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Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification.
Gil, Park, Lee, Kang, Kim, Kim, Kim, Kwon, Lim, Kang, Oh.
Chung-Ang University, Korea Research Institute of Bioscience and Biotechnology, Sungkyunkwan University, University of Science and Technology (UST Korea)
Journal of Biotechnology 2015, 206:66-74
Gene names: Glucose oxidase (GOD), Glycosyl phosphatidylinositol-anchored plasma membrane (Gas1p). Host systems: Saccharomyces cerevisiae [Yeast]. Gene species: Saccharomyces cerevisiae [Yeast]. Vectors: pD1211, pD1214-SSKT. Tags: His.
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.
Comments: "In order to select the best signal peptide for secretory expression of GOD, a DNA fragment encoding His-tagged GOD protein was amplified from pDLMOX-GOD(H) by PCR using H-GOD-F1 and H-GOD-R1 primers and cloned to the Electra vectors of S. cerevisiae secretion signal kit of DNA2.0 according to the protocol provided by the manufacture. After the resulting 11 vectors were introduced to S. cerevisiae L3262 strain, the culture media of the transformants were analyzed by Western blot. Culture medium of the transformant containing pD1214-KP-H-GOD generated the strongest band."