The extracellular matrix proteins laminin and fibronectin modify the AMPase activity of 5'-nucleotidase from chicken gizzard smooth muscle

Laminin and fibronectin, but not collagen, affect the AMPase activity of the purified transmembrane protein 5'-nucleotidase. Laminin stimulates whereas fibronectin inhibits the AMPase activity of this ectoenzyme. The AMPase-modulating effects by these components of the extracellular matrix require a preincubation period of several hours when detergent-solubilized 5'-nucleotidase is employed, they can, however, instantaneously be elicited with liposome-incorporated 5'-nucleotidase.     

Evidence for the direct interaction of chicken gizzard 5'-nucleotidase with laminin and fibronectin

The ectoenzyme 5'-nucleotidase purified from chicken gizzard is shown to specifically interact with laminin and fibronectin, components of the extracellular matrix, by a number of different techniques: (i) cosedimentation with laminin by sucrose gradient centrifugation; (ii) affinity adsorption to both laminin- and fibronectin-Sepharose 4-B; (iii) specific binding to both laminin and fibronectin dotted onto cellulose filters; and (iv) monoclonal antibodies against 5'-nucleotidase are shown to interfere with the interaction of 5'-nucleotidase with laminin and fibronectin. For all the techniques employed, the interactions were found to be specific, since 5'-nucleotidase did not bind to unrelated proteins such as bovine serum albumin or to monomeric actin. The interaction of purified chicken gizzard 5'-nucleotidase could be demonstrated for the hydrophobic enzyme solubilized in detergent and after its reconstitution into artificial phospholipid vesicles. The affinity adsorption experiments indicate that reconstituted enzyme binds more strongly to both laminin and fibronectin. The 5'-nucleotidase employed in this study is anchored to the plasma membrane by a glycan-phosphatidylinositol linker. After treatment with phosphatidylinositol-specific phospholipase C, the enzyme is transformed into a hydrophilic form, for which interactions with laminin and fibronectin could also be demonstrated by the dot-blot technique. Thus controlled cleavage of the phosphatidylinositol linker of 5'-nucleotidase could enable cells to rapidly alter their adhesiveness to certain components of the extracellular matrix.     

Chicken gizzard 5'-nucleotidase functions as a binding protein for the laminin/nidogen complex.

Soluble and reconstituted 5'-nucleotidase were used in the binding assays to the laminin/nidogen complex. They both are shown to interact specifically and in a saturable manner with the laminin/nidogen complex using a solid-phase binding assay. Dissociation constants in the region of 10(-8) M were determined for the association of soluble and membrane-bound 5'-nucleotidase. Scatchard analysis of the binding data indicate a stoichiometry of about 2.7 of the homodimeric soluble 5'-nucleotidase to the laminin/nidogen complex. The association of 5'-nucleotidase with laminin/nidogen occurs in the absence of divalent metal ions and does not require N-linked carbohydrate moieties of both laminin/nidogen and 5'-nucleotidase. 5'-Nucleotidase also associates with isolated laminin although with reduced affinity. No binding to isolated nidogen was observed. Peptides containing the RGD sequence did not influence the binding reaction. Monoclonal and polyclonal antibodies directed against 5'-nucleotidase and laminin specifically perturb the association of the reconstituted enzyme to laminin/nidogen. Sulfated polysaccharides such as heparinsulfate and dermatansulfate modulate the interaction of 5'-nucleotidase and laminin/nidogen in a complex biphasic manner and might also regulate the binding reaction in vivo. Immunohistochemistry shows a close spatial correlation of 5'-nucleotidase and laminin also in the epithelium of the small intestine pointing to an in vivo interaction of both glycoproteins.     

The extracellular matrix protein fibronectin is a substrate for kallikrein 7

Kallikrein 7 (hK7), a chymostatin-like serine protease, is overexpressed in pancreatic adenocarcinomas as well as other human cancers. Although it has been demonstrated to participate in normal desquamation by facilitating cell shedding at the skin surface, its role in human malignancies remains unclear. To investigate the ability of hK7 to degrade components of the extracellular matrix (ECM), recombinant hK7 was expressed and purified from cultured mammalian cells. Using a three-step chromatographic purification procedure, recombinant hK7 was obtained that displayed robust proteolytic activity against a fluorogenic peptide substrate following activation by thermolysin. We demonstrate that the active protease is able to cleave fibronectin in a time-dependent manner, but not laminin, using an in vitro degradation assay. These findings indicate that the aberrant expression and secretion of hK7 in human tumors may facilitate metastasis by directly degrading components of the extracellular matrix and may thus play an important role in tumorigenesis.     

The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices

Cells interact with extracellular fibronectin (FN) via adhesive fibronectin receptors (FNRs) that are members of the very late antigens (VLAs) subgroup of the integrin family. In stationary fibroblasts, the FNR is highly organized and distributed identically to extracellular FN fibrils. However, in highly migratory neural crest cells and embryonic somatic fibroblasts, this organization is lost and the FNR appears diffuse. Similarly, oncogenic transformation typically leads to disorganization of the FN receptor and loss of matrix FN. Two models can account for these observations. First, the FN matrix may organize the FN receptor at extracellular matrix contacts on the cell surface. Motile cells not depositing FN matrices thus lack organized receptors. Alternatively, as the FNR is required for optimal FN matrix assembly, (McDonald, J. A., B. J. Quade, T. J. Broekelmann, R. LaChance, K. Forseman, K. Hasegawa, and S. Akiyama. 1987. J. Biol. Chem. 272:2957-2967; Roman, J. R. M. LaChance, T. J. Broekelmann, C. J. R. Kennedy, E. A. Wayner, W. G. Carter, J. A. McDonald. 1989. J. Cell Biol. 108:2529-2543) and has putative cytoskeletal links, it could be organized from within the cell helping to position newly forming FN fibrils. To study this question, we developed peptide antibodies specifically recognizing the alpha 5 subunit of the FNR. Using these antibodies, we examined the organization of FN and of the FNR in normal, matrix assembly inhibited, and SV40-transformed human fibroblasts. On FN-coated substrates, the FNR is found in focal contacts rather than diffusely on the basal cell surface, suggesting FNR interaction with intracellular components. However, when FN fibrils are deposited, the FNR is co-distributed with these fibrils. Preventing FN matrix assembly prevents organization of the FNR. Moreover, when fibroblasts with well established FN matrices and co-distributed FNR are incubated briefly with monoclonal antibodies that block FNR binding to FN, the FNR is no longer co-distributed with the FN matrix. Thus, the FN receptor is organized in fibrils on the cell surface in response to extracellular FN. Because exogenous FN restores a FN matrix and receptor organization to SV40-transformed cells, the diffuse FN receptor phenotype appears to be related to loss of the FN matrix rather than to impaired FNR function. These results explain diffusely distributed FNRs in migratory neural crest and embryonic fibroblasts lacking well organized FN matrices and emphasize the existence of separate but related systems controlling FN deposition and recognition by receptor-armed cells.     

Limited proteolysis of chicken gizzard 5'-nucleotidase.

Chicken gizzard 5'-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5'-nucleotidase by limited proteolysis. A hydrophobic proteolytic fragment carrying the intact C-terminus, as well as two major hydrophilic products, were identified. We developed procedures for specific radiolabelling of the active center of 5'-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5'-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5'-Nucleotidase represents a binding protein for components of the extracellular matrix. The interaction between 5'-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5'-nucleotidase. In summary, we have further characterized chicken gizzard 5'-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5'-nucleotidase.     

5'-nucleotidase from bull seminal plasma, chicken gizzard and snake venom is a zinc metalloprotein

Using flame atomic absorption spectrometry the tight association of zinc to three different purified 5'-nucleotidases at a molar ratio of 2 could be proven. These 5'-nucleotidases purified from bull seminal plasma (BSP), chicken gizzard (CG) and snake venom (SV) are thus zinc metalloproteins. Removal of zinc results in the loss of their AMPase activity, which could be fully restored after readdition of zinc at a molar ratio of 2, for BSP and CG, and 1.5, for SV 5'-nucleotidase. Reactivation of their AMPase activity after the removal of zinc could also be obtained by addition of cobalt and copper ions, which were found to also bind with a molar ratio of 2 to the three 5'-nucleotidases tested.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

Tenascin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment

Tenascin is a large extracellular matrix (ECM) glycoprotein found in restricted tissue locations in the adult organism. It is copiously synthesized in regenerative organs or regenerating tissues and by certain tumors. We have analyzed the expression of tenascin in human long term bone marrow cultures as well as in cryostat sections of native bone marrow and found it strongly expressed by the stromal cells of the microenvironment. Two different protein subunits of 280 and 220 kD were detected by immunoblotting. These two forms are derived most likely from two different mRNA splice variants of 6 and 8 kb detected by Northern blotting. The in vivo analysis of cryostat sections showed a codistribution with other ECM molecules such as fibronectin and collagen type III in the microenvironment surrounding the maturing hematopoietic cells. Using two independent cell adhesion assays tenascin could be shown to function as a cytoadhesive molecule for hematopoietic cells. These data suggest a direct involvement of tenascin in the retention of hematopoietic progenitor cells in the stroma.     

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix

1. Download : Download high-res image (254KB)
2. Download : Download full-size image

     

Thrombospondin is an osteoblast-derived component of mineralized extracellular matrix

Thrombospondin, the most abundant protein of platelet alpha granules, is a biosynthetic product of a variety of connective tissue cells and a component of many extracellular matrices. In this study, thrombospondin distribution in bone was investigated using a monoclonal antibody specific for the human protein. Thrombospondin was localized in osteoid of undemineralized, frozen sections of fetal subperiosteal bone, and identified as a component of mineralized bone matrix of neonatal and/or young (growing) bone of many animal species by Western blot analysis. Adult human bone cells were demonstrated to contain mRNA for thrombospondin by hybridization of a cDNA thrombospondin probe to a 6.1 kb mRNA. Pulse-chase experiments indicated that the protein was synthesized and the majority was secreted from osteoblastic cells. Treatment of the cells with TGF-beta (0.01-10 ng/ml) slightly decreased total thrombospondin synthesis, but caused an increase in the retention on newly synthesized thrombospondin in the cell layer/matrix fraction. In cell attachment assays, thrombospondin mediated adhesion, but not spreading of adult human bone cells.     

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.     

A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA(+)FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA(+)FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.     

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion.We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors.The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.     

Cardiac extracellular matrix tenascin-C deposition during fibronectin degradation

Tenascin-C (TN-C) might aggravate left ventricular remodeling after myocardial infarction (MI). Our previous study demonstrated that ventricular remodeling after MI is linked with the degradation of fibronectin (FN). The aim of the present study was to determine whether cardiac extracellular matrix TN-C deposition after MI requires FN degradation. We found that treatment with angiotensin (ANG) II significantly down-regulated FN while remarkably up-regulated TN-C in co-cultured cardiomyocytes and fibroblasts. Inhibitors of matrix metalloproteinase (MMP)-2, MMP-3 or MMP-9 significantly attenuated ANG II-induced loss of FN and obviously blunted ANG II-induced re-expression of TN-C in co-cultured cells. Moreover, FN fragments dose-dependently induced the deposition of TN-C. In addition, MI induced a significant reduction of FN protein expression and a marked elevation of TN-C expression level at day 7 after MI compared with the sham group. The present findings suggest that cardiac TN-C matrix deposition after MI is induced by FN degradation, which is dependent on the activation of MMPs. These findings might contribute to gain mechanistic insights into the regulation of TN-C formation after MI.     

Regulation of fibronectin alternative splicing by a basement membrane-like extracellular matrix

Hepatocytes are the source of plasma fibronectin (FN) which lacks the alternatively spliced EDI segment, distinctive of oncofetal FN. When hepatic or other epithelial cells are cultured on plastic, EDI inclusion is triggered. Here we report that EDI inclusion is inhibited when hepatic cells are cultured on a basement membrane-like extracellular matrix (ECM), demonstrating a new role for the ECM in the control of gene expression. The effect is duplicated by collagen IV and laminin but not by collagen I; is not observed with another alternatively spliced FN exon (EDII); and correlates with a decrease in cell proliferation, consistently with high EDI inclusion levels observed in many physiological and pathological proliferative processes.     

Soluble fibronectin interaction with cell surface and extracellular matrix is mediated by carbohydrate-to-carbohydrate interaction

Cell adhesion and spreading on solid phase fibronectin (FN), coated on plate or presented in extracellular matrix, are mediated by integrin receptors alpha5beta1, alpha4beta1, etc., although binding of "soluble-form FN" to cell surface varies extensively depending on glycosylation status of FN per se. Deposition or incorporation at the cell surface or pericellular matrix of soluble-form FN from body fluids or synthesized de novo takes place through a yet-unknown (perhaps integrin-independent) mechanism. Here we present evidence that the mechanism involves carbohydrate-to-carbohydrate interaction. Binding or incorporation of soluble-form placental or hepatoma FN to cell surface or pericellular matrix is highly dependent on the specific glycosylation status of FN per se and combination with glycosylation status of the cell surface, and is greatly promoted by a certain type of coexisting (shedded) glycosphingolipid. A few lines of study indicate that the process is mediated by interaction of FN carbohydrate with cell surface carbohydrate. The great enhancement of the binding process by glycosphingolipid is based on dual interaction of glycosphingolipid carbohydrate with FN carbohydrate and with cell surface carbohydrate. Here we present an example of promotion of binding of soluble-form FN from placenta or from hepatoma cells, having a specific carbohydrate epitope termed "disialyl-I," to K562 or VA13 cell surface in the presence of glycosphingolipid Gg3, which interacts specifically with disialyl-I.     

Internalization of the fibronectin receptor is a constitutive process

Using a monoclonal antibody specific for the hamster fibronectin receptor (FnR), we have demonstrated that a portion of the CHO cell FnR population is constitutively endocytosed. Three independent techniques were used to study the internalization: 1) after saturation binding of an anti-FnR antibody (PB1) to cells at 4 degrees C, internalization was initiated by warming to 37 degrees C, and then acid/salt elution of membrane-bound ligand was used to quantitate the internalized 125I-PB1; 2) cell vesicular traffic was pharmacologically disrupted with monensin or chloroquine, and the subsequent reduction of the cell surface pool of FnR was monitored; and 3) selective immunoprecipitation was used to separate surface and internalized 125I-labeled FnR. These experiments indicate that about 30% of the cell surface FnR is endocytosed with a t1/2 of 7 min and that this internalization occurs regardless of the ligation state of the receptor. Other observations indicate that the larger fraction of the cell surface FnR pool (70-75%) is apparently shed from the cell upon ligation with antibody at 37 degrees C. This process occurs much more slowly than receptor internalization and leads to an overall reduction in the amount of cell surface FnR. Our results suggest physically or chemically distinct populations of FnR, one of which is unavailable for internalization and recycling.     

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.