title: Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal.
abstract: The folate and cobalamin (Cbl-) dependent enzyme methionine synthase (MS) is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl) or the combination of hydroxocobalamin (OHCbl) and S-adenosylmethionine (SAM). OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH) but could be rescued by provision of either glutathionylcobalamin (GSCbl) or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.
authors.affiliation: Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, 123 Al-Khoud, Oman.
authors.forename: Mostafa
authors.identifier: 0000-0002-8197-3378
authors.initials: M
authors.lastname: Waly
authors.affiliation: Natural Sciences Department, Regis College, Weston, MA 02493, USA.
authors.forename: Verna-Ann
authors.initials: VA
authors.lastname: Power-Charnitsky
authors.affiliation: Department of Neurology, Boston Children's Hospital, Boston, MA 02215, USA.
authors.forename: Nathaniel
authors.identifier: 0000-0001-8299-3963
authors.initials: N
authors.lastname: Hodgson
authors.affiliation: Department of Pharmaceutical Sciences, MCPHS University, Manchester, NH 03101, USA.
authors.forename: Alok
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Department of Medicine, New York University Medical School, New York, NY 10016, USA.
authors.forename: Tapan
authors.initials: T
authors.lastname: Audhya
authors.affiliation: Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA.
authors.forename: Yiting
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, FL 33328, USA.
authors.forename: Richard
authors.identifier: 0000-0002-7717-7037
authors.initials: R
authors.lastname: Deth
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects*; D045744:Cell Line, Tumor; D005978:Glutathione / Q000494:pharmacology*; D006801:Humans; D006879:Hydroxocobalamin / Q000494:pharmacology*; D019216:Metals, Heavy / Q000633:toxicity*; D008780:Methyltransferases / Q000096:biosynthesis*; D009363:Neoplasm Proteins / Q000378:metabolism*; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D013849:Thimerosal / Q000494:pharmacology*
chemical_list: D019216:Metals, Heavy; D009363:Neoplasm Proteins; D013849:Thimerosal; D008780:Methyltransferases; C022725:5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase; D005978:Glutathione; D006879:Hydroxocobalamin