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Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes.

ABSTRACT: A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.

SUBMITTER: Nakato E

PROVIDER: S-EPMC6521943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation.

Project description:Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.

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Heparan sulfate proteoglycans regulate autophagy in Drosophila.

Project description:Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.

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Heparan sulfate negatively regulates intestinal stem cell proliferation in Drosophila adult midgut.

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A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.

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Establishment of stable cell lines of Drosophila germ-line stem cells.

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Project description:S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12-HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12-HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single-point mutation. Using a HS oligosaccharide microarray, we demonstrated that the N43K mutant displayed markedly enhanced selectivity toward longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.

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Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines.

Project description:BACKGROUND:Cholangiocarcinoma (CCA) is a rare, aggressive and highly lethal tumor. The disease is very difficult to diagnose and multi-modality treatments are ineffective. To improve our understanding of the biological characteristics of CCA, and facilitate the identification of valid treatments, an in-depth characterization of two novel Chinese patient-derived primary CCA cell lines was performed. METHODS:Two CCA cell lines were developed and labelled ZJU-0826 and -1125. The two cell lines were characterized with respect to phenotypic, molecular, biomarker, functional and histological properties. RESULTS:Two novel cell lines were cultured for 2 years, and maintained for more than 100 passages. They retained their typical biliary epithelial morphology and ultrastructure. The population doubling times of ZJU-0826, and -1125 were 63.84 h and 44.73 h, respectively. The cells exhibited near-triploid karyotypes with complex structural aberrations. ZJU-1125 cells had mutations in TP53 exons. Short tandem repeats genotyping confirmed the human origin and difference between lines. An immunophenotype analysis showed that ZJU-0826 is positive for CD44, CD29, Pdx1, CD236, FoxA1, FoxA2, and Nanog, and ZJU-1125 positive for CD44, CD29, CD133, Pdx1, FoxA1, FoxA2, and Nanog. ZJU-1125 had greater invasion ability in vitro and tumorigenicity than those of ZJU-0826. CONCLUSIONS:Our results confirm the validity of the ZJU-0826 and -1125 as representative models for the elucidation of the molecular pathogenesis of perihilar CCA, and intrahepatic CCA in both in vitro and in vivo studies, respectively.

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| S-EPMC4378927 | biostudies-literature

Establishment and Characterization of Carboplatin-Resistant Retinoblastoma Cell Lines.

Project description:Carboplatin-based chemotherapy is the primary treatment option for the management of retinoblastoma, an intraocular malignant tumor observed in children. The aim of the present study was to establish carboplatin-resistant retinoblastoma cell lines to facilitate future research into the treatment of chemoresistant retinoblastoma. In total, two retinoblastoma cell lines, Y79 and SNUOT-Rb1, were treated with increasing concentrations of carboplatin to develop the carboplatin-resistant retinoblastoma cell lines (termed Y79/CBP and SNUOT-Rb1/CBP, respectively). To verify resistance to carboplatin, the degree of DNA fragmentation and the expression level of cleaved caspase-3 were evaluated in the cells, following carboplatin treatment. In addition, the newly developed carboplatin-resistant retinoblastoma cells formed in vivo intraocular tumors more effectively than their parental cells, even after the intravitreal injection of carboplatin. Interestingly, the proportion of cells in the G0/G1 phase was higher in Y79/CBP and SNUOT-Rb1/CBP cells than in their respective parental cells. In line with these data, the expression levels of cyclin D1 and cyclin D3 were decreased, whereas p18 and p27 expression was increased in the carboplatin-resistant cells. In addition, the expression levels of genes associated with multidrug resistance were increased. Thus, these carboplatin-resistant cell lines may serve as a useful tool in the study of chemoresistance in retinoblastoma and for the development potential therapeutics.

| S-EPMC9589373 | biostudies-literature

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