Bianca R. Tomasini-Johansson

A 49-Residue Peptide from Adhesin F1 of Streptococcus pyogenes Inhibits Fibronectin Matrix Assembly

F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC50 of ∼10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.

Extended Substrate Specificity of Rat Mast Cell Protease 5, a Rodent α-Chymase with Elastase-like Primary Specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of α-chymases from different species indicated that certain rodent α-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the α-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1′-(Xaa), and P2′-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat α-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human α-chymase.

Emerging roles of fibronectin in thrombosis

Fibronectin (FN) is a glycoprotein recognized originally in the 1940s as a contaminant of fibrinogen in Cohn fraction I of plasma. Decades of research demonstrated FN synthesis by a variety of cells and defined FN as an essential component of the extracellular matrix with roles in embryogenesis, development, and wound healing. More recently, FN has emerged as player in platelet thrombus formation and diseases associated with thrombosis including vascular remodeling, atherosclerosis, and cardiac repair following a myocardial infarct. We discuss the mechanisms by which this might occur and conclude that FN may have a unique role in thrombosis without affecting normal hemostasis and therefore may be a reasonable therapeutic target for the prevention of thrombotic diseases.

Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with Kd values of 5.2 and 59 nm to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the 2FNI module had little effect, whereas mAb 7D5 to the 4FNI module in the fibrin-binding region, 5C3 to the 9FNI module in the gelatin-binding region, or L8 to the G-strand of 1FNIII module adjacent to 9FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to 2FNI-5FNI of the fibrin-binding domain and 8FNI-9FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of 2FNI-5FNI and 8FNI-9FNI.

Specific Interactions between F1 Adhesin of Streptococcus pyogenes and N-terminal Modules of Fibronectin

Protein F1 is a surface protein ofStreptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8–30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1–7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.

Myofibroblasts exhibit enhanced fibronectin assembly that is intrinsic to their contractile phenotype

Background: Myofibroblasts have heightened expression of contractile genes and drive extracellular matrix formation during pulmonary fibrosis. Results: Enhanced fibronectin assembly by myofibroblasts requires smooth muscle α-actin expression. Conclusion: This study demonstrates a linkage between contractile gene expression and increased assembly of fibronectin fibrils by myofibroblasts. Significance: Targeting contractile gene expression in myofibroblasts may attenuate fibronectin matrix formation during fibrosis. Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

Ligation of the Fibrin-binding Domain by β-Strand Addition Is Sufficient for Expansion of Soluble Fibronectin

Background: Conversion of fibronectin from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. Results: Binding of polypeptides by β-strand addition to N-terminal modules 1–5FNI is linked to changes in distant integrin- and glycosaminoglycan-binding regions. Conclusion: Ligation of 1–5FNI is sufficient for fibronectin expansion. Significance: Allosteric interactions among regions of fibronectin control assembly into extracellular fibrils. How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. “Functional upstream domain” (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel β-strand addition to discontinuous sets of N-terminal FN type I modules, 2–5FNI of the fibrin-binding domain and 8–9FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of 2–5FNI, 8–9FNI, or the two sets in combination is important for inhibition, we tested “high affinity downstream domain” (HADD), which binds by β-strand addition to the continuous set of FNI modules, 1–5FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module (10FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after 9FNI or 3FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than 3FNIII limit β-strand addition to 1–5FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, 10FNIII and 12–14FNIII, increases accessibility of 1–5FNI. Allosteric loss of constraining interactions among 1–5FNI, 10FNIII, and 12–14FNIII likely enables assembly of FN into extracellular fibrils.

Plasma fibronectin concentration in inbred mouse strains

Dear Sir, Several observations indicate that the concentration of fibronectin in plasma is a determinant of platelet thrombus formation. Injured arterioles of mice engineered to have <2% or 50% normal plasma fibronectin concentration develop less stable platelet thrombi and occlude more slowly than arterioles of mice with normal plasma fibronectin concentration (1, 2). Fibronectin concentration is also a strong determinant of platelet thrombus build-up in the parallel flow chamber assay (3–5). The concentration of fibronectin varies widely in humans, from 230 to 650 µg/ml (6). High concentrations have been associated with venous thromboembolism (7, 8) and with coronary artery disease in some but not all populations (9–12). The latter association suggests that there is genetic interaction between fibronectin concentration in plasma and other determinants of cardiovascular disease. In order to relate studies in mice to observations in humans, we sought evidence for a similarly wide concentration range of plasma fibronectin in various genetic strains of mice. Rabbit antibodies to purified human plasma fibronectin were purified by affinity chromatography on immobilized human fibronectin. These antibodies recognized mouse fibronectin with high titer, and only fibronectin was detected by the antibodies in Western blots of mouse plasma proteins. A portion of the antibodies was labeled with biotin. Fibronectin from mouse plasma was isolated by affinity chromatography on gelatin as previously described (13). The protein was >95% pure by electrophoresis and used as a standard, assuming a 1 mg/ml solution to have an optical density (280 nm, 1 cm) of 1.3. Pooled citrated plasma from Swiss family mice was purchased (Pel-Freez, Rogers, AK) and served as reference plasma. The reference plasma, antibodies and purified mouse fibronectin standard were used to measure fibronectin concentration in the plasma by three different immunoassays. The concentration of fibronectin in the reference plasma was estimated to be about 240 µg/ml by Western blotting of diluted plasma in comparison to known amounts of purified fibronectin, 173 +/− 43 µg/ml (mean +/− SD) by a sandwich ELISA in which fibronectin was captured by immobilized anti-fibronectin and captured antigen was quantified by biotinylated anti-fibronectin followed by streptavidin-alkaline phosphatase (MPD, http://www.jax.org/phenome, Tomasini1 protocol), and 234 +/− 79 µg/ml in a competitive ELISA in which soluble fibronectin competed with immobilized fibronectin for binding of soluble anti-fibronectin. In addition, approximately 200 µg protein bound to gelatin-agarose from 1 ml of reference plasma and was shown to be nearly pure fibronectin by electrophoresis. The concentrations estimated by 4 methods, therefore, were consistent and centered on about 210 µg/ml. This value is 2.5-fold lower than concentrations previously reported for rodent plasma fibronectin (14–16). The reasons for the differences between our values and values reported in previous studies are unclear. Small portions of citrated plasma from 358 individual 9-week old mice from 24 strains from 7 genetic families were provided by Jackson Laboratory (Bar Harbor, ME). Similar plasma samples were used by Jackson Laboratory to search for strain-specific differences in prothrombin time, partial thromboplastin time, and fibrinogen (17) as part of the Mouse Phenome Project (MPD, Peters1). These plasma samples were assayed for fibronectin concentration by the sandwich ELISA (Table 1). The results are posted (MPD, Tomasini1) and can be viewed using the various tools at the website. The concentration for all animals was 200 +/− 54 µg/ml (mean +/− SD). There were no differences between males (199 +/− 66 µg/ml) and females (202 +/− 42 µg/ml). The 70 individuals from 5 strains of the Swiss family had a concentration of 179 +/− 46 µg/ml, which is similar to the concentration measured in the pooled reference plasma by sandwich ELISA as described above. Table 1 List of inbred mice arranged by genetic family, assayed for fibronectin (FN) concentration in plasma. The number of individuals per strain assayed is presented along with the mean concentration of plasma fibronectin (ND, not done). The variation in fibronectin concentration among mouse strains was similar to the variation reported for humans, ranging from 121 +/− 45 µg/ml for male SWR/J mice to 414 +/− 115 µg/ml for male SPRET/EiJ mice. Statistical analyses were performed on MPD based on unpaired t-tests between the mean values for each pair of strains. There are significant differences between male SWR/J and SPRET/EiJ mice (p < 0.01). One-Way Anova analysis with both parametric and non-parametric post-tests revealed significant differences (p < 0.001) between SPRET/EiJ or Balb/cByJ males with high concentration and males of 3 strains SWR/J, C57BL/6J, or AKR/J with low concentration. Other differences (p < 0.05) were seen between pairs of strains with intermediate concentrations. These differences would need to be tested prospectively with more animals to assess significance. The value for the reference plasma varied with a coefficient of variation (CV, standard deviation divided by the mean) of 0.25 for the 33 different times the plasma was tested over an 18-month period. Samples from individual mice of a given strain were assayed within a short period of time. To address the possibility that strain differences may be due to when a particular strain was tested, the sandwich ELISA was performed on pools composed of equal volumes of plasma from each of the males or females from 13 strains that had been tested individually. The strains were selected to represent the full range of mean individual values presented in Table 1. As shown in Figure 1 of the Supplemental Data, fibronectin concentrations of the strain and sex-specific pools correlated with the mean fibronectin concentrations of the individuals that composed the pools (slope = 0.86, r = 0.84, p < 0.0001, n = 26). The strain that was used for knock-out of fibronectin was C57BL/6J (14), a strain with among the lowest concentrations of fibronectin. Based on 140 +/− 17 µg/ml for wild-type male individuals of this strain (Table 1), plasma of C57BL/6J mice heterozygote for the knock-out would be predicted to have a fibronectin concentration of about 70 µg/ml. Indeed, the concentration of fibronectin in citrated plasma of 3 such heterozygous male mice, generously provided by Dr. Denisa Wagner of Harvard Medical School, was 77 µg/ml (individual values of 86, 89, and 56 µg/ml). Such low concentrations are poorly supportive of thrombus formation in flow chamber assays (3, 18), consistent with the long occlusion times found in fibronectin heterozygote knock-out mice after ferric chloride injury of arterioles (2). We found marked variations among individuals within certain strains (noted in SDs in Table 1).To learn whether in-strain variability was unique to fibronectin, we calculated the CVs of measurements of fibronectin and fibrinogen from values posted on MPD, Tomasini1 and Peters1, respectively. As depicted in Figure 2 of the Supplemental Data, the range in CVs for fibronectin concentration varied from 0.08 to 0.56 in different strains and was similar to the range in CVs (0.03 to 0.53) for fibrinogen concentration, thus suggesting that intrinsic variations in the concentrations of both plasma proteins exist among individuals in certain strains. There was no strain-dependent correlation between CVs for fibronectin and CVs for fibrinogen (r= −0.01, n=24), as well as no correlation between the concentrations of fibronectin and fibrinogen (r=0.38, p=0.1, n=24). Thus, there does not appear to be a single factor, e.g., concentration of a cytokine, that accounts for either differences or variabilities of the two proteins in various strains. This survey of plasma fibronectin concentration in diverse mouse strains can serve as a reference in strain selection for mouse models of diseases, such as thrombosis, in which plasma fibronectin may be an important component of the genetic background. In addition, it suggests that variation in fibronectin concentration may be in part genetic and opens the possibility for identification of pathways and genetic regulatory domains responsible for control of concentration by Quantitative Trait Locus analysis of F2 animals from crosses of strains with high and low concentration and for discovery of genetic interaction between fibronectin and other determinants of disease. Data on many relevant parameters are posted on MPD for the same strains. We encourage the hemostasis/thrombosis community to add to this data set.

PEGylated pUR4/FUD peptide inhibitor of fibronectin fibrillogenesis decreases fibrosis in murine Unilateral Ureteral Obstruction model of kidney disease

Fibronectin is a blood and extracellular matrix glycoprotein that plays important roles in wound healing and fibrosis since it controls the deposition of collagen and other extracellular matrix molecules and is a substrate for infiltrating lymphocytes. Using a high-affinity fibronectin-binding peptide (FUD/pUR4) that inhibits fibronectin deposition into extracellular matrix (ECM), we tested the ability of a PEGylated FUD/pUR4 (PEG-FUD) to inhibit fibrosis in the Unilateral Ureteral Obstruction (UUO) kidney disease model. Fibronectin fibrillogenesis assays, using human fibroblasts and human proximal tubular epithelial cultures, showed that PEG-FUD can inhibit fibronectin fibrillogenesis in vitro with an IC50 similar to unconjugated FUD, in the order of 20–35 nM. In contrast, a mutated FUD (mFUD) conjugated to PEG that lacked activity did not inhibit fibronectin assembly, even at 20 μM. The in vivo activity of PEG-FUD was tested in the murine UUO model by daily subcutaneous injection of 12.5 mg/kg for 7 days until harvest at day 10. Control treatments included saline, PEG, unconjugated FUD, and PEG-mFUD. Immunoblotting studies showed that fibronectin was enriched in the extracellular matrix fractions of extracted UUO kidneys, compared to contralateral untreated kidneys. In vivo, PEG-FUD significantly decreased fibronectin by ~70% in UUO kidneys as determined by both IHC and immunoblotting, respectively. In contrast, neither PEG-mFUD, PEG, nor saline had any significant effect. PEG-FUD also decreased collagens I and III and CD45-expressing cells (leukocytes) by ~60% and ~50%, as ascertained by picrosirius red staining and IHC, respectively. Immunoblotting studies also showed that the fibronectin remaining after PEG-FUD treatment was intact. Utilizing a custom-made polyclonal antibody generated against pUR4/FUD, intact PEG-FUD was detected by immunoblotting in both the ECM and lysate fractions of UUO kidneys. No adverse reaction or event was noted with any treatment. In summary, these studies suggest that PEG-FUD reached the kidneys without degradation, and decreased fibronectin incorporation into interstitial tissue. Decreased fibronectin was accompanied by a decrease in collagen and leukocyte infiltration. We propose that PEG-FUD, a specific inhibitor of fibronectin assembly, may be a candidate therapeutic for the treatment of fibrosis in kidney diseases.

Microtiter assays for quantitation of assembly of plasma and cellular fibronectin

Fibronectin (FN) is a plasma glycoprotein produced by hepatocytes that circulates at near micromolar concentration and assembles into extracellular matrix fibrils at cell surfaces along with locally produced cellular FN. We describe two microplate assays that quantify assembly of human FN by cells in monolayer culture. One assay measures fluorescence due to incorporation of ALEXA488-plasma FN into matrices of fibroblasts and has been used successfully in high-throughput screens. The other measures fluorescence due to binding of fluorochrome-labeled antibody to the EDA domain of cellular FN synthesized and deposited by various cell types. The assays take advantage of the tight association of assembled FN with cell monolayers and adherence of cell monolayers to wells of microtiter plates. The assays are straightforward, adapt to 96- and 384-well formats, and use reagents that are also suitable to image FN that is assembled into fibrils.