title: Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification.
abstract: Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. In this study, we show that heterologous expression of YlMpo1p can perform and enhance mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, the level of mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants. When YlMpo1p overexpression is applied to glyco-engineered S. cerevisiae in which the synthesis of immunogenic glycans is abolished, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in yeast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability.
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Gil
authors.affiliation: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, Republic of Korea.
authors.forename: Jeong-Nam
authors.initials: JN
authors.lastname: Park
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Kyung Jin
authors.initials: KJ
authors.lastname: Lee
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Ji-Yeon
authors.initials: JY
authors.lastname: Kang
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Yeong Hun
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Republic of Korea.
authors.forename: Seonghun
authors.initials: S
authors.lastname: Kim
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Sang-Yoon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Republic of Korea.
authors.forename: Ohsuk
authors.initials: O
authors.lastname: Kwon
authors.affiliation: SKKU Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Yong Taik
authors.initials: YT
authors.lastname: Lim
authors.affiliation: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, Republic of Korea. Electronic address: hyunkang@cau.ac.kr.
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305-806, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Republic of Korea. Electronic address: dboh@kribb.re.kr.
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
mesh_terms: D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D008360:Mannosephosphates / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D010766:Phosphorylation; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C105147:MNN4 protein, S cerevisiae; D008360:Mannosephosphates; D008565:Membrane Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C027693:mannose-6-phosphate; D008358:Mannose
keywords: Enzyme replacement therapy; Lysosomal storage disease; Mannose-6-phosphate modification; Mannosylphosphorylation; ScMNN4; YlMPO1