Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amyloid precursor protein (APP) is a glycoprotein consisting of at least four isoforms derived from a single gene by a process of alternative splicing. The membrane-bound forms of APP have been suggested to have adhesive properties and to mediate neural cell adhesion. Previous studies have demonstrated the ability of Fab' fragments of antibodies to extracellular domains of APP to inhibit neural cell binding to a collagen substrate, suggesting a physiological role for the collagen-binding properties of APP. The binding of APP has been demonstrated to be specific for type IV collagen, and no binding to other extracellular matrix components, including fibronectin and laminin, was detected. The APP-collagen binding appeared to be mediated by a heparin-bridge mechanism, since the binding was abolished by the addition of excess heparan or heparinase. These results were observed by both a homogenate-collagen binding assay and a cell-surface adhesion assay, thus providing further evidence for the adhesion role of APP. They also pose the question of the possible role of the heparin-binding properties of APP in the genesis of the neuritic plaques characteristic of Alzheimer's disease.
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PMID:APP-collagen interaction is mediated by a heparin bridge mechanism. 152 Apr

O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modification and the formation of antithrombin binding regions in the biosynthesis of heparin/heparan sulfate. The resulting GlcNSO3(3-OSO3) units are largely restricted to heparin chains with high affinity for antithrombin (HA heparin). Low affinity (LA) heparin fails to serve as a substrate in the 3-O-sulfotransferase reaction yet contains potential 3-O-sulfate acceptor sites (Kusche, M., Torri, G., Casu, B., and Lindahl, U. (1990) J. Biol. Chem. 265, 7292-7300), as verified in the present study using a novel sequencing procedure. O-Desulfated, re-N-sulfated LA heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of LA heparin, both yielded labeled HA components following incubation with solubilized mouse mastocytoma microsomal enzymes and [35S]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfo-transferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region. Indeed, the addition of LA heparin precluded enzymatic 3-O-sulfation of a synthetic pentasaccharide substrate. The Km for the pentasaccharide was determined to approximately be 6 microM. Incubations of mixed pentasaccharide substrate and saccharide inhibitors revealed Ki values for intact LA heparin and for a heparin octasaccharide fraction of approximately 1.3 and approximately 0.7 microM, respectively. Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate and GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-sulfotransferase. By contrast, chondroitin sulfate or dermatan sulfate showed no significant inhibitory activity. It is proposed that the regulation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan sulfate depends on the topological organization of the membrane-bound enzyme machinery in the intact cell.
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PMID:Biosynthesis of heparin/heparan sulfate. The D-glucosaminyl 3-O-sulfotransferase reaction: target and inhibitor saccharides. 774 62

Matrix production by smooth muscle cells (SMC) appears to play a major role in the intimal thickening process. Proteoglycans (PG) are the predominant extracellular matrix component of early restenotic lesions. As angiotensin II (A II) has been proposed as a mediator of restenotic process, we hypothesized that A II may directly affect PG synthesis by SMC. SMC were cultured in the presence of [35S]sulfate and angiotensin II, and both the secreted and membrane-bound proteoglycans were analyzed. A II (1 to 100 nM) evoked a dose- and time-dependent increase in both cell- and media-associated PG production, an effect abrogated by the A II receptor antagonist, saralasin. SMC constitutively synthesize small amounts of PG with a molecular mass of 170-250 kDa. After treatment with A II, the abundance of PG is increased, as well as its molecular mass (230-300 kDa). Selective degradation by chondroitinases and heparinase identified chondroitin and dermatan sulfate PG as the predominant form being induced. These results demonstrate that the effect of A II is not general and is specific to certain classes of PGs. In order to further examine the specificity of the A II effect, we compared the synthesis of PG induced by A II with that induced by platelet-derived growth factors AA and BB (PDGF-AA and -BB), insulin-like growth factor I (IGF-I), and tumor necrosis factor alpha (TNF alpha). This comparison demonstrated that the profile of PG induced by A II is different from the other factors examined. Taken together, these data indicate that A II may not only function as a hypertrophic factor for SMC, but in addition may also be a potent modulator of specific PG synthesis by these same cells, which could significantly contribute to the formation of atherosclerotic and restenotic lesions.
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PMID:Stimulation of rat vascular smooth muscle cell glycosaminoglycan production by angiotensin II. 784 Aug 14

Previous studies established that uterine epithelial cells and cell lines express cell surface heparin/heparan sulfate (HP/HS)-binding proteins (Wilson, O., Jacobs, A. L., Stewart, S., and Carson, D. D. (1990) J. Cell. Physiol. 143, 60-67; Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). The accompanying paper (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823) describes the cloning of a full-length cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of human epithelia. A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepare a rabbit polyclonal antibody. This antibody reacted with a protein with an apparent Mr of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 x g particulate fraction of RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations > or = 0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence. HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans. NaCl-solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of 125I-HP with transblots of the NaCl-solubilized HIP preparations separated by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP. Indirect immunofluorescence studies demonstrated that HIP is expressed on the surfaces of intact RL95 cells. Binding of HIP antibodies to RL95 cell surfaces at 4 degrees C was saturable and blocked by preincubation with the peptide antigen. Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies directed against HIP but not irrelevant antibodies. Finally, indirect immunofluorescence studies demonstrate that HIP is expressed in both lumenal and glandular epithelium of normal human endometrium throughout the menstrual cycle. In addition, HIP expression increases in the predecidual cells of post-ovulatory day 13-15 stroma. Collectively, these data indicate that HIP is a membrane-associated HP-binding protein expressed on the surface of normal human uterine epithelia and uterine epithelial cell lines.
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PMID:Cell surface expression of HIP, a novel heparin/heparan sulfate binding protein, of human uterine epithelial cells and cell lines. 866 17

A microassay was developed to detect human herpesvirus 6 (HHV-6) binding to its cellular receptor using flow cytometry. Comparable results were obtained either by using HHV-6 preparations conjugated with fluorescein isothiocyanate or by indirect immunofluorescent labeling of membrane-bound virus using as primary antibody a monoclonal antibody specific for the HHV-6 gp60/110 envelope glycoprotein. Virus attachment to the plasma membrane was specific and saturable. As expected, among cell lines of various origin, maximum binding was detected on human T-lymphoid cells (HSB-2). Papain digestion of HSB-2 cells prevented HHV-6 attachment and reduced significantly virus infection, indicating the involvement of a protein-based receptor in the attachment step. After removal of the protease, virus receptors were resynthesized and their regeneration was prevented partially by cycloheximide, an inhibitor of protein synthesis. Unexpectedly, only high concentrations (mg/ml) of soluble heparan sulfate and heparin inhibited HHV-6 binding and infection. Under the same conditions, few micrograms (per ml) of heparin suppressed completely herpes simplex type 1 (HSV-1) attachment to the same cell line. Treatment of HSB-2 cells with heparitinase and heparinase, at doses that reduced significantly HSV-1 attachment, had little effect on HHV-6 binding to the cell membrane, indicating a different requirement of heparan sulfate-containing glycosaminoglycans for the two herpesviruses. These data suggest that protein components of the cellular membrane play an essential role in HHV-6 binding and infection while heparan sulfate-glycos-aminoglycans appear to be involved only partially in virus-receptor interaction.
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PMID:Early interactions of human herpesvirus 6 with lymphoid cells: role of membrane protein components and glycosaminoglycans in virus binding. 1107 78

Our tumor necrosis factor-alpha (TNF-alpha) analog LK-805 (E107K) exhibited twofold higher specific cytotoxicity on the mouse fibroblast L-929 cell line than its native counterpart. In addition, significantly lowered systemic toxicity was observed in tumor-bearing mouse models treated with this analog. Due to a charge reversal and clustering of three lysines in the exposed tip region of LK-805, we assumed that additional ionic interactions between the positively charged TNF analog and the negatively charged components of the cell surface were created, which might contribute to improved properties of LK-805. To prove this hypothesis, we designed truncated forms of TNF-alpha and analog LK-805 and performed three independent sets of experiments: measurement of cytotoxic activity in the presence of excess heparan sulfate, determination of cytotoxic activity on heparinase-treated L-929 cells, and binding of various TNF-alpha proteins onto the heparin-sepharose affinity column. Cytotoxicity studies of both kinds confirmed the pivotal role of the E107K mutation for interaction with heparan sulfate proteoglycans on the cell surface of L-929 cells. However, heparin-binding studies revealed that intact, full-length N-termini of TNF-alpha or its analogs were necessary for high retention on the heparin affinity column, whereas the three-lysine containing tip of LK-805 by itself was not enough for binding. Obviously, immobilized heparin does not represent an adequate model for membrane-bound heparan sulfate proteoglycans of L-929 cells.
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PMID:Increased in vitro cytotoxicity of TNF-alpha analog LK-805 is based on the interaction with cell surface heparan sulfate proteoglycan. 1248 60

Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane with typical features of shed vesicle membranes. Shed vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Addition of vesicles to endothelial cells stimulated chemotaxis and urokinase plasminogen activator production, which were blocked by anti-FGF-2 antibodies. Treatment of intact vesicles with 2.0 m NaCl or heparinase, which release FGF-2 from membrane-bound proteoglycans, did not abolish their stimulatory effect on endothelial cells, indicating that FGF-2 is carried inside vesicles. The comparison of the stimulatory effects of shed vesicles and vesicle-free conditioned medium showed that vesicles represent a major reservoir of FGF-2. Thus, FGF-2 can be released from cells through vesicle shedding.
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PMID:Shedding of membrane vesicles mediates fibroblast growth factor-2 release from cells. 1452 6

The soluble (Gs) and membrane-bound (Gm) forms of human respiratory syncytial virus (HRSV) attachment protein were purified by immunoaffinity chromatography from cultures of HEp-2 cells infected with vaccinia virus recombinants expressing either protein. Sucrose gradient centrifugation indicated that Gs, which is secreted into the culture medium, remains monomeric, whereas Gm is an oligomer, probably a homotetramer. Nevertheless, Gs was capable of binding to the surface of cells in vitro, as assessed by a flow cytometry-based binding assay. The attachment of Gs to cells was inhibited by previous heparinase treatment of living cells, and Gs did not bind to CHO cell mutants defective in proteoglycan biosynthesis. Thus, Gs, as previously reported for the G protein of intact virions, binds to glycosaminoglycans presented at the cell surface as proteoglycans. Deletion of a previously reported heparin binding domain from Gs protein substantially inhibited its ability to bind to cells, but the remaining level of binding was still sensitive to heparinase treatment, suggesting that other regions of the Gs molecule may contribute to attachment to proteoglycans. The significance of these results for HRSV infection is discussed.
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PMID:The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans. 1501 75

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, adheres to, invades and multiplies in both professional phagocytes and epithelial cells. Adherence to epithelial cells is predominantly mediated by the 28-kDa heparin-binding haemagglutinin adhesin (HBHA), which is also required for the extrapulmonary dissemination of the bacilli. To study the cellular mechanisms that might result in HBHA-mediated extrapulmonary dissemination, we used a transwell model of cellular barrier and fluorescence microscopy and found that HBHA induces a reorganization of the actin filament network in confluent endothelial cells, but does not affect the tight junctions that link them. When coupled to colloidal gold particles, HBHA-mediated a rapid attachment of the particles to the membrane of human laryngeal epithelial cells (non polarized HEp-2 cells) and human type II pneumocytes (polarized A-549 pneumocytes). After attachment, the particles were internalized in membrane-bound vacuoles that migrated across the polarized pneumocytes to reach the basal side. Attachment of the HBHA-coated particles was not observed when the epithelial cells were pretreated with heparinase III, a lyase that specifically cleaves the heparan sulfate chains borne by the proteoglycans. Furthermore, no binding was observed when the gold particles were coated with HBHA lacking its C-terminal heparin-binding domain. These observations indicate that HBHA induces receptor-mediated endocytosis through the recognition of heparan sulfate-containing proteoglycans by the heparin-binding domain of the adhesin. In addition, the transcellular migration of the endocytic vacuoles containing HBHA-coated particles suggests that HBHA induces epithelial transcytosis, which may represent a macrophage-independent extrapulmonary dissemination mechanism leading to systemic infection by M. tuberculosis.
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PMID:Mycobacterium tuberculosis heparin-binding haemagglutinin adhesin (HBHA) triggers receptor-mediated transcytosis without altering the integrity of tight junctions. 1591 62

Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis.
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PMID:Heparan sulfate proteoglycans mediate factor XIIa binding to the cell surface. 2558 88


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