Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae

• Published in: Biochimica et biophysica acta Vol. 1860; no. 9; pp. 1809 - 1820
• Main Authors: Saito, Fumie, Sakamoto, Izumi, Kanatani, Akio, Chiba, Yasunori
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2016
• Subjects: antibodies; beta-fructofuranosidase; biosynthesis; Core 3-type O-glycan; culture media; genes; Glycan engineering; glycoproteins; Glycoproteins - genetics; Glycoproteins - metabolism; glycosylation; Glycosylation - drug effects; Glycosyltransferase; glycosyltransferases; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Golgi Apparatus - drug effects; Golgi Apparatus - genetics; Golgi Apparatus - metabolism; Humans; Ions - pharmacology; manganese; Manganese - pharmacology; manganese chloride; Manganese ion; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Mucin-1 - genetics; Mucin-1 - metabolism; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; polysaccharides; Polysaccharides - genetics; Polysaccharides - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; UDPglucose 4-Epimerase - genetics; UDPglucose 4-Epimerase - metabolism; Up-Regulation - drug effects; Up-Regulation - genetics; uridine diphosphate; Yeast; yeasts; Glycan engineering; Yeast; R3A-1c; Glycosyltransferase; GALNT1; TMD; ER; β3Gn-T6; Manganese ion; BEMAD; Gal; ppGalNAc-T1; B3GNT6; Core 3-type O-glycan; MUC1ap; GlcNAc; βNGase; ABEE
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2016.05.038

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. 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. 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 Mn2+ 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. Physiological Mn2+ concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn2+ supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production. This study reveals that control of Mn2+ concentration is important for production of specific mammalian-type glycans in S. cerevisiae. •A synthetic pathway of core 3-type O-glycan is constructed in Saccharomyces cerevisiae.•Multiple expression of exogenous glycosyltransferases depletes Mn2+ in the cell.•Addition of Mn2+ restores β3Gn-T6 expression in the core 3-producing strain.•Extra Mn2+ makes it possible to synthesize core 3-type O-glycan in S. cerevisiae.