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



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.



This study reveals that control of Mn(2+) concentration is important for production of specific mammalian-type glycans in S. cerevisiae.
Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; GlycoGene Inc., Avenue Takanawa Rm. 412, 3-25-27, Takanawa, Minato-ku, Tokyo 108-0074, Japan.
Fumie
F
Saito
AcroScale Inc., 3-8-1 Kokubun-cho, Aoba-ku, Sendai, Miyagi 980-0803, Japan.
Izumi
I
Sakamoto
GlycoGene Inc., Avenue Takanawa Rm. 412, 3-25-27, Takanawa, Minato-ku, Tokyo 108-0074, Japan.
Akio
A
Kanatani
Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address: y-chiba@aist.go.jp.
Yasunori
Y
Chiba
D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D018396:Mucin-1 / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D006023:Glycoproteins; D007477:Ions; D026901:Membrane Transport Proteins; D018396:Mucin-1; D011134:Polysaccharides; C121945:UDP-N-acetylglucosamine transporter; D008345:Manganese; D016695:Glycosyltransferases; D017351:N-Acetylglucosaminyltransferases; C454460:UDP-GlcNAc GalNAc-peptide beta1,3-N-acetylglucosaminyltransferase; D014534:UDPglucose 4-Epimerase
Core 3-type O-glycan; Glycan engineering; Glycosyltransferase; Manganese ion; Yeast