| 1. |
- Chen, Yen Hsi, et al.
(författare)
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The GAGOme : a cell-based library of displayed glycosaminoglycans
- 2018
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 15:11, s. 881-888
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Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.
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| 2. |
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| 3. |
- Larson, Daniel, et al.
(författare)
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Bioaccumulation of microcystins in the food web : a field study of four Swedish lakes
- 2014
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Ingår i: Inland Waters. - 2044-2041 .- 2044-205X. ; 4:1, s. 91-104
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Tidskriftsartikel (refereegranskat)abstract
- The transfer of microcystins (MC) up the food chain was measured in 4 lakes in central Sweden; Ekoln, Lilla Ullfjärden, Valloxen, and Storsjön. In lakes Ekoln and Valloxen, Microcystis aeruginosa was the dominant cyanobacterium, while the oscillatorian species Planktothrix prolifica form dense blooms in Lake Lilla Ullfjärden. The cyanobacterial composition in Lake Storsjön was more diverse with several Microcystis andDolichospermum species. All dominant taxa are well-known producers of hepatotoxic MC. The highest recorded MC in the water samples from Lake Lilla Ullfjärden was measured in the bloom of P. prolifica (35 µg L−1). The highest MC content was measured in invertebrates; however, the MC concentration was usually low in fish. Maximum levels were 9 µg g−1 dry weight (dw) in zooplankton, 10 µg g−1 dw in benthos, and 2.7 µg g−1 dw in fish (smelt) liver. In fish muscle the highest recorded levels were 0.10–0.18 µg g−1 dw in bleak and smelt from Lake Lilla Ullfjärden and in pike-perch and roach from Lake Storsjön. Based on the World Health Organization’s tolerable daily intake value of 0.04 µg kg−1 body weight, we conclude that any risk related to MC from human consumption of fish from these lakes is minimal.
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| 4. |
- Mastio, Roberto, et al.
(författare)
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Fluorescently labeled xylosides offer insight into the biosynthetic pathways of glycosaminoglycans
- 2021
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 11:60, s. 38283-38292
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Tidskriftsartikel (refereegranskat)abstract
- Five novel xylosides tagged with the fluorescent probe Pacific Blue™ were synthesized and found to act as substrates for β4GalT7, a bottleneck enzyme in the biosynthetic pathways leading to glycosaminoglycans. By confocal microscopy of A549 cells, we showed that the xylosides were taken up by the cells, but did not enter the Golgi apparatus where most of the glycosaminoglycan biosynthesis occurs. Instead, after a possible double galactosylation by β4GalT7 and β3GalT6, the biosynthesis was terminated. We hypothesize this is due to the charge of the fluorescent probe, which is required for fluorescent ability and stability under physiological conditions.
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| 5. |
- Persson, Andrea, et al.
(författare)
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LC-MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:26, s. 10202-10219
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Tidskriftsartikel (refereegranskat)abstract
- Structural characterization of glycosaminoglycans remains a challenge but is essential for determining structure-function relationships between glycosaminoglycans and the biomolecules with which they interact and for gaining insight into the biosynthesis of glycosaminoglycans. We have recently reported that xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, has cytotoxic effects and shown that it differs in disaccharide composition from nontoxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we developed a novel LC-MS/MS approach based on reversed-phase dibutylamine ion-pairing chromatography and negative-mode higher-energy collision dissociation and used it in combination with cell growth studies and disaccharide fingerprinting. This strategy enabled detailed structural characterization of linkage regions, internal oligosaccharides, and nonreducing ends, revealing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also sialylation of the linkage region and previously undescribed methylation and sulfation of the nonreducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared to be essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.
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| 6. |
- Thorsheim, Karin, et al.
(författare)
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Naphthyl Thio- and Carba-xylopyranosides for Exploration of the Active Site of ß-1,4-Galactosyltransferase 7 (ß4GalT7)
- 2017
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 23:71, s. 18057-18065
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Tidskriftsartikel (refereegranskat)abstract
- Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for -1,4-galactosyltransferase7 (4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for 4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba--xylopyranoside in the d-configuration proved to be a good substrate for 4GalT7, whereas the enantiomer in the l-configuration showed no activity. Further investigations by X-ray crystallography, NMR spectroscopy, and molecular modeling provided a rationale for the pronounced activity of the sulfur analogues. Favorable - interactions between the 2-naphthyl moiety and a tyrosine side chain of the enzyme were observed for the thio analogues, which open up for the design of efficient GAG primers and inhibitors.
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| 7. |
- Willén, Daniel, et al.
(författare)
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Azide-Functionalized Naphthoxyloside as a Tool for Glycosaminoglycan Investigations
- 2021
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Ingår i: Bioconjugate Chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 32:12, s. 2507-2515
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Tidskriftsartikel (refereegranskat)abstract
- We present a xylosylated naphthoxyloside carrying a terminal azide functionality that can be used for conjugation using click chemistry. We show that this naphthoxyloside serves as a substrate for β4GalT7 and induces the formation of soluble glycosaminoglycan (GAG) chains with physiologically relevant lengths and sulfation patterns. Finally, we demonstrate its usefulness by conjugation to the Alexa Fluor 647 and TAMRA fluorophores and coupling to a surface plasmon resonance chip for interaction studies with the hepatocyte growth factor known to interact with the GAG heparan sulfate.
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| 8. |
- Willén, Daniel, et al.
(författare)
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Synthesis of Double-Modified Xyloside Analogues for Probing the β4GalT7 Active Site
- 2018
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Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 83:3, s. 1259-1277
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Tidskriftsartikel (refereegranskat)abstract
- Monosubstituted naphthoxylosides have been shown to function as substrates for, and inhibitors of, the enzyme β4GalT7, a key enzyme in the biosynthetic pathway leading to glycosaminoglycans and proteoglycans. In this article, we explore the synthesis of 16 xyloside analogues, modified at two different positions, as well as their function as inhibitors of and/or substrates for the enzyme. Seemingly simple compounds turned out to require complex synthetic pathways. A meta-analysis of the synthetic work shows that, regardless of the abundance of methods available for carbohydrate synthesis, even simple modifications can turn out to be problematic, and double modifications present additional challenges due to conformational, steric, and stereoelectronic effects.
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| 9. |
- Willén, Daniel
(författare)
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Tools for understanding the glycosaminoglycan biosynthesis
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- For cells to function correctly within an organism, they need different systems to communicate with each other. One crucial part of cell signaling is the proteoglycans (PGs) and glycosaminoglycans (GAGs), which are macromolecules that bind different signaling molecules and proteins necessary for processes such as growth and proliferation. Therefore, PGs and GAGs are involved in pathological processes like cancer and bacterial or viral infection. A tetrasaccharide linker connects several types of GAGs to the PG core protein, with the first carbohydrate being a xylose. Xylose is, due to its scarcity in mammalian cells, an attractive target for therapeutics. In GAG biosynthesis, the enzyme β4GalT7 galactosylates the xylose. Therefore, this enzyme could be interesting to target using synthetic xylosides that could act as substrates or inhibitors. These xylosides could allow us to understand and control the biosynthesis of GAGs. This thesis is focused on the design and synthesis of modified xylosides and how we can use these as tools to study the formation of GAGs. We decided to alter the carbohydrate itself to investigate if it is possible to design effective substrates or inhibitors for β4GalT7. We also decided to modify the aglycon, the part attached to the carbohydrate, to answer questions related to how cells process GAGs. This work has resulted in several new substances that, that enter cells, and work well as substrates and inhibitors of β4GalT7 to provide answers to how GAGs are structured, how they move within the cell, and how they interact with other biomolecules such as viral proteins. The results of this work pave the way for the use of synthetic xylosides to answer several questions about GAG-related processes and open up the possibility for creating tools for influencing and studying cells' expression of GAGs.
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