Interaction of fibronectin with heparin in model extracellular matrices: role of arginine residues and sulfate groups

The interaction of heparin with the NH2-terminal domain of human plasma fibronectin was studied by using matrix-driven translocation, an assay for the adhesion of extracellular macromolecules with cell or particle surfaces within artificial collagen matrices. Partial desulfation of heparin rendered it ineffective in competitively inhibiting the interaction of the fibronectin NH2-terminal domain with heparin-coated particles, suggesting a role for sulfate groups of heparin in the interaction. Analysis of the fibronectin domain in terms of its primary structure, its proposed organization into "type I modules", and its hydrophilic and flexible segments led to the identification of several arginine-containing sites of potential interaction with the sulfate groups of heparin. Modification of increasing numbers of arginine side chains with 1,2-cyclohexanedione under mild conditions eventually led to decreases in translocation-promoting activity, and of heparin binding capacity as measured in a gel-shift assay, but the major portions of these functions were retained even when the four most accessible arginines (attributed to sites in and adjacent to the large loops of the type I modules) were modified. With the modification of additional arginines (attributed to sites in the small loops), both functions were lost. The peptide Gly-Arg-Gly, corresponding to a repeated determinant at the tips of two small loops, inhibited translocation, but arginine alone did not. Cleavage of the large loops by CNBr also led to loss of translocation-promoting activity. The correspondence between the molecular determinants of matrix-driven translocation and those previously found for mesenchymal morphogenesis indicates the utility of this system in the analysis of adhesive interactions of biological importance.     

Protein structural requirements for Ca2+ binding to the light chain of factor X. Studies using isolated intact fragments containing the gamma-carboxyglutamic acid region and/or the epidermal growth factor-like domains

Coagulation factor X is a multidomain proenzyme of a serine protease. Calcium ions bind to the vitamin K-dependent gamma-carboxyglutamic acid (Gla) residues and to a site in the NH2-terminal of two epidermal growth factor (EGF)-like domains. To study structure-function relationships in the NH2-terminal part of factor X and to determine the structure of isolated domains, we have developed methods that allow the subsequent isolation of the first or both EGF-like domains with or without an attached Gla domain from controlled proteolytic digests of the protein. The Ca2(+)-induced changes of the intrinsic protein fluorescence were measured to elucidate whether the isolated fragments retain their native conformation. Changes in the fluorescence caused by Ca2+ binding were found to result from perturbations of the environment of the Trp residue in position 41. Calcium ion binding to the Gla-containing region linked to the NH2-terminal EGF-like domain was identical with that to intact factor X, indicating a native orientation of the ligand binding groups in the fragment. In contrast, the isolated Gla peptide had a lower affinity for Ca2+, suggesting that the NH2-terminal EGF-like domain serves as a scaffold for the folding of the Gla region. Similarly, the presence of the Gla region was found to increase the affinity of the Gla-independent site in the first EGF-like domain for Ca2+. The metal ion-induced resistance against chymotryptic cleavage COOH-terminal of Tyr-44 in intact factor X is similar in the isolated fragment that contains the Gla region linked to one EGF-like domain, indicating a native conformation of the fragment in the presence of Ca2+. Furthermore, the Gla-independent metal ion binding site binds Ca2+ but does not appear to bind Mg2+.     

Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit

Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca2+-requiring form (derived microCANP) by limited autolysis in the presence of Ca2+. The NH2-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived microCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5% of that of the native dimeric enzyme. The Ca2+-sensitivity of one hybrid CANP reconstituted from the large subunit of derived microCANP and the small subunit of native mCANP was similar to that of derived microCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived microCANP required a high concentration of Ca2+ for activity, like native mCANP. These results indicate that the Ca2+-sensitivity of derived microCANP is determined by the structural change of the large subunit resulting from loss of its NH2-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca2+-requirement.     

Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes. Translocation of the toxin across the membrane.

Adenylate cyclase (AC) toxin from Bordetella pertussis penetrates eukaryotic cells and upon activation by calmodulin generates unregulated levels of intracellular cAMP. The process of toxin penetration into sheep erythrocytes was resolved into three consecutive steps including insertion, translocation, and intracellular cleavage. Insertion of the toxin into the cell membrane occurred over a wide temperature range (4-36 degrees C). In contrast, translocation of the toxin, i.e. transfer of the NH2-terminal catalytically active fragment across the membrane, occurred only above 20 degrees C and was highly temperature-dependent. While a single exposure of the toxin to Ca2+ was sufficient for its insertion into the plasma membrane, toxin translocation required exogenous Ca2+ at mM concentrations. Translocation was not affected by pretreatment of cells with trypsin, N-ethylmaleimide, and sodium carbonate at alkaline pH. The NH2-terminal fragment of the toxin was cleaved in the cell releasing the 45-kDa active AC into the cytosol. The cleavage was blocked by treatment of cells with N-ethylmaleimide. It is hypothesized that the COOH-terminal portion of the toxin creates in the membrane a channel through which the NH2-terminal fragment is translocated.     

The actin filament-severing domain of plasma gelsolin

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.     

Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX

Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bj?rk, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.     

Domain structure of human plasma and cellular fibronectin. Use of a monoclonal antibody and heparin affinity to identify three different subunit chains

The domain structure of human plasma fibronectin was investigated by using heparin-binding and antibody reactivity of fibronectin and its proteolytically derived fragments. Digestion of human plasma fibronectin with a combination of trypsin and cathepsin D produced six major fragments. Affinity chromatography showed that one fragment (Mr 45 000) binds to gelatin and three fragments (Mr 31 000, 36 000, and 61 000) bind to heparin. The 31K fragment corresponds to NH2-terminal fragments isolated from other species. The 36K and 61K fragments are derived from a region near the C-terminus of the molecule and appear to be structurally related as demonstrated by two-dimensional peptide maps. A protease-sensitive fragment (Mr 137 000), which binds neither gelatin nor heparin but which has been shown previously to be chemotactic for cells [Postlethwaite, A. E., Keski-Oja, J., Balian, G., & Kang, A. H. (1981) J. Exp. Med. 153, 494-499], separates the NH2-terminal heparin- and gelatin-binding fragments from the C-terminal 36K and 61K heparin-binding fragments. A monoclonal antibody to fibronectin that recognized the 61K heparin-binding fragment was used to isolate a sixth fragment (Mr 34 000) that did not bind to heparin or gelatin and that represents a difference between the 61K and 36K heparin-binding fragments. Cathepsin D digestion produced an 83K heparin-binding, monoclonal antibody reactive fragment that contains the interchain disulfide bond(s) linking the two fibronectin chains at their C-termini. The data indicate that plasma fibronectin is a heterodimeric molecule consisting of two very similar but not identical chains (A and B). In contrast, enzymatic digestion of cellular fibronectin produced a 50K heparin-binding fragment lacking monoclonal antibody reactivity which suggests that the cellular fibronectin subunit is similar to the plasma A chain in enzyme susceptibility but contains a larger heparin-binding domain. A model relating the differences in the three fibronectin polypeptides to differences in published cDNA sequences is presented.     

Modulation of troponin T molecular conformation and flexibility by metal ion binding to the NH2-terminal variable region

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

A proteolytic NH2-terminal truncation of cardiac troponin I that is up-regulated in simulated microgravity

In a tail suspension rat model, we investigated changes in myofilament protein during cardiac adaptation in simulated microgravity. Contractile force and velocity of cardiac muscle were decreased in the tail suspension rats as compared with the control. Ca(2+)-dependent actomyosin ATPase activity was also decreased; however, sensitivity of cardiac muscle to Ca(2+) activation was unchanged. There was no change in expression of myosin heavy chain, tropomyosin, troponin T, or troponin I isoforms in hearts of tail suspension rats. A novel finding is a fragment of cardiac troponin I (cTnI) that had increased amounts in the heart of tail suspension rats. Binding of this cTnI fragment by a monoclonal antibody that specifically recognizes the COOH terminus indicates an intact COOH terminus. NH(2)-terminal sequence analysis of the cTnI fragment revealed truncations primarily of amino acids 1-26 and 1-27 and smaller amounts of 1-30, including Ser(23) and Ser(24), which are substrates of protein kinase A phosphorylation. This cTnI fragment is present in normal cardiac muscle and incorporated into myofibrils, indicating a role in regulating contractility. This proteolytic modification of cTnI up-regulated during simulated microgravity suggests a potential role of the NH(2)-terminal segment of cTnI in functional adaptations of cardiac muscle.     

The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Interaction of the Two Structural Domains of Calmodulin with Mature and Immature Rat Brain Microtubules

The inhibitory effect of calmodulin on the assembly of mature and immature rat brain microtubules was compared with that of the two major structural domains of this protein, the COOH-terminal fragment (amino acids 78-148) and the NH2-terminal fragment (amino acids 1-77), to determine the calmodulin structural domain responsible for the inhibitory effect on microtubule assembly. Microtubules prepared during the early stages of brain development, i.e., during intensive neurite outgrowth, are more sensitive to inhibition by the Ca2(+)-calmodulin complex than those obtained from adult brain. Significant inhibition of immature microtubule assembly was observed with both fragments in the absence of Ca2+, but the effects were more important when Ca2+ was present. With adult brain microtubules, the two fragments remained without effect on assembly in the absence of Ca2+, whereas some inhibition was seen in its presence but only with the COOH-terminal polypeptide. Under all these conditions, the COOH-terminal fragment was always more active than the NH2-terminal fragment on microtubule polymerization, albeit to a lesser extent than native calmodulin.     

Absence of a cleavable signal sequence in Sindbis virus glycoprotein PE2.

Partial NH2-terminal sequence analysis has been performed on some products that result from the translation of 26 S mRNA of Sindbis virus either in vivo or in vitro. In vivo products were obtained after pulse-labeling of virus-infected cells. In vitro products were obtained after cell-free translation either in the absence or presence of microsomal membrane vesicles from dog pancreas. The sequence data indicate that the selective translocation across the microsomal membrane required for a distinct portion of one of the integral viral envelope proteins (PE2) is not accompanied by cleavage of its putative signal sequence. Furthermore, the NH2-terminal sequence of a proteolytic derivative (PE'2) that contains the bulk of PE2 and that is generated after exposure of the microsomal vesicles to proteolytic enzymes is identical to that of intact PE2, strongly suggesting that only a COOH-terminal portion of PE2 is excluded from translocation across the microsomal membrane.     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.     

Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.     

On the origin of the positive band in the far-ultraviolet circular dichroic spectrum of fibronectin

The genesis of the positive bands in the far-ultraviolet circular dichroic spectra of human plasma fibronectin and its 31-kDa NH2-terminal heparin-binding fragment was studied. Spectra of ester derivatives of tyrosine, tryptophan, and phenylalanine and of model mixtures of these derivatives in which they are present in the same ratios as in the proteins indicate that all the aromatic side chains make substantial contributions to composite positive bands with maxima several nanometers below those of the proteins. In the presence of solvent perturbants such as polyethylene glycol and ethylene glycol, the bands of the model mixtures are red-shifted to the approximate positions they have in the spectra of the proteins. No additional red shift is seen with solvent perturbation of the proteins, suggesting that the conditions leading to this effect are satisfied within the proteins. A separate effect of solvent perturbation, an increase in amplitude of the positive band, occurs equally in solutions of free aromatic amino acid derivatives and in the proteins. This effect is used to estimate the relative accessibility of the chromophores of fibronectin and its fragment to different perturbants. The possible influence of protein secondary structure on the amplitude of the positive circular dichroic band is discussed.     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Latent fibronectin-degrading serine proteinase activity in N-terminal heparin-binding domain of human plasma fibronectin

The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262-265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond K85-D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca(2+)-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.     

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.