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ABSTRACT: The lysosomal membrane protein type 2 is a novel identified lysosomal sorting receptor for β-glucocerebrosidase (GC). Mutations in both genes underlie human pathologies causing action myoclonus-renal failure syndrome (AMRF) and Gaucher disease (GD), respectively. We now demonstrate that the lumenal acidification mediated by the vacuolar (H(+) )-ATPase triggers the dissociation of LIMP-2 and GC in late endosomal/lysosomal compartments. Moreover, we identified a single histidine residue in LIMP-2 that is necessary for LIMP-2 and GC binding. This residue is in close proximity to a proposed coiled-coil domain, which determines the binding to GC and may function as a critical pH sensor.
Traffic 04/2012; 13(8):1113-23. · 4.92 Impact Factor
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ABSTRACT: α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyl-linked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions, and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages, and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-Mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term enzyme replacement therapy partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides, and cholesterol. The present study demonstrates cerebellar alterations in α-mannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures.
Journal of Neuropathology and Experimental Neurology 01/2011; 70(1):83-94. · 4.26 Impact Factor
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ABSTRACT: Whereas we have a profound understanding about the function and biogenesis of the protein constituents in the lumen of the lysosomal compartment, much less is known about the functions of proteins of the lysosomal membrane. Proteomic analyses of the lysosomal membrane suggest that, apart from the well-known lysosomal membrane proteins, additional and less abundant membrane proteins are present. The identification of disease-causing genes and the in-depth analysis of knockout mice leading to mutated or absent membrane proteins of the lysosomal membrane have demonstrated the essential role of these proteins in lysosomal acidification, transport of metabolites resulting from hydrolytic degradation and interaction and fusion with other cellular membrane systems. In addition, trafficking pathways of lysosomal membrane proteins are closely linked to the biogenesis of this compartment. This is exemplified by the recent finding that LIMP-2 (lysosomal integral membrane protein type-2) is responsible for the mannose 6-phosphate receptor-independent delivery of newly synthesized β-glucocerebrosidase to the lysosome. Similar to LIMP-2, which could also be linked to vesicular transport processes in certain polarized cell types, the major constituents of the lysosomal membrane, the glycoproteins LAMP (lysosome-associated membrane protein)-1 and LAMP-2 are essential for regulation of lysosomal motility and participating in control of membrane fusion events between autophagosomes or phagosomes with late endosomes/lysosomes. Our recent investigations into the role of these proteins have not only increased our understanding of the endolysosomal system, but also supported their major role in cell physiology and the development of different diseases.
Biochemical Society Transactions 12/2010; 38(6):1420-3. · 3.71 Impact Factor
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Nature Genetics 01/2010; 42(1):3. · 35.53 Impact Factor
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Alexander Schneede,
Christine K Schmidt,
Maarit Hölttä-Vuori,
Jörg Heeren,
Marion Willenborg, Judith Blanz,
Mykola Domanskyy,
Bernadette Breiden,
Susanne Brodesser,
Jobst Landgrebe,
Konrad Sandhoff,
Elina Ikonen,
Paul Saftig,
Eeva-Liisa Eskelinen
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ABSTRACT: The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP(-/-)), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP(-/-) cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP(-/-) cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.
Journal of Cellular and Molecular Medicine 11/2009; 15(2):280-95. · 4.13 Impact Factor
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ABSTRACT: Action myoclonus-renal failure syndrome (AMRF) is caused by mutations in the lysosomal integral membrane protein type 2 (LIMP-2/SCARB2). LIMP-2 was identified as a sorting receptor for beta-glucocerebrosidase (beta-GC), which is defective in Gaucher disease. To date, six AMRF-causing mutations have been described, including splice site, missense and nonsense mutations. All mutations investigated in this study lead to a retention of LIMP-2 in the endoplasmic reticulum (ER) but affect the binding to beta-GC differentially. From the three nonsense mutations, only the Q288X mutation was still able to bind to beta-GC as efficiently as compared with wild-type LIMP-2, whereas the W146SfsX16 and W178X mutations lost their beta-GC-binding capacity almost completely. The LIMP-2 segment 145-288, comprising the nonsense mutations, contains a highly conserved coiled-coil domain, which we suggest determines beta-GC binding. In fact, disruption of the helical arrangement and amphiphatic nature of the coiled-coil domain abolishes beta-GC binding, and a synthetic peptide comprising the coiled-coil domain of LIMP-2 displays pH-selective multimerization properties. In contrast to the reduced binding properties of the nonsense mutations, the only missense mutation (H363N) found in AMRF leads to increased binding of beta-GC to LIMP-2, indicating that this highly conserved histidine modifies the affinity of LIMP-2 to its ligand. With the present study, we demonstrate that disruption of the coiled-coil structure or AMRF disease-causing mutations abolish beta-GC binding, indicating the importance of an intact coiled-coil structure for the interaction of LIMP-2 and beta-GC.
Human Molecular Genetics 11/2009; 19(4):563-72. · 7.64 Impact Factor
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Judith Blanz,
Stijn Stroobants,
Renate Lüllmann-Rauch,
Willy Morelle,
Meike Lüdemann,
Rudi D'Hooge,
Helena Reuterwall,
Jean Claude Michalski,
Jens Fogh,
Claes Andersson,
Paul Saftig
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ABSTRACT: Despite the progress in the treatment of lysosomal storage disorders (LSDs) mainly by enzyme replacement therapy, only limited success was reported in targeting the appropriate lysosomal enzyme into the brain. This prevents efficient clearance of neuronal storage, which is present in many of these disorders including alpha-mannosidosis. Here we show that the neuropathology of a mouse model for alpha-mannosidosis can be efficiently treated using recombinant human alpha-mannosidase (rhLAMAN). After intravenous administration of different doses (25-500 U/kg), rhLAMAN was widely distributed among tissues, and immunohistochemistry revealed lysosomal delivery of the injected enzyme. Whereas low doses (25 U/kg) led to a significant clearance (<70%) in visceral tissues, higher doses were needed for a clear effect in central and peripheral nervous tissues. A distinct reduction (<50%) of brain storage required repeated high-dose injections (500 U/kg), whereas lower doses (250 U/kg) were sufficient for clearance of stored substrates in peripheral neurons of the trigeminal ganglion. Successful transfer across the blood-brain barrier was evident as the injected enzyme was found in hippocampal neurons, leading to a nearly complete disappearance of storage vacuoles. Importantly, the decrease in neuronal storage in the brain correlated with an improvement of the neuromotor disabilities found in untreated alpha-mannosidosis mice. Uptake of rhLAMAN seems to be independent of mannose-6-phosphate receptors, which is consistent with the low phosphorylation profile of the enzyme. These data suggest that high-dose injections of low phosphorylated enzymes might be an interesting option to efficiently treat LSDs with CNS involvement.
Human Molecular Genetics 09/2008; 17(22):3437-45. · 7.64 Impact Factor
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Samuel F Berkovic,
Leanne M Dibbens,
Alicia Oshlack,
Jeremy D Silver,
Marina Katerelos,
Danya F Vears,
Renate Lüllmann-Rauch, Judith Blanz,
Ke Wei Zhang,
Jim Stankovich, [......],
Lata Vadlamudi,
Richard A Macdonell,
Bree L Hodgson,
Marta A Bayly,
Judy Savige,
John C Mulley,
Gordon K Smyth,
David A Power,
Paul Saftig,
Melanie Bahlo
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ABSTRACT: Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.
The American Journal of Human Genetics 04/2008; 82(3):673-84. · 10.60 Impact Factor
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ABSTRACT: beta-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. beta-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of beta-glucocerebrosidase was secreted. Missorting of beta-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of beta-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a beta-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for beta-glucocerebrosidase.
Cell 12/2007; 131(4):770-83. · 32.40 Impact Factor
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ABSTRACT: ClC-2 is a broadly expressed plasma membrane chloride channel that is modulated by voltage, cell swelling, and pH. A human mutation leading to a heterozygous loss of ClC-2 has previously been reported to be associated with epilepsy, whereas the disruption of Clcn2 in mice led to testicular and retinal degeneration. We now show that the white matter of the brain and spinal cord of ClC-2 knock-out mice developed widespread vacuolation that progressed with age. Fluid-filled spaces appeared between myelin sheaths of the central but not the peripheral nervous system. Neuronal morphology, in contrast, seemed normal. Except for the previously reported blindness, neurological deficits were mild and included a decreased conduction velocity in neurons of the central auditory pathway. The heterozygous loss of ClC-2 had no detectable functional or morphological consequences. Neither heterozygous nor homozygous ClC-2 knock-out mice had lowered seizure thresholds. Sequencing of a large collection of human DNA and electrophysiological analysis showed that several ClC-2 sequence abnormalities previously found in patients with epilepsy most likely represent innocuous polymorphisms.
Journal of Neuroscience 07/2007; 27(24):6581-9. · 7.11 Impact Factor
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ABSTRACT: The ability of KCNQ (Kv7) channels to form hetero-oligomers is of high physiological importance, because heteromers of KCNQ3 with KCNQ2 or KCNQ5 underlie the neuronal M-current, which modulates neuronal excitability. In KCNQ channels, we recently identified a C-terminal subunit interaction (si) domain that determines their subunit-specific assembly. Within this si domain, there are two motifs that comprise approximately 30 amino acid residues each and that exhibit a high probability for coiled-coil formation. Transfer of the first or the second coiled-coil (TCC) domain from KCNQ3 into the KCNQ1 scaffold resulted in chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3, both of which coimmunoprecipitated with KCNQ2. However, only KCNQ1(TCC2)Q3 enhanced KCNQ2 currents and surface expression or exerted a strong dominant-negative effect on KCNQ2. Deletion of TCC2 within KCNQ2 yielded functional homomeric channels but prevented the current augmentation measured after coexpression of KCNQ2 and KCNQ3. In contrast, deleting TCC1 within KCNQ2 did not give functional homomeric KCNQ2 or heteromeric KCNQ2/KCNQ3 channels. Mutations that disrupted the predicted coiled-coil structure of TCC1 in KCNQ2 or KCNQ3 abolished channel activity after expressing these constructs singly or in combination, whereas helix-breaking mutations in TCC2 of KCNQ2 gave functional homomeric channels but prevented the heteromerization with KCNQ3. In contrast, KCNQ3 carrying a coiled-coil disrupting mutation in TCC2 hetero-oligomerized with KCNQ2. Our data suggest that the TCC1 domains of KCNQ2 and KCNQ3 are required to form functional homomeric as well as heteromeric channels, whereas both TCC2 domains facilitate an efficient transport of heteromeric KCNQ2/KCNQ3 channels to the plasma membrane.
Journal of Neuroscience 05/2006; 26(14):3757-66. · 7.11 Impact Factor
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ABSTRACT: In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.
Molecular Biology of the Cell 10/2002; 13(9):3355-68. · 4.94 Impact Factor
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ABSTRACT: Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated
protein and an important constituent of the lysosomal membrane
Nature 08/2000; 406(6798):902-906. · 36.28 Impact Factor
