| 1. |
- Batool, Tahira, et al.
(författare)
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Overexpression of heparanase attenuated TGF-beta-stimulated signaling in tumor cells
- 2017
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 7:3, s. 405-413
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) mediates the activity of various growth factors including TGF-beta. Heparanase is an endo-glucuronidase that specifically cleaves and modifies HS structure. In this study, we examined the effect of heparanase expression on TGF-beta 1-dependent signaling activities. We found that overexpression of heparanase in human tumor cells (i.e., Fadu pharyngeal carcinoma, MCF7 breast carcinoma) attenuated TGF-beta 1-stimulated Smad phosphorylation and led to a slower cell proliferation. TGF-beta 1-stimulated Akt and Erk phosphorylation was also affected in the heparanase overexpression cells. This effect involved the enzymatic activity of heparanase, as overexpression of mutant inactive heparanase did not affect TGF-beta 1 signaling activity. Analysis of HS isolated from Fadu cells revealed an increase in sulfation of the HS that had a rapid turnover in cells overexpressing heparanase. It appears that the structural alterations of HS affect the ability of TGF-beta 1 to signal via its receptors and elicit a growth response. Given that heparanase expression promotes tumor growth in most cancers, this finding highlights a crosstalk between heparanase, HS, and TGF-beta 1 function in tumorigenesis.
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| 2. |
- Batool, Tahira, et al.
(författare)
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Upregulated BMP-Smad signaling activity in the glucuronyl C5-epimerase knock out MEF cells
- 2019
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Ingår i: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 54, s. 122-129
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Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of glucuronic acid to iduronic acid in the process of heparan sulfate biosynthesis. Targeted interruption of the gene, Glce,in mice resulted in neonatal lethality with varied defects in organ development. To understand the molecular mechanisms of the phenotypes, we used mouse embryonic fibroblasts (MEF) as a model to examine selected signaling pathways. Our earlier studies found reduced activities of FGF-2, GDNF, but increased activity of sonic hedgehog in the mutant cells. In this study, we focused on the bone morphogenetic protein (BMP) signaling pathway. Western blotting detected substantially elevated endogenous Smad1/5/8 phosphorylation in the Hsepi mutant (KO) MEF cells, which is reverted by re-expression of the enzyme in the KO cells. The mutant cells displayed an enhanced proliferation and elevated alkaline phosphatase activity, marking higher differentiation, when cultured in osteogenic medium. The high level of Smad1/5/8 phosphorylation was also found in primary calvarial cells isolated from the KO mice. Analysis of the genes involved in the BMP signaling pathway revealed upregulation of a number of BMP ligands, but reduced expression of several Smads and BMP antagonist (Grem1) in the KO MEF cells. The results suggest that Hsepi expression modulates BMP signaling activity, which, at least partially, is associated with defected molecular structure of heparan sulfate expressed in the cells.
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| 3. |
- Cui, Hao, et al.
(författare)
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Re-expression of glucuronyl C5-epimerase in the mutant MEF cells increases heparan sulfate epimerization but has no influence on the Golgi localization and enzymatic activity of 2-O-sulfotransferase
- 2021
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Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:8, s. 1018-1025
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure-function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.
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| 4. |
- Ek, Pia, et al.
(författare)
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Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269, s. 5016-5023
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Tidskriftsartikel (refereegranskat)abstract
- Protein histidine phosphorylation in eukaryotes has beensparsely studied compared to protein serine/threonine andtyrosine phosphorylation. In an attempt to rectify this byprobing porcine liver cytosol with the phosphohistidinecontainingpeptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide(phosphopeptide I), we observed a phosphataseactivity that was insensitive towards okadaic acid andEDTA. This suggested the existence of a phosphohistidinephosphatase different from protein phosphatase 1, 2Aand 2C. A 1000-fold purification to apparent homogeneitygave a 14-kDa phosphatase with a specific activity of 3lmolÆmin)1Æmg)1 at pH 7.5 with 7 lM phosphopeptide Ias substrate. Partial amino-acid sequence determination ofthe purified porcine enzyme by MS revealed similaritywith a human sequence representing a human chromosome9 gene of hitherto unknown function. Molecularcloning from a human embryonic kidney cell cDNAlibraryfollowed by expression and purification, yielded aprotein with a molecular mass of 13 700 Da, and anEDTA-insensitive phosphohistidine phosphatase activityof 9 lmolÆmin)1Æmg)1 towards phosphopeptide I. Nodetectable activity was obtained towards a set of phosphoserine-,phosphothreonine-, and phosphotyrosine peptides.Northern blot analysis indicated that the humanphosphohistidine phosphatase mRNA was present preferentiallyin heart and skeletal muscle. These resultsprovide a new tool for studying eukaryotic histidinephosphorylation/dephosphorylation.
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| 5. |
- Fang, Jianping, et al.
(författare)
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Enzyme overexpression - an exercise toward understanding regulation of heparan sulfate biosynthesis
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.
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| 6. |
- Gustafsen, Camilla, et al.
(författare)
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Heparan sulfate proteoglycans present PCSK9 to the LDL receptor
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9: LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.
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| 7. |
- Hjertström, Elina, 1981-
(författare)
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Roles of Heparan Sulfate in Amyloid-β Pathology and Hypoxia
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS) is a highly sulfated polysaccharide expressed on the cell surface and in the extracellular matrix, interacting with a large number of proteins. HS is implicated in human diseases, including different types of cancer and amyloid diseases such as Alzheimer's disease (AD). The aims of this thesis were to gain deeper insights into AD and cancer progression by elucidating the roles of HS in amyloid-β (Aβ) pathology and hypoxia. The toxic Aβ-peptide is a key molecule in AD due to its ability to aggregate and form amyloid plaques in the brains of diseased patients. It has been reported that HS accumulates with Aβ in these amyloid plaques. We have found that HS is differentially accumulated with Aβ species within the amyloid plaques in the brains of AD patients. We also identified that the HS in the plaques originated from glial cells. Further, we investigated the role of HS in Aβ toxicity using cell models that either lack HS or express abnormal HS. The results show that cell surface HS mediates Aβ internalization and cytotoxicity. Upregulation of heparanase, an endo-glucuronidase that specifically cleaves HS chains, in human cancers increases the potential of tumor cells to metastasize. Spalax, an animal model for hypoxic tolerance, expresses high levels of heparanase. Analysis of HS from different Spalax organs revealed a high sulfation degree and an atypical domain structure, likely modulated by high heparanase expression in the organs. Cells cultured under hypoxic conditions showed a similar HS domain structure and had an increase in heparanase mRNA. We propose that hypoxia-induced heparanase expression is relevant for tumor progression, a process often associated with oxygen deficiency. Altogether, the findings in this thesis are important for future development of therapeutics aiming at interfering with HS functions in AD and cancer.
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| 8. |
- Jendresen, Charlotte, et al.
(författare)
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Systemic LPS-induced A beta-solubilization and clearance in A beta PP-transgenic mice is diminished by heparanase overexpression
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-beta (A(beta) is the main constituent of amyloid deposits in Alzheimer's disease (AD). The neuropathology is associated with neuroinflammation. Here, we investigated effects of systemic lipopolysaccharide (LPS)-treatment on neuroinflammation and A beta deposition in A beta PP-mice and doubletransgenic mice with brain expression of A beta PP and heparanase, an enzyme that degrades HS and generates an attenuated LPS-response. At 13 months of age, the mice received a single intraperitoneal injection of 50 mu g LPS or vehicle, and were sacrificed 1.5 months thereafter. A beta in the brain was analyzed histologically and biochemically after sequential detergent extraction. Neuroinflammation was assessed by CD45 immunostaining and mesoscale cytokine/chemokine ELISA. In single-transgenic mice, LPS-treatment reduced total A beta deposition and increased Tween-soluble A beta. This was associated with a reduced CXCL1, IL-1 beta, TNF-alpha-level and microgliosis, which correlated with amyloid deposition and total A beta. In contrast, LPS did not change A beta accumulation or inflammation marker in the doubletransgenic mice. Our findings suggest that a single pro-inflammatory LPS-stimulus, if given sufficient time to act, triggers A beta-clearance in A beta PP-transgenic mouse brain. The effects depend on HS and heparanase.
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| 9. |
- Jia, Juan, 1979-, et al.
(författare)
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Heparanase cleavage of heparin modulates protease storage in mast cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Overexpression of heparanase caused extensive degradation of heparan sulfate (HS), and elimination of heparanase resulted in non-degraded HS chains in mice. In this study, we have investigated the impact of heparanase in the processing of heparin and storage of proteases in mast cells. We used fetal skin mast cells (FSMCs) isolated from wild type (WT) embryos and embryos either overexpressing human heparanase (hpa-tg), or lacking heparanase (Hpse-KO). FSMCs from hpa-tg embryos produced substantially shorter heparin chains than did WT counterparts, whereas FSMCs from Hpse-KO embryos expressed longer chains than WT cells. Extensive fragmentation of heparin in hpa-tg FSMC caused losing of proteases in the cells; in contrast, increased storage of proteases was observed in Hpse-KO cells. These results provide the first in vivo evidence demonstrating that heparanase is responsible for processing of mast cell heparin. Control of heparin degradation by heparanase in mast cell may contribute to modulating protease storage in the cells.
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| 10. |
- Jia, Juan, 1979-
(författare)
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Structure and functions of heparan sulfate/heparin – Importance of glucuronyl C5-epimerase and heparanase
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS) and heparin are linear polysaccharide chains covalently O-linked to serine residues within the core proteins, so called HS proteoglycans (PGs) or heparin PG. HSPGs are produced by almost all mammalian cells and known to play important roles in developmental processes, physiological and pathological conditions; whereas heparin PG is produced by mast cells and best known as an anticoagulant in clinic. Biosynthesis of HS/heparin occurs in Golgi compartment and involves many enzymes, one of which is glucuronyl C5-epimerase (Hsepi) that catalyzes the conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). Heparanase is an enzyme involved in metabolism of HS; it cleaves the linkage between GlcA and glucosamine residues in HS/heparin chains. Heparanase is expressed essentially by all cells and found up-regulated in many metastatic tumors. This thesis focuses on the structure and functions of HS/heparin through studies on the implications of Hsepi and heparanase. My study demonstrated that the modification catalyzed by Hsepi is critical for HS-dependent function of growth factors, especially FGF2; heparanase is involved in regulation of HS biosynthesis and matrix metalloproteinases expression; moreover, my experimental data demonstrated the functions of heparin in mast cells, showing cleavage of heparin by heparanase contributes to modulation of protease storage in mast cells.
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