Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions. The initial modification step, catalysed by the enzyme glucosaminyl N-deacetylase/N-sulphotransferase-2, NDST-2, is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.

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 IC(50) of approximately 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.

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.

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5min following ligand addition at concentrations as low as 5nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with α 6 and β 1 integrin subunits at the base of the extensions. α 5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectinfibronectin interactions during progression of assembly.

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.

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 ␣-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.

Background: Conversion of fibronectin from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. Results: Binding of polypeptides by ␤-strand addition to N-terminal modules 1-5 FNI is linked to changes in distant integrin-and glycosaminoglycan-binding regions. Conclusion: Ligation of 1-5 FNI is sufficient for fibronectin expansion. Significance: Allosteric interactions among regions of fibronectin control assembly into extracellular fibrils.

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of ␣-chymases from different species indicated that certain rodent ␣-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 ␣-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His 6 purification tag and an enterokinasesusceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serineprotease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-AlaAla-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 ␣-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human ␣-chymase.Chymases constitute a family of mast cell (MC) 1 serine proteases with chymotrypsin-like substrate specificity, i.e. they cleave a substrate at the C-terminal side of aromatic amino acids. When comparing the structures of a variety of leukocyte serine proteases, chymases form a separate branch, indicating a common ancestor for this subfamily. Based on the phylogenetic tree, chymases can be subdivided into ␣-and ␤-chymases (1). A single gene encoding an ␣-chymase is present in most investigated mammalian species, but genes encoding ␤-chymases seem to be present in rodents only. The rodent ␣-chymases rat mast cell protease 5 (rMCP-5) (2) (initially designated rMCP-3 (3)) and mouse mast cell protease 5 (mMCP-5) (4) are 95% identical at the protein level and have similar expression patterns, indicating that they are functional homologues (3, 5). mMCP-5 and rMCP-5 are predominantly expressed in connective tissue MCs but also early in MC development (6 -8).The substrate specificities of mMCP-5/rMCP-5 have never been studied, and thus, the biological function of the rodent ␣-chymases is unknown. In fact, neither of these chymases has been purified to allow any biochemical characterization. Instead, most data today on ␣-chymases originate from studies on the human chymase (HC). HC expressi...