Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Human gingiva was stained with cupromeronic blue according to Scott's critical electrolyte concentration technique in order to localize glycosaminoglycans (GAG) in the electron microscope. Identification was performed by digestion with chondroitinase AC, ABC and heparinase. The GAG were localized in three compartments of the connective tissue: the supra-alveolar fiber apparatus, the loose connective tissue and the basement membranes. In the supra-alveolar fiber apparatus, consisting mainly of densely packed parallel collagen fibrils, dermatan sulfate GAG are regularly attached to the d-band of the collagen fibrils. The precipitates (6-7 nm in diameter) aggregate to thicker precipitates (up to 16 nm), thus possibly providing stability to the fiber system. In the loose connective tissue with sparse collagen fibrils dermatan and chondroitin sulfate GAG form very large precipitates (up to 30 nm in diameter and 400 nm length) which interconnect the few collagen fibrils. The basement membranes of the epithelium and capillary endothelium contain heparan sulfate GAG as fine precipitates (4-6 nm in diameter) which form a meshwork. These findings are consistent with the Scott model (1) for the interactions among glycans and glycans and collagen fibrils in connective tissues.
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PMID:Ultrastructural localization of glycosaminoglycans in human gingival connective tissue using cupromeronic blue. 777 52

In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mucinlike glycoproteins from cat tracheal gland cells in primary culture. 821 86

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.
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PMID:Novel purification and detailed characterization of perlecan isolated from the Engelbreth-Holm-Swarm tumor for use in an animal model of fibrillar A beta amyloid persistence in brain. 888 31

Collagen XIV, a fibril-associated collagen with interrupted triple helices, is expressed in differentiated soft connective tissues and in cartilage. However, a cellular receptor for this protein has not been identified. Here we show that human placental collagen XIV, isolated by a mild and simple two-step method, serves as adhesive protein for a variety of mesenchymal and some epithelial cells. Cell adhesion could be inhibited by preincubation of the collagen XIV substrate with heparin or with the chondroitin/dermatan sulfate proteoglycan decorin and by pretreatment of cells with chondroitinase ABC or heparinase III, suggesting a cell membrane proteoglycan as receptor. Affinity chromatography of 125I-labeled fibroblast cell surface proteins on collagen XIV-Sepharose yielded a chondroitin/dermatan sulfate proteoglycan with a molecular mass of 97-105 kDa after chondroitinase ABC digestion and of 60-70 kDa after further treatment with N-glycosidase F. The eluates contained also some high-molecular-weight material that was susceptible to digestion with heparinase but no detectable integrins. Immunoprecipitation with a specific monoclonal antibody identified the prominent chondroitin/dermatan sulfate proteoglycan as a member of the CD44 family. The interaction between collagen XIV and cells appears to be finely tuned, since matrix-associated glycosaminoglycans, and particularly proteoglycans like decorin, could compete with cells for the binding site(s) on collagen XIV under physiological conditions.
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PMID:A chondroitin/dermatan sulfate form of CD44 is a receptor for collagen XIV (undulin). 898 22

Heparan sulfate (HS) proteoglycans play a key role in cell proliferation induced by basic fibroblast growth factor (FGF-2) and other heparin-binding growth factors. To modulate the involvement of HS, we have used a synthetic, nonsulfated polyanionic aromatic compound (RG-13577) that mimics functional features of heparin/HS. FGF-2-stimulated proliferation of vascular endothelial cells was markedly inhibited in the presence of 5-10 microg/ml compound RG-13577 (poly-4-hydroxyphenoxy acetic acid; Mr approximately 5 kD). Direct interaction between RG-13577 and FGF-2 was demonstrated by the ability of the former to compete with heparin on binding to FGF-2. RG-13577 inhibited FGF-2 binding to soluble- and cell surface-FGF receptor 1 (FGFR1). Unlike heparin, RG-13577 alone failed to mediate dimerization of FGF-2. Moreover, it abrogated heparin-mediated dimerization of FGF-2 and FGFR1, as well as FGF-2 mitogenic activity in HS-deficient F32 lymphoid cells. The antiproliferative effect of compound RG-13577 was associated with abrogation of FGF-2-induced tyrosine phosphorylation of FGFR1 and of cytoplasmic proteins involved in FGF-2 signal transduction, such as p90 and mitogen-activated protein kinase. A more effective inhibition of tyrosine phosphorylation was obtained after removal of the cell surface HS by heparinase. In contrast, tyrosine phosphorylation of an approximately 200-kD protein was stimulated by RG-13577, but not by heparin or FGF-2. RG-13577 prevented microvessel outgrowth from rat aortic rings embedded in a collagen gel. Development of nontoxic polyanionic compounds may provide an effective strategy to inhibit FGF-2-induced cell proliferation associated with angiogenesis, arteriosclerosis, and restenosis.
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PMID:Modulation of fibroblast growth factor-2 receptor binding, dimerization, signaling, and angiogenic activity by a synthetic heparin-mimicking polyanionic compound. 912

Vascular smooth muscle cells (SMCs) are very quiescent in the mature vessel and exhibit a remarkable phenotype-dependent diversity in gene expression that may reflect the growth responsiveness of these cells under a variety of normal and pathological conditions. In this report, we describe the expression pattern of Oct-1, a member of a family of transcription factors involved in cell growth processes, in cultured and in in vivo SMCs. Oct-1 mRNA was undetectable in the contractile-state in vivo SMCs; was induced upon disruption of in vivo SMC-extracellular matrix interactions; and was constitutively expressed by cultured SMCs. Oct-1 transcripts were repressed when cultured SMCs were plated on Engelbreth-Holm-Swarm tumor-derived basement membranes (EHS-BM) but were rapidly induced after disruption of SMC-EHS-BM contacts; reexpression was regulated at the transcriptional level. To identify the EHS-BM component involved in the active repression of Oct-1 mRNA expression, SMCs were plated on laminin, type IV collagen, fibronectin, or perlecan matrices. Oct-1 mRNA levels were readily detectable when SMCs were cultured on matrices composed of laminin, type IV collagen, or fibronectin but were repressed when SMCs were cultured on perlecan matrices. Finally, the Oct-1-suppressing activity of EHS-BM was sensitive to heparinase digestion but not to chondroitinase ABC or hyaluronidase digestion, suggesting that the heparan sulfate side chains of perlecan play a biologically important role in negatively regulating the expression of Oct-1 transcripts.
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PMID:Perlecan regulates Oct-1 gene expression in vascular smooth muscle cells. 920 11

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.
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PMID:The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation. 968 20

Two N-terminal ends of human type XVIII collagen chains have recently been identified. The two chains have different signal peptides and variant N-terminal noncollagenous NC1 domains of 493 (NC1-493) and 303 (NC1-303) amino acid residues, respectively, but share 301 residues of their NC1 domains as well as the collagenous and C-terminal noncollagenous portions of the molecule. Antibodies were produced against the NC1 region common to both human alpha1(XVIII) chain variants and against NC1 sequences specific to the long variant and were used in combination with in situ hybridization to localize this collagen in a number of human tissues. They were also used for Western blotting, which resulted in detection of overlapping high-molecular weight bands above the 200-kd standard in a kidney extract. Heparin lyase II and heparin lyase III digestions of kidney and placenta extracts indicated that at least in these tissues, type XVIII collagen contains heparin sulfate glycosaminoglycan side chains. Type XVIII collagen was found to be a ubiquitous basement membrane component, occurring prominently at vascular and epithelial basement membranes throughout the body. Comparison of the expression of the NC1-493 and NC1-303 variants revealed marked differences. The short variant was found in most conventional basement membranes, including blood vessels and the various epithelial structures, and around muscular structures. The long variant was expressed very strongly in liver, where it was virtually the only variant in the liver sinusoids, and it occurred only in minor amounts elsewhere. Thus, the 192 N-terminal residues specific to the long variant apparently confer some functional property needed above all in the liver sinusoids, but also at certain other locations.
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PMID:The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans. 970 20

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.
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PMID:Characterization of type XI collagen-glycosaminoglycan interactions. 1108 37

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.
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PMID:Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling. 1264 3


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