A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.

TitleA bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.
Publication TypeJournal Article
Year of Publication2012
AuthorsTiels, P., Baranova E., Piens K., De Visscher C., Pynaert G., Nerinckx W., Stout J., Fudalej F., Hulpiau P., Tännler S., Geysens S., Van Hecke A., Valevska A., Vervecken W., Remaut H., and Callewaert N.
JournalNat Biotechnol
Volume30
Issue12
Pagination1225-31
Date Published2012 Dec
ISSN1546-1696
Keywordsalpha-Glucosidases, Animals, Arthrobacter, Bacterial Proteins, Biological Transport, Active, Biotechnology, Catalytic Domain, Disease Models, Animal, Glycogen Storage Disease Type II, Glycoside Hydrolases, Humans, Lysosomal Storage Diseases, Lysosomes, Mannosephosphates, Mice, Mice, Knockout, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Pichia, Protein Conformation, Recombinant Proteins, Yarrowia
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.

DOI10.1038/nbt.2427
Alternate JournalNat. Biotechnol.
PubMed ID23159880
Research group: