agarose beads. This synthetic peptide will be useful in the elucidation of the molecular details of the attachment of cells to fibronectin and could allow manipulation of the adhesive properties of cell culture surfaces and prosthetic materials.

The cell attachment domain of fibronectin. Determination of the primary structure.

The complete amino acid sequence of the cell attachment domain of human plasma fibronectin (Pierschbacher, M. D., Hayman, E. G., and Ruoslahti, E. (1981) Cell 26, 259-267) has been determined by automated sequential degradation of a peptic fragment comprising this region and of peptides derived from this fragment by digestion with thermolysin, staphylococcal V8 protease, cyanogen bromide cleavage, and partial acid hydrolysis. The fragment contains 108 residues with isoleucine and methionine as the NH2- and carboxyl-terminal amino acids, respectively. No cysteines are present. The calculated molecular weight of the cell attachment fragment, based on the amino acid sequence, is 11,482, which is in good agreement with the molecular weight estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and ultracentrifugation. There are no homologies in this fragment with other published sequences. The implications of the structure of the cell attachment fragment to the molecular mechanism of cell-fibronectin interaction are discussed.

Molecular and Biological Interactions of Fibronectin

Fibronectin is a high molecular weight glycoprotein present at cell surfaces and in various body fluids. It is involved in cellular adhesion. The frequent absence of fibronectin from the surface of malignant cells may contribute to the invasive growth of tumors. To develop a better understanding of the functions of fibronectin, we have studied its structure by making use of proteolytic fragmentation and monoclonal antibodies. Fragments of plasma fibronectin which retain one or several of the affinities of intact fibronectin have been purified using affinity chromatography on collagen and heparin, and on monoclonal antibodies. These studies reveal a structure in which the different functions are located in separate protease-resistant molecular domains. The NH2-terminal domain contains the binding sites for fibrin, actin, and Staphylococci; this is followed by a collagen-binding domain, the cell attachment domain, and at the COOH-terminal end, the heparin-binding domain. These binding sites allow fibronectin to participate in multiple interactions with collagens and proteoglycans. The insoluble complexes formed by these 3 components that can be generated in vitro may represent a model for the basic structure of extracellular matrices. The role of fibronectin in such a matrix may be to contribute to the stability of the complex and to provide adhesion sites for cells. Disturbances in the interactions involving fibronectin could be potentially important to understanding diseases of connective tissues and in malignancy.

Molecular and biological interactions of fibronectin.

Fibronectin is a high molecular weight glycoprotein present at cell surfaces and in various body fluids. It is involved in cellular adhesion. The frequent absence of fibronectin from the surface of malignant cells may contribute to the invasive growth of tumors. To develop a better understanding of the functions of fibronectin, we have studied its structure by making use of proteolytic fragmentation and monoclonal antibodies. Fragments of plasma fibronectin which retain one or several of the affinities of intact fibronectin have been purified using affinity chromatography on collagen and heparin, and on monoclonal antibodies. These studies reveal a structure in which the different functions are located in separate protease-resistant molecular domains. The NH2-terminal domain contains the binding sites for fibrin, actin, and Staphylococci; this is followed by a collagen-binding domain, the cell attachment domain, and at the COOH-terminal end, the heparin-binding domain. These binding sites allow fibronectin to participate in multiple interactions with collagens and proteoglycans. The insoluble complexes formed by these 3 components that can be generated in vitro may represent a model for the basic structure of extracellular matrices. The role of fibronectin in such a matrix may be to contribute to the stability of the complex and to provide adhesion sites for cells. Disturbances in the interactions involving fibronectin could be potentially important to understanding diseases of connective tissues and in malignancy.