title: Mannose 6-phosphate-independent Lysosomal Sorting of LIMP-2.
abstract: The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for β-glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP-2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP-2 with the cation-independent M6P receptor (MPR) results in M6P-dependent targeting of LIMP-2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P-dependent delivery of LIMP-2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP-2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P-forming N-acetylglucosamine (GlcNAc)-1-phosphotransferase, LIMP-2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP-2 levels within lysosomes purified from liver of wild-type (wt) and GlcNAc-1-phosphotransferase-defective mice. Heterologous expression of the luminal domain of LIMP-2 in wild-type, LIMP-2-deficient and GlcNAc-1-phosphotransferase-defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP-2. Finally, cathepsin Z, a known GlcNAc-1-phosphotransferase substrate, but not LIMP-2, could be precipitated with M6P-specific antibodies. These data prove M6P-independent lysosomal sorting of LIMP-2 and subsequently GC in vivo.
authors.affiliation: Biochemisches Institut der Christian Albrechts Universität zu Kiel, Otto-Hahn-Platz 9, 24118, Kiel, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Blanz
authors.affiliation: Biochemisches Institut der Christian Albrechts Universität zu Kiel, Otto-Hahn-Platz 9, 24118, Kiel, Germany.
authors.forename: Friederike
authors.initials: F
authors.lastname: Zunke
authors.affiliation: Arbeitsbereich Molekularbiologie, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Markmann
authors.affiliation: Biochemisches Institut der Christian Albrechts Universität zu Kiel, Otto-Hahn-Platz 9, 24118, Kiel, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Damme
authors.affiliation: Arbeitsbereich Molekularbiologie, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.affiliation: Biochemisches Institut der Christian Albrechts Universität zu Kiel, Otto-Hahn-Platz 9, 24118, Kiel, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
authors.affiliation: Biochemie III/ Fakultät für Chemie, Universität Bielefeld, Universitätsstr. 25, 33615, Bielefeld, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schwake
mesh_terms: D000818:Animals; D018955:CD36 Antigens / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D005962:Glucosylceramidase / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism
chemical_list: D018955:CD36 Antigens; D051907:Lysosomal Membrane Proteins; D008360:Mannosephosphates; C489452:Scarb2 protein, mouse; C027693:mannose-6-phosphate; D017855:Transferases (Other Substituted Phosphate Groups); C548056:GNPTAB protein, mouse; D005962:Glucosylceramidase
keywords: Gaucher disease; LIMP-2/SCARB2; mannose 6-phosphate; mannose 6-phosphate receptor; β-glucocerebrosidase