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  • Title: Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae.
    Author: Saito F, Sakamoto I, Kanatani A, Chiba Y.
    Journal: Biochim Biophys Acta; 2016 Sep; 1860(9):1809-20. PubMed ID: 27259834.
    Abstract:
    BACKGROUND: Production of various mucin-like glycoproteins could be useful for development of antibodies specific to disease-related glycoproteins as well as for the biosynthesis of clinically useful glycoproteins. A Saccharomyces cerevisiae strain capable of in vivo production of mucin-type core 1 structure (Galβ1-3GalNAcα1-O-Ser/Thr) has been reported, but a strain producing core 3 structure (GlcNAcβ1-3GalNAcα1-O-Ser/Thr) has not been constructed. METHODS: To generate core 3-producing strain, genes encoding uridine diphosphate (UDP)-Gal-4-epimerase, UDP-GalNAc transporter, UDP-GlcNAc transporter, and two glycosyltransferases were integrated into the genome. A Mucin-1-derived acceptor peptide (MUC1ap) was expressed as an acceptor. The amount of the resulting modified peptide was analyzed by HPLC. RESULTS: Introduction of a codon-optimized UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 6 (β3Gn-T6) gene yielded increases in β3Gn-T6 activity but did not alter the level of core 3 production. The highest in vitro activity of β3Gn-T6 was observed at Mn(2+) concentrations of 10mM and above. Supplementation of MnCl2 to the culture medium yielded increases of up to 25% in the accumulation of core 3 on the MUC1ap. The yeast invertase from the core 3-producing strain was less extensively N-glycosylated; however, it was partially restored by the addition of MnCl2 to the medium. CONCLUSIONS: Physiological Mn(2+) concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn(2+) supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production. GENERAL SIGNIFICANCE: This study reveals that control of Mn(2+) concentration is important for production of specific mammalian-type glycans in S. cerevisiae.
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