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A Glycoengineered Enzyme with Multiple Mannose-6-Phosphates Is Internalized into Diseased Cells to Restore Its Activity in Lysosomes

조회수 : 19 등록일 : 2018.11.05 00:00

저자 : Kim, S (Kim, Sanggil); Lee, HS (Lee, Hyun Soo)
출처 : CELL CHEMICAL BIOLOGY
출판일 : 2018.10.18


A Glycoengineered Enzyme with Multiple Mannose-6-Phosphates Is Internalized into Diseased Cells to Restore Its Activity in Lysosomes


Hyun, JY (Hyun, Ji Young)1 ] Kim, S (Kim, Sanggil)2 ] Lee, HS (Lee, Hyun Soo)2 ] Shin, I (Shin, Injae)1 ]


[ 1 ] Yonsei Univ, Ctr Biofunct Mol, Dept Chem, Seoul 03722, South Korea
[ 2 ] Sogang Univ, Dept Chem, Seoul 04107, South Korea



In this study we developed an efficient method to prepare glycoengineered beta-N-acetylhexosaminidase containing multiple mannose-6-phosphates (M6Ps) by combining genetic code expansion with bioorthogonal ligation techniques. We found that multiple M6P-conjugated enzymes were produced with a high efficiency by using combined techniques. Importantly, glycoengineered enzymes entered lysosomes of patient-derived primary cells, which lack endogenous lysosomal beta-N-acetylhexosaminidase, more readily than commercialized human beta-hexosaminidase. Moreover, glycoengineered enzymes successfully removed GM2-ganglioside stored in lysosomes of diseased cells, indicating that its activity is restored in diseased cells. We also synthesized and applied a lysosome-targeting fluorogenic substrate to monitor endogenous and supplemental glycoengineered beta-N-acetylhexosaminidase activities in lysosomes. The results of this study indicate that the present strategy, which relies on genetic code expansion and bioorthogonal ligation techniques, is highly attractive to generate multi-M6P-containing lysosomal enzymes that can be used to study lysosomal storage disorders associated with lysosomal enzyme deficiencies.





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