scholarly journals Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1

1987 ◽  
Vol 245 (2) ◽  
pp. 543-550 ◽  
Author(s):  
D H Farrell ◽  
D D Cunningham
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Order Rate Constant ◽  
Subsequent Treatment ◽  
Protease Nexin ◽  
Inhibition Of Thrombin

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

scholarly journals Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts

1985 ◽  
Vol 232 (1) ◽  
pp. 161-168 ◽  
Author(s):  
S Johansson ◽  
K Hedman ◽  
L Kjellén ◽  
J Christner ◽  
A Vaheri ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Human Articular Cartilage ◽  
Sulphate Proteoglycan ◽  
Fibroblast Cultures ◽  
Core Proteins ◽  
The Matrix

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called ‘link proteins’ were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.

scholarly journals Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells

1999 ◽  
Vol 343 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Hans-Joachim LüKE ◽  
Peter PREHM
Keyword(s):  
Cell Surface ◽  
Plasma Membranes ◽  
Human Fibroblasts ◽  
Metastatic Potential ◽  
Melanoma Cells ◽  
High Molecular Mass ◽  
Chain Elongation ◽  
Hyaluronan Synthase ◽  
Surface Receptors ◽  
Melanoma Cell Lines

The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others.

scholarly journals Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs

1984 ◽  
Vol 217 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Y Ohhashi ◽  
F Hasumi ◽  
Y Mori
Keyword(s):  
Cell Surface ◽  
Guinea Pigs ◽  
Lysosomal Enzymes ◽  
Culture Medium ◽  
Chondroitin Sulphate ◽  
Alkali Treatment ◽  
Heparan Sulphate ◽  
Polymorphonuclear Leucocytes ◽  
Dermatan Sulphate ◽  
Cell Cell

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.

Widespread use of Lactobacillus OppA, a surface located protein, as an adhesin that recognises epithelial cell surface glycosaminoglycans

2019 ◽  
Vol 10 (4) ◽  
pp. 463-472 ◽  
Author(s):  
C. Martín ◽  
S. Escobedo ◽  
J.E. Suárez ◽  
L.M. Quirós
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Lactobacillus Reuteri ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Surface Protein ◽  
Epithelial Cell Surface ◽  
Surface Proteome ◽  
Reduced Adherence

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.

scholarly journals The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies

2002 ◽  
Vol 366 (2) ◽  
pp. 491-500 ◽  
Author(s):  
Frank DIETZ ◽  
Sebastian FRANKEN ◽  
Kenya YOSHIDA ◽  
Hideji NAKAMURA ◽  
Joachim KAPPLER ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Factor Activity ◽  
Hek 293 ◽  
Green Fluorescent

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1μg/ml. The expression of the fusion proteins, bHRP-4–green fluorescent protein and mHDGF–green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42nM. Comparison of the bHRP-4 amino acid sequence with HRP-1–3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.

Glycosaminoglycan expression in intestinal epithelial skin-fibroblastic cell cocultures. Fibroblastic cell-mediated effects of glucocorticoids

1989 ◽  
Vol 92 (4) ◽  
pp. 679-685
Author(s):  
F. Bouziges ◽  
P. Simon-Assmann ◽  
C. Leberquier ◽  
K. Haffen ◽  
M. Kedinger
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Cell Cultures ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Cell Types ◽  
Molecular Forms ◽  
Fibroblastic Cell ◽  
Endodermal Cells ◽  
Molecular Weight Form

The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS. Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures. The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.

scholarly journals Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: colocalization of cell surface urokinase with vinculin.

1988 ◽  
Vol 106 (4) ◽  
pp. 1241-1247 ◽  
Author(s):  
C A Hébert ◽  
J B Baker
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Actin Binding ◽  
Double Labeling ◽  
Amino Acid Peptide ◽  
Adhesion Sites ◽  
Fibrosarcoma Cells

Several cell types display binding sites for [125I]urokinase (Vassalli, J.-D., D. Baccino, D. Belin. 1985. J. Cell Biol. 100:86-92) which in certain cases are occupied with endogenous urokinase. These sites appear to focus urokinase at cell surfaces and hence may participate in tissue matrix destruction and cell invasion. Recently Pöllänen et al. (1987) demonstrated that the cell surface urokinase of human fibroblasts and fibrosarcoma cells is deposited underneath the cells in strands, apparently at sites of cell-to-substratum contact. Here, using immunofluorescence double labeling, we show that the urokinase strands present on human foreskin fibroblasts are colocalized with strands of vinculin, an intracellular actin-binding protein that is deposited at cell-to-substratum focal adhesion sites. Thus, this indicates linkage of the plasminogen/plasmin system both to sites of cell adhesion and to the cytoskeleton. The urokinase strands on HT 1080 fibrosarcoma cells are more numerous and have shapes that are more tortuous than those on normal fibroblasts. In intact HT 1080 cells, colocalized vinculin strands are obscured by an intense background of soluble vinculin but are apparent on isolated ventral plasma membranes. Certain properties of the urokinase strands suggest that they are related to the [125I]urokinase-binding sites that have been described by several groups: (a) incubating fibroblasts with dexamethasone for 48 h or at pH 3 at 5 degrees C for 10 min greatly decreases the number and intensity of the urokinase strands; (b) strands reappear when glucocorticoid-treated cells are incubated with exogenous 54-kD (but not 35-kD) urokinase, and this process is inhibited by a previously described 16-amino acid peptide that blocks [125I]urokinase binding to the cells.

Localization of actin, beta-spectrin, 43 × 10(3) Mr and 58 × 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes

1990 ◽  
Vol 97 (4) ◽  
pp. 615-626
Author(s):  
M.P. Daniels
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Plasma Membranes ◽  
Subsequent Treatment ◽  
Brain Extract ◽  
Early Stages ◽  
Cytoplasmic Face ◽  
Alpha Bungarotoxin ◽  
Immunofluorescence Labeling

I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4–6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 × 10(3) Mr and 58 × 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.

Sign in / Sign up

Export Citation Format

Share Document