|Title||A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||Tiels, P., E. Baranova, K. Piens, C. De Visscher, G. Pynaert, W. Nerinckx, J. Stout, F. Fudalej, P. Hulpiau, S. Tännler, S. Geysens, A. Van Hecke, A. Valevska, W. Vervecken, H. Remaut, and N. Callewaert|
|Date Published||2012 Dec|
|Keywords||alpha-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|
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.
|Alternate Journal||Nat. Biotechnol.|
A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.