Abstract
Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with α-mannosidase to produce in yeast a form of the Pompe disease enzyme α-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of α-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Arthrobacter / enzymology
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Arthrobacter / genetics
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Biological Transport, Active
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Biotechnology
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Catalytic Domain / genetics
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Disease Models, Animal
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Glycogen Storage Disease Type II / drug therapy
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Glycogen Storage Disease Type II / enzymology
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Glycogen Storage Disease Type II / genetics
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Glycoside Hydrolases / chemistry
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Glycoside Hydrolases / genetics
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Glycoside Hydrolases / metabolism*
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Humans
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Lysosomal Storage Diseases / drug therapy
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Lysosomal Storage Diseases / enzymology
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Lysosomal Storage Diseases / genetics
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Lysosomes / enzymology*
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Mannosephosphates / metabolism*
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Mice
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Mice, Knockout
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Pichia / enzymology
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Pichia / genetics
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Protein Conformation
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Yarrowia / enzymology
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Yarrowia / genetics
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alpha-Glucosidases / deficiency
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alpha-Glucosidases / genetics
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alpha-Glucosidases / metabolism
Substances
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Bacterial Proteins
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Mannosephosphates
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Recombinant Proteins
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mannose-6-phosphate
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Glycoside Hydrolases
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alpha-Glucosidases
Associated data
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GENBANK/JF809872
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GENBANK/JF809873
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GENBANK/JF809874
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GENBANK/JF809875
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GENBANK/JF809876