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

Nature Biotechnology volume 30, pages 1225–1231 (2012)Cite this article

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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.

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Figure 1: Project, technology concepts and strategy.
Figure 2: Y.lipolytica N-glycan analysis and CcGH92_4 and CcGH92_5 activity and substrate specificity.
Figure 3: Structure of the C. cellulans CcGH92_5 GH92 domain.
Figure 4: Enzyme uptake by Pompe disease patient fibroblasts and therapeutic activity in Pompe disease mouse model.

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EMBL/GenBank/DDBJ

European Nucleotide Archive

NCBI Reference Sequence

Protein Data Bank

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Acknowledgements

This work was supported by an R&D grant from IWT-Flanders (projects 080775 and 110379), the Marie Curie Excellence Grant MEXT-014292 under EU Framework Program 6 and by grant G.0.327.11.N.10 of FWO-Vlaanderen. P.T. holds a doctor-assistant position at Ghent University. C.D.V. holds a fellowship of the Institute for the Advancement of Scientific and Technological Research in Industry (IWT). H.R. is supported by VIB grant PRJ9 and the Odysseus program of the FWO-Vlaanderen. We thank J.-M. Nicaud (CNRS-INRA-INAPG UMR2585, France) for donating Y. lipolytica Po1d lnuga, A. Reuser for providing GAA KO mice and TNO Triskelion for executing the in vivo part of the mouse studies. We acknowledge M. Zawisza, D. Bracke, T. Herman, H. Van Put, S. Poiz and I. Dewerte for their excellent technical assistance during the project and thank the Protein Service Facility (Y. Leoen and J. Haustraete) for their assistance, as well as Y.-C. Lin for handling the genome sequencing data. We dedicate this paper to the memory of the late Prof. Dr. Yoshifumi Jigami, one of the pioneers in glyco-engineering of yeasts.

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Author notes

  1. Petra Tiels, Ekaterina Baranova and Kathleen Piens: These authors contributed equally to this work.

Authors and Affiliations

  1. Department for Molecular Biomedical Research, Unit for Medical Biotechnology, VIB, Ghent, Belgium.,

    Petra Tiels, Charlotte De Visscher, Wim Nerinckx, Annelies Van Hecke & Nico Callewaert

  2. Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium

    Petra Tiels, Charlotte De Visscher, Wim Nerinckx, Annelies Van Hecke & Nico Callewaert

  3. Department for Structural Biology, Structural & Molecular Microbiology, VIB, Brussels, Belgium.,

    Ekaterina Baranova & Han Remaut

  4. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Ekaterina Baranova & Han Remaut

  5. Oxyrane, Ghent, Belgium

    Kathleen Piens, Gwenda Pynaert, Jan Stout, Franck Fudalej, Simon Tännler, Steven Geysens, Albena Valevska & Wouter Vervecken

  6. Department for Molecular Biomedical Research, Bio-IT core facility, VIB, Ghent, Belgium.,

    Paco Hulpiau

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Contributions

P.T.: Y. lipolytica MNN4 strain generation, experiments leading to the discovery of enzyme activities in C. cellulans, production of these in E. coli and characterization and thermostability assays. E.B.: CcGH92_5 purification and structure determination. K.P.: CcGH92_4 and CcGH92_5 characterization and enzyme process development, GAA enzyme characterization. C.D.V.: P. pastoris PNO1 and GLA strain generation and characterization. G.P.: cell uptake assays, mouse study bio-analysis. W.N.: docking studies. J.S.: GAA purification, mouse study statistics. F.F.: fermentation, GAA strain generation. P.H.: bio-informatics analysis of CcGH92_5 sequence homologs. S.T.: fermentation development for GAA and CcGH92_4 and CcGH92_5. S.G.: glycan analysis. A.V.H.: Y. lipolytica MNN4 strain generation, production of C. cellulans enzymes in E. coli and thermostability assays. A.V.: Y. lipolytica MNN4 & GAA strain generation. W.V.: Y. lipolytica och1 and MNN4 strain generation, scientific supervision at Oxyrane. H.R.: supervision and analysis of CcGH92 structure determination. N.C.: project initiation and scientific supervision. N.C., P.T., K.P., G.P., C.D.V., W.V. and H.R. co-wrote the manuscript.

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Correspondence to Wouter Vervecken, Han Remaut or Nico Callewaert.

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Competing interests

N.C., W.V., P.T., K.P., A.V., G.P, S.G., J.S. and H.R. are named inventors on patent applications claiming use of the technology described in this publication. W.V., A.V., G.P., S.G. S.T., F.F., J.S. and K.P. hold stock options in Oxyrane UK, a company which develops biopharmaceuticals based partially on technology resulting from the work reported here.

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Tiels, P., Baranova, E., Piens, K. et al. A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes. Nat Biotechnol 30, 1225–1231 (2012). https://doi.org/10.1038/nbt.2427

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