In Vitro N-Glycan Mannosyl-Phosphorylation of a Therapeutic Enzyme by Using Recombinant Mnn14 Produced from Pichia pastoris
2
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward
Abstract
Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward
참고문헌 (21)
- 오두병, "Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases", BMB Reports, 48(8), pp.438~444 (2015), 생화학분자생물학회
- Chiba Y, "Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease", Glycobiol, 12, pp.821~828 (2002)
- Tiels P, "A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes", Nat. Biotechol, 30, pp.1225~1231 (2012)
- Zhu, Y, "Conjugation of mannose 6-phosphate-containing oligosaccharides to acid alpha-glucosidase improves the clearance of glycogen in Pompe mice", J. Biol. Chem, 279, pp.50336~50341 (2004)
- Zhou Q, "Strategies for neoglycan conjugation to human acid alphaglucosidase", Bioconjug. Chem, 22, pp.741~751 (2011)
- Zhou Q, "Glycan structure determinants for cation-independent mannose 6-phosphate receptor binding and cellular uptake of a recombinant protein", Bioconjug. Chem, 24, pp.2025~2035 (2013)
- Gil JY, "Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification", J. Biotechnol, 206, pp.66~74 (2015)
- Kim YH, "Abolishment of N-glycan mannosylphosphorylation in glycoengineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes", Appl. Microbiol. Biotechnol, 101, pp.2979~2989 (2017)
- Laroy W, "Glycome mapping on DNA sequencing equipment", Nat. Protocols, 1, pp.397~405 (2006)
- Choi HY, "N-glycan remodeling using mannosidase inhibitors to increase highmannose glycans on acid α-glucosidase in transgenic rice cell cultures", Sci. Rep, 8, pp.16130~ (2018)
- Kang JY, "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans", Anal. Biochem, 501, pp.1~3 (2016)
- Shimma YI, "Expression of human glycosyltransferase gene in yeast as a tool for enzymatic synthesis of sugar chain", Glycoconj. J, 21, pp.75~78 (2004)
- McVie-Wylie AJ, "Biochemical and pharmacological characterization of different recombinant acid alpha-glucosidase preparations evaluated for the treatment of Pompe disease", Mol. Genet. Metab, 94, pp.448~455 (2008)
- Togawa T, "Comparative study on mannose-6-phosphate residue contents of recombinant lysosomal enzymes", Mol. Genet. Met, 111, pp.369~373 (2014)
- Kang JY, "Lysosomal targeting enhancement by conjugation of glycopeptides containing mannose-6-phosphate glycans derived from glyco-engineered yeast", Sci. Rep, 8, pp.8730~ (2018)
- Zhu Y, "Carbohydrate-remodelled acid alpha-glucosidase with higher affinity for the cation-independent mannose 6-phosphate receptor demonstrates improved delivery to muscles of Pompe mice", Biochem. J, 389, pp.619~628 (2005)
- Kudo M, "Structural requirements for efficient processing and activation of recombinant human UDP-Nacetylglucosamine : Lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase", J. Biol. Chem, 281, pp.11761~11768 (2006)
- Meel EV, "Multiple domains of GlcNAc-1-phosphotransferase mediate recognition of lysosomal enzymes", J. Biol. Chem, 291, pp.8295~8307 (2016)
- Liu L, "Engineering of GlcNAc-1-phosphotransferase for production of highly phosphorylated lsysosomal enzymes for enzyme replacement therapy", Mol. Thr. (Meth. Clin. Dev. ), 5, pp.59~65 (2017)
- Wang XH, "MNN6, a member of the KRE2", MNT1 family, is the gene for mannosylphosphate transfer in Saccharomyces cerevisiae, 1997, pp.272, 18117~18124
- Park JN, "Essential role of YlMPO1, a novel Yarrowia lipolytica homologue of Saccharomyces cervisiae MNN4, in mannosylphosphorylation of N-and O-linked glycans", Appl. Env. Microbiol, 77, pp.1187~1195 (2011)