Xylosyltransferase-deficient human HEK293 cells show a strongly reduced proliferation capacity and viability |
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| Institution: | 1. Department of Cellular and Molecular Medicine, Centre of Biological Research, CIB-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain;2. CIBER de Enfermedades Raras (CIBERER), Madrid, Spain;1. Department of Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy;2. Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy;1. Department of Medical Genetics, Peking University School of Basic Medical Sciences, Beijing 100191, China;2. Peking University Center for Human Disease Genomics, Beijing 100191, China;3. Department of Immunology, Peking University School of Basic Medical Science, Key Laboratory of Medical Immunology, Beijing 100191, China;1. Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32545 Bad Oeynhausen, Germany;2. MVZ Labor Limbach Nürnberg, Lina-Ammon-Strasse 28, 90471 Nürnberg, Germany;3. Department of Chemistry and Molecular and Cell Biology Howard Hughes Medical Institute University of California, Berkeley, CA 94720, USA;1. Department of Biochemistry, Hoshi University School of Pharmacy, 2-4-41, Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan;2. Department of Microbiology, Hoshi University School of Pharmacy, 2-4-41, Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan;3. Department of Clinical and Analytical Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan;4. SBI Pharmaceuticals Co., Ltd., 1-6-1, Roppongi, Minato-ku, Tokyo, 106-6020, Japan;5. Division of Glycobiologics, Intractable Disease Research Center, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan |
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| Abstract: | Human xylosyltransferases-I and –II (XT-I and XT-II) catalyze the initial and rate-limiting step in proteoglycan (PG)-biosynthesis. Because PG are major components of the extracellular matrix (ECM), an alternated XT expression is associated with the manifestation of ECM-related diseases.While Drosophila melanogaster and Caenorhabditis elegans only harbor one XT-isoform, all higher organisms contain two isoforms, which are expressed in a tissue-specific manner. The reason for the appearance of two isoenzymes remains unexplained and remarkable, as all other enzymes involved in the synthesis of the tetrasaccharid linker, which connects the PG core protein with attached glycosaminoglycans, only show one isoform. In human, mutations in the XYLT genes cause diseases affecting the homeostasis of the ECM, such as skeletal dysplasias.We investigated for the first time whether already XT-I-deficient human embryonic kidney (HEK293) cells can compensate for decreased expression levels of both XT-isoforms. A siRNA-mediated XYLT2 mRNA knockdown led to reduced cellular proliferation rates and a partially increased cellular senescence of treated HEK293 cells. These results were verified by conducting a stable CRISPR/Cas9-mediated XYLT2 knockout, which revealed that only cells expressing at least partially functional XT-II proteins remain proliferative. Our study, therefore, shows for the first time that cells lacking both XT-isoforms are not viable and clearly indicates the importance of the XT concerning the cellular metabolism. |
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| Keywords: | CRISPR/Cas9 Extracellular matrix HEK293 cells Xylosyltransferase |
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