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

DOI: 10.1038/nbt.2427 DOI Help
PMID: 23159880 PMID Help

Authors: Petra Tiels (VIB; Ghent University) , Ekaterina Baranova (MRC Mitochondrial Biology Unit) , Kathleen Piens (Oxyrane) , Charlotte De Visscher (VIB; Ghent University) , Gwenda Pynaert (Oxyrane) , Wim Nerinckx (VIB; Ghent University) , Jan Stout (Oxyrane) , Franck Fudalej (Oxyrane) , Paco Hulpiau (VIB) , Simon Tännler (Oxyrane) , Steven Geysens (Oxyrane) , Annelies Van Hecke (VIB; Ghent University) , Albena Valevska (Oxyrane) , Wouter Vervecken (Oxyrane) , Han Remaut (VIB; Vrije Universiteit Brussel) , Nico Callewaert (VIB; Ghent University)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Nature Biotechnology , VOL 30 (12) , PAGES 1225 - 1231

State: Published (Approved)
Published: November 2012
Diamond Proposal Number(s): 7351

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 alpha-mannosidase to produce in yeast a form of the Pompe disease enzyme alpha-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of alpha-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.

Journal Keywords: Arthrobacter; Bacterial; Biological; Active; Biotechnology; Catalytic; Disease; Animal; Glycogen; Glycoside; Humans; Lysosomal; Lysosomes; Mannosephosphates; Mice; Mice; Knockout; Models; Molecular; Mutagenesis; Site-Directed; Pichia; Protein; Recombinant; Yarrowia; alpha-Glucosidases

Subject Areas: Biology and Bio-materials, Medicine


Instruments: I02-Macromolecular Crystallography

Other Facilities: SLS (PXIII), ESRF (BM30A)

Added On: 03/04/2013 16:38

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