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.     

人早期胎盘绒毛纤维连接蛋白的分离纯化及鉴定

人妊娠５－８周的胎盘绒毛经匀浆后,用２ｍｏ１／Ｌｕｒｅａ－ＰＢＳ提取,通过Ｈｅｐａｒｉｎ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱层析,再经ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ凝胶过滤层析,得到人早期胎盘纤维连接蛋白（ｅａｒｌｙｐｌａｃｅｎｔａｆｉｂｒｏｎｅｃｔｉｎ,ｅｐＦＮ）。经还原及非还原ＳＤＳ－ＰＡＧＥ和免疫印迹电泳分析,ｅｐＦＮ分子量约５００ｋＤ,是由两个２５０ｋＤ亚基组成,与人足月胎盘纤维连接蛋白（ｔｅｒｍｐｌａｃｅｎｔａｆｉｂｒｏｎｅｃｔｉｎ,简称ｔｐＦＮ）相似,而大于人血浆纤维连接蛋白（ｐｌａｓｍａｆｉｂｒｏｎｅｃｔｉｎ,ｐＦＮ）。ｅｐＦＮ与抗人ｐＦＮ抗体及抗人羊水纤维连接蛋白（ａｍｎｉｏｔｉｃｆｌｕｉｄｆｉｂｒｏｎｅｃｔｉｎ,简称ａｍＦＮ）的三个主要功能区单抗均可发生反应。与五种植物凝集素结合力实验表明,ｅｐＦＮ在糖基组成上与ｐＦＮ和ｔｐＦＮ均不相同。     

A novel method for purification of plasma fibronectin

Plasma fibronectin was purified from a gelatin-affinity chromatography column by elution with glucose. This procedure was effective only if the gelatin was particulate when it was attached to the Sepharose 4B. Glucose could not elute fibronectin from the gelatin if the gelatin was melted before it was attached to the Sepharose 4B. This new purification technique has the advantage of using very mild conditions for the isolation of plasma fibronectin.     

A rapid method for fibronectin purification on nitrocellulose membranes suitable for tissue culture

We have developed a simple method for plasma fibronectin purification based on the well-known gelatin binding property of fibronectin. In this procedure we immobilize the melted gelatin to nitrocellulose membranes; these are then used to affinity-purify the fibronectin from the plasma sample. The fibronectin is eluted from the membrane by treatment with 8 M urea. The procedure described here gives a yield of up to 60% (from presumed fibronectin concentration) and the fibronectin obtained is homogeneous in SDS-PAGE and biologically active, as assessed by a cell migration assay. The method is rapid, simple, inexpensive, does not require the use of chromatographic equipment and is suitable for tissue culture applications.     

明胶亲和层析介质分离纯化猪血浆纤维结合蛋白

目的：研究猪血浆中纤维结合蛋白的分离纯化方法,得到高纯度、高活性的目标蛋白。方法：以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,考察了不同洗脱方式对纯化结果的影响,对产品进行了纯度、生物活性的测定。结果：通过对纯化工艺条件的摸索,采用含不同浓度尿素缓冲液进行分步洗脱,可以得到纯度为95％以上的产品,回收率达到50．96％;经BHK细胞培养实验证明其具有与人血浆纤维结合蛋白同样的生物活性。结论：采用了较温和的洗脱方式以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,提高了动物资源的可利用性。     

Identification of novel and distinct binding sites within tenascin-C for soluble and fibrillar fibronectin

Interactions between fibronectin and tenascin-C within the extracellular matrix provide specific environmental cues that dictate tissue structure and cell function. The major binding site for fibronectin lies within the fibronectin type III-like repeats (TNfn) of tenascin-C. Here, we systematically screened TNfn domains for their ability to bind to both soluble and fibrillar fibronectin. All TNfn domains containing the TNfn3 module interact with soluble fibronectin. However, TNfn domains bind differentially to fibrillar fibronectin. This distinct binding pattern is dictated by the fibrillar conformation of FN. TNfn1-3, but not TNfn3-5, binds to immature fibronectin fibrils, and additional TNfn domains are required for binding to mature fibrils. Multiple binding sites for distinct regions of fibronectin exist within tenascin-C. TNfn domains comprise a binding site for the N-terminal 70-kDa domain of fibronectin that is freely available and a binding site for the central binding domain of fibronectin that is cryptic in full-length tenascin-C. The 70-kDa and central binding domain regions are key for fibronectin matrix assembly; accordingly, binding of several TNfn domains to these regions inhibits fibronectin fibrillogenesis. These data highlight the complexity of protein-protein binding, the importance of protein conformation on these interactions, and the implications for the physiological assembly of complex three-dimensional matrices.     

Isolation and biological characterization of active fragments of the adhesive glycoprotein fibronectin

Cellular fibronectin is a major cell surface glycoprotein that can mediate the adhesion of cells to collagen in vitro. To analyze its mechanism of action, we have undertaken experiments to isolate fragments of fibroblast fibronectin that retain different active sites. In this paper, we describe the purification of three chymotryptic fragments with apparent molecular weights of 40,000, 160,000 and 205,000 from chicken cellular fibronectin. These fragments were electrophoretically pure and retained different biologically active sites, as determined by a series of bioassays and competitive inhibition experiments. The 40K fragment was identified as the collagen-binding fragment. The 160K fragment was found to contain the cell surface-binding site(s) of cellular fibronectin. The 205K fragment contained both collagen-binding and cell surface-binding sites, and apparently represents the sum of the 40K and 160K fragments. When native fibronectin is cleaved to the 205K fragment, a polypeptide region containing all interchain disulfide bonds is lost. This alteration was accompanied by decreased hemagglutinating activity and loss of the capacity to restore a normal morphology to transformed cells, whereas cell attachment to collagen and cell spreading activities remained. Our results directly support the idea that the fibronectin molecule consists of separate structural domains containing different biological characteristics.     

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.     

Shape,conformation and stability of fibronectin fragments determined by electron microscopy,circular dichroism and ultracentrifugation

Human plasma fibronectin digested with cathepsin D yields a number of fragments: an N-terminal fibrin-binding 30K³ fragment, a gelatin-binding 40K fragment, a 70K fragment containing the 30K and 40K domains, a central 95/105K fragment and a heparin-binding 140K fragment. The 140K fragment is linked by disulphide bonds and forms the C-terminal ends of the fibronectin. Electron microscopy of rotary-shadowed specimens revealed the 40K, 70K and 95/105K fragments as thin rods (diameter about 2.2 nm) with lengths of 13, 25 and 25 nm. respectively. These dimensions agree with the distances between flexible, proteasesusceptible regions in individual fibronectin strands determined previously. The 140K fragment appeared as a V-shaped structure with two arms 21.5 nm long emerging at a preferred angle of about 70 °. The distribution function of this angle was identical to that observed for the angle between the two arms of intact fibronectin. The free energy required for a distortion of this angle by 60 ° was estimated to be 8 kJ/mol. Reduced and alkylated fibronectin was visualized as single strands (about 55 nm long) with kinks of variable angles corresponding to sites of increased flexibility. The hydrodynamic shapes of the fragments in solution were similar to the shapes observed by electron microscopy, indicating that the individual domains of fibronectin maintain their native structure after proteolytic separation. This was also demonstrated by circular dichroism (c.d.) studies. The c.d. spectrum and the thermal melting curve of native fibronectin were well represented by a linear combination of the contributions of the fragments, indicating conformational independence of the domains. The data support earlier findings that fibronectin consists of two thin strands (length about 60 nm), which are composed of several domains separated by flexible and protease-susceptible intervening regions. This very extended structure agrees with the small sedimentation constant found for fibronectin under conditions where electrostatic interactions between domains are repulsive or depressed. Probably because of such interactions the sedimentation constant is larger at neutral pH and low ionic strength, but even under these conditions a very asymmetric shape is still maintained.     

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.     

Mutual inhibitory activities of tumor-degenerating factor (TDF) and fibronectin

It was reported in our previous studies that the "spongy degeneration-like" changes of human tumor cells were detected by coculture with human embryonic fibroblasts in vitro. It was discovered that the degenerative changes were mediated by a factor secreted from human embryonic fibroblasts. This factor was named the tumor-degenerating factor (TDF). The present study found that fibronectin inhibited TDF activity while TDF inhibited cell attachment mediated by fibronectin. It was possible that these mutual inhibitions were due to the direct binding of the TDF molecule to the fibronectin molecule. Since it is well known that fibronectin is composed of multiple domains which differ in their biological activities, this study also attempted to clarify which domain(s) inhibit TDF activity, through the use of trypsin, thermolysin and 2-nitro-5-thiocyanobenzoic acid (NTCB). It is concluded that multiple domains of fibronectin are required for the inhibition of TDF activity.     

Domain organization of the extracellular region of CD45.

CD45 is a large, heavily glycosylated, transmembrane protein phosphotyrosine phosphatase found on all nucleated cells of haematopoietic origin. In lymphocytes, the cytoplasmic phosphatase is necessary for efficient signalling through the antigen receptor but in contrast little is known about the interactions of the extracellular region of the molecule. This consists of a mucin-like region, a novel cysteine-containing region and a region containing three putative fibronectin type III domains. To confirm this organization and to identify parts potentially important for function, we have expressed fragments of the extracellular domain of rat CD45 as recombinant soluble proteins. Proteins corresponding to two, three and four domains of CD45 were expressed in secreted forms. Single domains and constructs for proteins with truncations of the predicted domains were not expressed. This is consistent with the proposed structural organization. Determination of the positions of the disulphide bonds in the N-terminal cysteine-containing region and the first fibronectin type III domain identified novel disulphide bonds within the fibronectin type III domain and an unusual inter-domain disulphide linkage. Circular dichroism spectroscopy indicated that this region of rat CD45 has mainly beta-strand secondary structure and no alpha-helical content. These studies support the proposed domain organization of CD45.     

Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation

The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase.

A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.     

Cryptic domains of tenascin-C differentially control fibronectin fibrillogenesis

The three-dimensional organization of the ubiquitous extracellular matrix glycoprotein fibronectin regulates cell fate and morphogenesis during development; in particular tubule formation that constitutes the vasculature, lung and kidney. Tenascin-C is a matrix protein with a restricted expression pattern; it is specifically up-regulated at sites of fibronectin fibril assembly during development and in remodeling adult tissues. Here we demonstrate that specific domains of tenascin-C inhibit fibronectin matrix assembly whereas full-length tenascin-C does not. These domains act via distinct mechanisms: TNfn1-8 blocks fibrillogenesis by binding to fibronectin fibrils and preventing intermolecular fibronectin interactions whilst FBG acts independently of binding to fibronectin and instead is internalized and causes cytoskeletal re-organization. We also show that TNfn1-8 disrupts epithelial cell tubulogenesis. Our data demonstrate that tenascin-C contains cryptic sites which can control tissue levels of fibrillar fibronectin either by preventing de novo fibril assembly or reducing levels of deposited fibronectin. Exposure of these domains during tissue remodeling may provide a novel means of controlling fibronectin assembly and tubulogenic processes dependent on the assembly of this matrix.     

Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain

Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.     

Purification of the beta subunit of the fibronectin receptor

In this work we describe a method for purification of the beta subunit of the mouse fibronectin receptor (GP135). Cellular glycoproteins were isolated from a detergent extract of SR-Balb tumor cell membranes by two steps of affinity chromatography on lentil lectin-Sepharose and wheat-germ-agglutinin--agarose. This material was subsequently bound to an Affi gel 102 column and eluted by increasing salt concentration. Most of the GP135 was eluted at 80 mM sodium chloride together with a few other components. A final step of hydroxyapatite chromatography in sodium dodecyl sulphate allowed elution of GP135 as a single chromatographic peak. Fractions containing GP135 were identified at each chromatographic step by immunoblotting with a specific antiserum. By this procedure GP135 was purified to homogeneity as judged by SDS-PAGE analysis of 125I-labelled material.     

Collagen binding by the mannose receptor mediated through the fibronectin type II domain

The macrophage mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of an N-terminal cysteine-rich domain related to the R-type carbohydrate-recognition domain of ricin, a fibronectin type II domain and eight to ten domains related to C-type carbohydrate-recognition domains. The mannose receptor acts as a molecular scavenger, clearing harmful glycoconjugates or micro-organisms through recognition of their defining carbohydrate structures. Cell-adhesion assays, as well as collagen-binding assays, have now been used to show that the mannose receptor can also bind collagen and that the fibronectin type II domain mediates this activity. Neither of the two types of sugar-binding domain in the receptor is involved in collagen binding. Fibroblasts expressing the mannose receptor adhere to type I, type III and type IV collagens, but not to type V collagen, and the adherence is inhibited by isolated mannose receptor fibronectin type II domain. The fibronectin type II domain shows the same specificity for collagen as the whole receptor, binding to type I, type III and type IV collagens. This is the first activity assigned to the fibronectin type II domain of the mannose receptor. The results suggest additional roles for this multifunctional receptor in mediating collagen clearance or cell-matrix adhesion.     

ADAM13 disintegrin and cysteine-rich domains bind to the second heparin-binding domain of fibronectin

ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.     

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates. At the same time, heparin or two monoclonal antibodies against the heparin-binding domain were able to inhibit the haptotactic migration to CH-271 or fibronectin, though not to C-274 or a mixture of C-274 and H-271. This suggests that although heparin did not affect tumor cell adhesion to the cell-binding domain near the heparin-binding domain in CH-271 or fibronectin, it did lead to a modulation of cell motility. It seems likely that the regulatory mechanism may depend on interaction between heparin-like molecules on the cell surface and the heparin-binding domain in fibronectin, rather than on simple steric hindrance or on the masking of the cell-binding domain caused by the binding of heparin to heparin-binding domain.