A Glycoengineered Enzyme with Multiple Mannose-6-Phosphates Is Internalized into Diseased Cells to Restore Its Activity in Lysosomes.
Cell Chem Biol
; 25(10): 1255-1267.e8, 2018 10 18.
Article
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| MEDLINE
| ID: mdl-30146240
ABSTRACT
In this study we developed an efficient method to prepare glycoengineered ß-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 ß-N-acetylhexosaminidase, more readily than commercialized human ß-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 ß-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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Beta-N-Acetilhexosaminidasas
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Ingeniería de Proteínas
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Enfermedades por Almacenamiento Lisosomal
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Gangliósido G(M2)
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Lisosomas
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Manosafosfatos
Límite:
Animals
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Female
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Humans
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Male
Idioma:
En
Año:
2018
Tipo del documento:
Article