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Miao HQ Navarro E Patel S Sargent D Koo H Wan H Plata A Zhou Q Ludwig D Bohlen P Kussie P 《Protein expression and purification》2002,26(3):425-431
Heparanase is an endoglucuronidase that plays an important role in tumor invasion and metastasis. A full-length heparanase gene was cloned from a mouse embryo cDNA library and determined to encode a protein of 535 amino acids that is 77% identical to human heparanase. The full-length mouse gene was stably expressed in NS0 myeloma cells. The recombinant mouse heparanase protein was purified to homogeneity from cell lysates by a combination of Con-A affinity chromatography, heparin affinity chromatography, and size exclusion chromatography. The purified protein consisted of a non-covalent heterodimer of 50- and 8-kDa polypeptides, similar to the human homolog. The protein was enzymatically active in assays using radiolabeled ECM and heparan sulfate as substrates. The maximum heparanase activity was observed at acidic conditions; however, significant activity was also detected at neutral pH. The enzymatic activity of mouse heparanase was blocked by known heparanase inhibitors. 相似文献
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Radiolabeled heparan sulfate immobilized on microplate as substrate for the detection of heparanase activity 总被引:1,自引:0,他引:1
We developed a quantitative assay to monitor the enzymatic activity of heparanase, a protein responsible for the degradation of heparan sulfate (HS) present on cell surface and extracellular matrix. Our assay is based on a new procedure to immobilize radiolabeled HS to a solid support by a single end which is adaptable to a microplate format, thus allowing the rapid analysis of numerous samples. First, HS was radiolabeled by partial de-N-acetylation and re-N-acetylation with [3H] acetic anhydride, second, after reductive amination at the reducing terminus, it was covalently linked to an amino-reactive biotin analog, and third it was immobilized on a streptavidin-coated plate. The degradation of our solid-phase tritiated HS by heparanase was monitored by measuring the soluble radioactivity released in the well. The heparanase-induced release of radioactivity was linear with respect either to time or to the amount of enzyme and was inhibited by heparin or high ionic strength. The linearity of this assay for time and enzyme concentrations could be useful to determine the potency of heparanase inhibitors. Moreover, this assay was shown to be suitable for monitoring HS-degrading activity of either heparanase endogenously expressed by the HCT 116 tumor cell line or recombinant forms of this protein. 相似文献
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Heparanase is an endo-β-d-glucuronidase responsible for the cleavage of heparan sulfate, participating in extracellular matrix degradation and remodeling. Heparanase activity is well correlated with the potential for metastasis and angiogenesis in a large number of tumor-derived cell types, directly implicating the involvement of heparanase in tumor progression. Here, we provide the first evidence that the hydrophobic C-terminus region of heparanase has specific roles in intracellular trafficking, secretion, activation, and heparanase-mediated tumor cell migration. Furthermore, partial deletion of this hydrophobic C-terminus region, substitution within the hydrophobic C-terminus region to hydrophilic amino acids, and experiments of single amino acid mutations further point out the importance of the hydrophobic C-terminus region. Therefore, our findings suggest that the hydrophobic C-terminus region of heparanase is a determinant for its intracellular trafficking to the Golgi apparatus, followed by secretion, activation, and tumor cell migration. 相似文献
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A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors. 相似文献
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A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely β-d-GlcA(2S)-(1→4)-α-d-GlcN(NS,6S)-(1→4)-β-d-GlcA-(1→4)-α-d-GlcN(NS,6S)-OMe, was synthesized in a convergent manner via coupling of a pair of the disaccharide building blocks as a key step. 相似文献
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Edward Hammond 《Analytical biochemistry》2010,396(1):112-1918
The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a Km of 46 μM and a kcat of 3.5 s−1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a Ki of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies. 相似文献
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S. Xie X. Zhang Z. Zhou X. Li Y. Huang J. Zhang J. Weng 《Plant biology (Stuttgart, Germany)》2018,20(1):59-66
- The process of alternative splicing is critical for the regulation of growth and development of plants. Thus far, little is known about the role of alternative splicing in the regulation of maize (Zea mays L.) endosperm development.
- RNA sequencing (RNA‐seq) data of endosperms from two maize inbred lines, Mo17 and Ji419, at 15 and 25 days after pollination (DAP), respectively, were used to identify genes that were alternatively spliced during endosperm development. Intron retention (IR) in GRMZM2G005887 was further validated using PCR and re‐sequencing technologies.
- In total, 49,000 alternatively spliced events and ca. 20,000 alternatively spliced genes were identified in the two maize inbred lines. Of these, 30 genes involved in amino acid biosynthesis and starch biosynthesis were identified, with IR occurring only in a specific sample, and were significantly co‐expressed with ten well‐known genes related to maize endosperm development. Moreover, IR in GRMZM2G005887, which encodes a cysteine synthase, was confirmed to occur only in the endosperm of Mo17 at 15 DAP, resulting in the retention of a 121‐bp fragment in its 5′ untranslated region. Two cis‐acting regulatory elements, CAAT‐box and TATA‐box were observed in the retained fragment in Mo17 at 15 DAP; this could regulate the expression of this gene and influence endosperm development.
- The results suggest that the 30 genes with IR identified herein might be associated with maize endosperm development, and are likely to play important roles in the developing maize endosperm.
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Chun-Chun Cheng Yen-Hua Lee Shau-Ping Lin Wei-Chun HuangFu I-Hsuan Liu 《Journal of biomedical science》2014,21(1):21
Background
Stem cell-fate is highly regulated by stem cell niche, which is composed of a distinct microenvironment, including neighboring cells, signals and extracellular matrix. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells and are potentially applicable in wide variety of pathological conditions. However, the niche microenvironment for BM-MSCs maintenance has not been clearly characterized. Accumulating evidence indicated that heparan sulfate glycosaminoglycans (HS-GAGs) modulate the self-renewal and differentiation of BM-MSCs, while overexpression of heparanase (HPSE1) resulted in the change of histological profile of bone marrow. Here, we inhibited the enzymatic activity of cell-autonomous HPSE1 in BM-MSCs to clarify the physiological role of HPSE1 in BM-MSCs.Results
Isolated mouse BM-MSCs express HPSE1 as indicated by the existence of its mRNA and protein, which includes latent form and enzymatically active HPSE1. During in vitro osteo-differentiations, although the expression levels of Hpse1 fluctuated, enzymatic inhibition did not affect osteogenic differentiation, which might due to increased expression level of matrix metalloproteinase 9 (Mmp9). However, cell proliferation and colony formation efficiency were decreased when HPSE1 was enzymatically inhibited. HPSE1 inhibition potentiated SDF-1/CXCR4 signaling axis and in turn augmented the migratory/anchoring behavior of BM-MSCs. We further demonstrated that inhibition of HPSE1 decreased the accumulation of acetylation marks on histone H4 lysine residues suggesting that HPSE1 also modulates the chromatin remodeling.Conclusions
Our findings indicated cell-autonomous HPSE1 modulates clonogenicity, proliferative potential and migration of BM-MSCs and suggested the HS-GAGs may contribute to the niche microenvironment of BM-MSCs. 相似文献13.
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Krishnaveni MS Hansen JL Seeger W Morty RE Sheikh SP Eickelberg O 《Biochemical and biophysical research communications》2006,351(3):651-657
Transforming growth factor (TGF)-beta ligands signal through transmembrane type I and type II serine/threonine kinase receptors, which form heteromeric signalling complexes upon ligand binding. Type II TGF-beta receptors (TbetaRII) are reported to exist as homodimers at the cell surface, but the oligomerization pattern and dynamics of TbetaRII splice variants in live cells has not been demonstrated thus far. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET), we demonstrate that the mouse TbetaRII receptor splice variant TbetaRII-B is capable of forming ligand-independent homodimers and heterodimers with TbetaRII. The homomeric interaction of mouse (m)TbetaRII-B isoforms, however, is less robust than the heteromeric interactions of mTbetaRII-B with wild-type TbetaRII, which indicates that these receptors may be more likely to heterodimerize when both receptors are expressed. Moreover, we demonstrate that mTbetaRII-B is a signalling receptor with ubiquitous tissue expression. Our study thus demonstrates previously unappreciated complex formation of TGF-beta type II receptors, and suggests that mTbetaRII-B can direct TGF-beta-induced signalling in vitro and in vivo. 相似文献
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Interleukin 23 (IL-23) is a new member of the IL-12 family that plays a critical role in promoting the proliferation of memory T helper 1 cells. The heterodimerized IL-23 receptor is composed of a shared IL-12 receptor beta 1 (IL-12Rβ1) and an IL-12Rβ2-related molecule called IL-23R. The standard form of IL-23R is encoded by at least 12 exons. Here, we demonstrate that at least six spliced isoforms of IL-23R (IL-23R1 to 6) can be generated through alternative splicing. The splicing strategies for the IL-23R gene are complicated and most often result in the deletion of exon 7 and/or exon 10. Translation prediction revealed that these spliced variants result in either premature termination to give rise to a diverse form of receptor ectodomain, or a frameshift to generate various lengths of the IL-23R endodomain. Differential expressions of IL-23R spliced variants are observed in natural killer and CD3+CD4+ T cells. The expressions of these spliced variants are also prevalently and complicatedly regulated in tumor cell lines. Interestingly, only IL-23R2 and/or IL-23R4 variants are predominantly detected in certain human lung carcinomas, but not in their resected normal margin tissues. Thus, our results indicate that the regulation of alternative splicing on the IL-23R gene is complicated, and the preferential expression of certain IL-23R spliced variants may be a contributive factor to the pathogenesis of certain cancers. 相似文献
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细胞外基质和基底膜的降解是癌细胞穿透组织屏障发生转移的重要步骤。硫酸乙酰肝素蛋白聚糖是细胞外基质和基底膜的组成成分,其多糖侧链可以被葡萄糖苷内切酶--肝素酶,特异性识别并切割,以破坏细胞外基质和基底膜的完整性,促进肿瘤转移。临床上肿瘤患者肝素酶高表达与肿瘤恶性程度和转移发生密切相关。深入了解硫酸乙酰肝素、肝素酶及它们与肿瘤转移相关的作用机制有助于我们寻找肿瘤治疗的新思路。本文将从硫酸乙酰肝素的合成调控、功能、肝素酶的转录和活性调节、肝素酶表达与肿瘤患者的临床特征,以及硫酸乙酰肝素、肝素酶与肿瘤转移的关系进行综述。 相似文献
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