title: A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.
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.
authors.affiliation: Unit for Medical Biotechnology, Department for Molecular Biomedical Research, VIB, Ghent, Belgium.
authors.forename: Petra
authors.initials: P
authors.lastname: Tiels
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Baranova
authors.forename: Kathleen
authors.initials: K
authors.lastname: Piens
authors.forename: Charlotte
authors.initials: C
authors.lastname: De Visscher
authors.forename: Gwenda
authors.initials: G
authors.lastname: Pynaert
authors.forename: Wim
authors.initials: W
authors.lastname: Nerinckx
authors.forename: Jan
authors.initials: J
authors.lastname: Stout
authors.forename: Franck
authors.initials: F
authors.lastname: Fudalej
authors.forename: Paco
authors.initials: P
authors.lastname: Hulpiau
authors.forename: Simon
authors.initials: S
authors.lastname: Tännler
authors.forename: Steven
authors.initials: S
authors.lastname: Geysens
authors.forename: Annelies
authors.initials: A
authors.lastname: Van Hecke
authors.forename: Albena
authors.initials: A
authors.lastname: Valevska
authors.forename: Wouter
authors.initials: W
authors.lastname: Vervecken
authors.forename: Han
authors.initials: H
authors.lastname: Remaut
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
mesh_terms: D000818:Animals; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active; D001709:Biotechnology; D020134:Catalytic Domain / Q000235:genetics; D004195:Disease Models, Animal; D006009:Glycogen Storage Disease Type II / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016464:Lysosomal Storage Diseases / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics; D000520:alpha-Glucosidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008360:Mannosephosphates; D011994:Recombinant Proteins; C027693:mannose-6-phosphate; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases