title: Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor.
abstract: The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2-β-GCase lysosomal targeting.
authors.affiliation: 1] Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK [2].
authors.forename: Yuguang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: 1] Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK [2].
authors.forename: Jingshan
authors.initials: J
authors.lastname: Ren
authors.affiliation: Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK.
authors.forename: Sergi
authors.initials: S
authors.lastname: Padilla-Parra
authors.affiliation: Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Fry
authors.affiliation: Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK.
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D005962:Glucosylceramidase / Q000378:metabolism*; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology / Q000528:radiation effects; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D051116:Receptors, Scavenger / Q000378:metabolism*
chemical_list: D051907:Lysosomal Membrane Proteins; D017527:Receptor, IGF Type 2; D051116:Receptors, Scavenger; C074299:SCARB2 protein, human; D005962:Glucosylceramidase