scholarly journals Thrombin adhesive properties: induction by plasmin and heparan sulfate.

1993 ◽  
Vol 123 (5) ◽  
pp. 1279-1287 ◽  
Author(s):  
R Bar-Shavit ◽  
Y Eskohjido ◽  
J W Fenton ◽  
J D Esko ◽  
I Vlodavsky
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Dermatan Sulfate ◽  
Basement Membranes ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Adhesive Properties ◽  
Adhesive Protein ◽  
Adhesive Molecule

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.

scholarly journals Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycans Are Targeted by Bleomycin in Cancer Cells

2017 ◽  
Vol 43 (3) ◽  
pp. 1220-1234 ◽  
Author(s):  
Xiulian Li ◽  
Ying Lan ◽  
Yanli He ◽  
Yong Liu ◽  
Heng Luo ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Cancer Cells ◽  
Isotope Labeling ◽  
Cancer Drug ◽  
Severe Side Effect ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Positively Charged

Background/Aims: Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside of cells by free diffusion. We previously reported that the cell surface negatively charged glycosaminoglycans (GAGs) may be involved in the cellular uptake of bleomycin. We also observed that a class of positively charged small molecules has Golgi localization once inside of the cells. We therefore hypothesized that bleomycin might perturb Golgi-operated GAG biosynthesis. Methods: We used stable isotope labeling coupled with LC/MS analysis of GAG disaccharides simultaneously from bleomycin-treated and non-treated cancer cells. To further understand the cytotoxicity of bleomycin and its relationship to GAGs, we used sodium chlorate to inhibit GAG sulfation and commercially available GAGs to compete for cell surface GAG/bleomycin interactions in seven cell lines including CHO745 defective in both heparan sulfate and chondroitin sulfate biosynthesis. Results: we discovered that heparan sulfate GAG was significantly undersulfated and the quantity and disaccharide compositions of GAGs were changed in bleomycin-treated cells in a concentration- and time-dependent manner. We revealed that bleomycin-induced cytotoxicity was directly related to cell surface GAGs. Conclusion: GAGs were targeted by bleomycin both at cell surface and at Golgi. Thus, GAGs might be the biological relevant molecules that might be related to the bleomycin-induced fibrosis in certain cancer patients, a severe side effect with largely unknown molecular mechanism.

scholarly journals Chondroitin Sulfate/Dermatan Sulfate-Protein Interactions and Their Biological Functions in Human Diseases: Implications and Analytical Tools

2021 ◽  
Vol 9 ◽  
Author(s):  
Bin Zhang ◽  
Lianli Chi
Keyword(s):  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Protein Interactions ◽  
Dermatan Sulfate ◽  
Interaction Analysis ◽  
Structural Complexity ◽  
Protein Characterization ◽  
Biological Functions ◽  
Gag Protein ◽  
Core Proteins

Chondroitin sulfate (CS) and dermatan sulfate (DS) are linear anionic polysaccharides that are widely present on the cell surface and in the cell matrix and connective tissue. CS and DS chains are usually attached to core proteins and are present in the form of proteoglycans (PGs). They not only are important structural substances but also bind to a variety of cytokines, growth factors, cell surface receptors, adhesion molecules, enzymes and fibrillary glycoproteins to execute series of important biological functions. CS and DS exhibit variable sulfation patterns and different sequence arrangements, and their molecular weights also vary within a large range, increasing the structural complexity and diversity of CS/DS. The structure-function relationship of CS/DS PGs directly and indirectly involves them in a variety of physiological and pathological processes. Accumulating evidence suggests that CS/DS serves as an important cofactor for many cell behaviors. Understanding the molecular basis of these interactions helps to elucidate the occurrence and development of various diseases and the development of new therapeutic approaches. The present article reviews the physiological and pathological processes in which CS and DS participate through their interactions with different proteins. Moreover, classic and emerging glycosaminoglycan (GAG)-protein interaction analysis tools and their applications in CS/DS-protein characterization are also discussed.

scholarly journals Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues.

1990 ◽  
Vol 38 (10) ◽  
pp. 1479-1486 ◽  
Author(s):  
K J McCarthy ◽  
J R Couchman
Keyword(s):  
Chondroitin Sulfate ◽  
Dermatan Sulfate ◽  
Core Protein ◽  
Rat Kidney ◽  
Basement Membranes ◽  
Rat Tissues ◽  
Adult Rat ◽  
Unique Distribution ◽  
Reichert's Membrane

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.

Heparanase modulates heparinoids anticoagulant activities via non-enzymatic mechanisms

2007 ◽  
Vol 98 (12) ◽  
pp. 1193-1199 ◽  
Author(s):  
Lars Muhl ◽  
Etty Zwang ◽  
Neta Ilan ◽  
Yair Herishanu ◽  
Varda Deutsch ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Factor Vii ◽  
Factor X ◽  
Heparin Binding ◽  
Antagonistic Action ◽  
Endothelial Cell Surface ◽  
Coagulant Activity ◽  
Coagulation Protein ◽  
Enzymatic Mechanisms

SummaryA key element for the physiological restriction of blood coagulation at the endothelial cell surface is its non-thrombogenic property, mainly attributed to cell surface heparan sulfate proteoglycans. Heparanase is an endo-β-D-glucuronidase with specific heparan sulfate degrading activity, which is produced and stored in platelets, and is released upon their activation. We examined the effects of heparanase pro-enzyme on coagulation functions, predominantly under physiological conditions. While heparanase pro-enzyme does not directly affect coagulation protein activities, it has profound effects on heparinoid-mediated regulation of coagulation responses, apparently via mechanisms that do not involve its enzymatic activity. Heparanase pro-enzyme reverses the anti-coagulant activity of unfractionated heparin on the coagulation pathway as well as on thrombin activity. In addition, heparanase pro-enzyme abrogated the factor X inhibitory activity of low-molecular-weight heparin (LMWH). The pro-coagulant effects of the non-active heparanase were also exerted by its major functional heparin-binding peptide. Finally, the effects of heparanase on the activity of factor VII activating protease that is auto-activated by heparinoids indicated a complete antagonistic action of heparanase in this system. Altogether, heparanase pro-coagulant activities that were also demonstrated in plasma samples from patients under LMWH treatment,point to a possible use of this molecule as antagonist for heparinoid treatment.

scholarly journals Cell-substratum adhesion in chick neural retina depends upon protein-heparan sulfate interactions.

1985 ◽  
Vol 100 (4) ◽  
pp. 1192-1199 ◽  
Author(s):  
G J Cole ◽  
D Schubert ◽  
L Glaser
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Neural Retina ◽  
Heparan Sulfate Proteoglycan ◽  
Retinal Cell ◽  
Sulfate Proteoglycan ◽  
Heparin Binding ◽  
Embryonic Chick ◽  
Retinal Cells ◽  
Inert Surfaces

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.

scholarly journals Interaction of an Outer Membrane Protein of Enterotoxigenic Escherichia coli with Cell Surface Heparan Sulfate Proteoglycans

2002 ◽  
Vol 70 (3) ◽  
pp. 1530-1537 ◽  
Author(s):  
James M. Fleckenstein ◽  
James T. Holland ◽  
David L. Hasty
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycans ◽  
Eukaryotic Cells ◽  
Heparin Binding ◽  
Wild Type ◽  
E Coli

ABSTRACT We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.

scholarly journals Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

1992 ◽  
Vol 117 (6) ◽  
pp. 1331-1341 ◽  
Author(s):  
SL Drake ◽  
DJ Klein ◽  
DJ Mickelson ◽  
TR Oegema ◽  
LT Furcht ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Melanoma Cell ◽  
Synthetic Peptide ◽  
Core Protein ◽  
Melanoma Cells ◽  
Heparin Binding ◽  
Mouse Melanoma ◽  
Mouse Melanoma Cell

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.

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