Genetic LAMP2 deficiency accelerates the age-associated formation of basal laminar deposits in the retina.

The early stages of age-related macular degeneration (AMD) are characterized by the accumulation of basal laminar deposits (BLamDs). The mechanism for BLamDs accumulating between the retinal pigment epithelium (RPE) and its basal lamina remains elusive. Here we examined the role in AMD of lysosome-associated membrane protein-2 (LAMP2), a glycoprotein that plays a critical role in lysosomal biogenesis and maturation of autophagosomes/phagosomes. LAMP2 was preferentially expressed by RPE cells, and its expression declined with age. Deletion of the Lamp2 gene in mice resulted in age-dependent autofluorescence abnormalities of the fundus, thickening of Bruch's membrane, and the formation of BLamDs, resembling histopathological changes occurring in AMD. Moreover, LAMP2-deficient mice developed molecular signatures similar to those found in human AMD-namely, the accumulation of APOE, APOA1, clusterin, and vitronectin-adjacent to BLamDs. In contrast, collagen 4, laminin, and fibronectin, which are extracellular matrix proteins constituting RPE basal lamina and Bruch's membrane were reduced in Lamp2 knockout (KO) mice. Mechanistically, retarded phagocytic degradation of photoreceptor outer segments compromised lysosomal degradation and increased exocytosis in LAMP2-deficient RPE cells. The accumulation of BLamDs observed in LAMP2-deficient mice was eventually followed by loss of the RPE and photoreceptors. Finally, we observed loss of LAMP2 expression along with ultramicroscopic features of abnormal phagocytosis and exocytosis in eyes from AMD patients but not from control individuals. Taken together, these results indicate an important role for LAMP2 in RPE function in health and disease, suggesting that LAMP2 reduction may contribute to the formation of BLamDs in AMD.

Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson's disease.

Mitochondrial and lysosomal dysfunction have been implicated in substantia nigra dopaminergic neurodegeneration in Parkinson's disease (PD), but how these pathways are linked in human neurons remains unclear. Here we studied dopaminergic neurons derived from patients with idiopathic and familial PD. We identified a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidized dopamine accumulation and ultimately resulting in reduced glucocerebrosidase enzymatic activity, lysosomal dysfunction, and α-synuclein accumulation. This toxic cascade was observed in human, but not in mouse, PD neurons at least in part because of species-specific differences in dopamine metabolism. Increasing dopamine synthesis or α-synuclein amounts in mouse midbrain neurons recapitulated pathological phenotypes observed in human neurons. Thus, dopamine oxidation represents an important link between mitochondrial and lysosomal dysfunction in PD pathogenesis.

Long-term enzyme replacement therapy improves neurocognitive functioning and hippocampal synaptic plasticity in immune-tolerant alpha-mannosidosis mice.

Alpha-mannosidosis is a glycoproteinosis caused by deficiency of lysosomal acid alpha-mannosidase (LAMAN), which markedly affects neurons of the central nervous system (CNS), and causes pathognomonic intellectual dysfunction in the clinical condition. Cognitive improvement consequently remains a major therapeutic objective in research on this devastating genetic error. Immune-tolerant LAMAN knockout mice were developed to evaluate the effects of enzyme replacement therapy (ERT) by prolonged administration of recombinant human enzyme. Biochemical evidence suggested that hippocampus may be one of the brain structures that benefits most from long-term ERT. In the present functional study, ERT was initiated in 2-month-old immune-tolerant alpha-mannosidosis mice and continued for 9months. During the course of treatment, mice were trained in the Morris water maze task to assess spatial-cognitive performance, which was related to synaptic plasticity recordings and hippocampal histopathology. Long-term ERT reduced primary substrate storage and neuroinflammation in hippocampus, and improved spatial learning after mid-term (10weeks+) and long-term (30weeks+) treatment. Long-term treatment substantially improved the spatial-cognitive abilities of alpha-mannosidosis mice, whereas the effects of mid-term treatment were more modest. Detailed analyses of spatial memory and spatial-cognitive performance indicated that even prolonged ERT did not restore higher cognitive abilities to the level of healthy mice. However, it did demonstrate marked therapeutic effects that coincided with increased synaptic connectivity, reflected by improvements in hippocampal CA3-CA1 long-term potentiation (LTP), expression of postsynaptic marker PSD-95 as well as postsynaptic density morphology. These experiments indicate that long-term ERT may hold promise, not only for the somatic defects of alpha-mannosidosis, but also to alleviate cognitive impairments of the disorder.

Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy.

Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.

Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development.

While lysosomes are degradative compartments and one of the defenses against invading pathogens, they are also hubs of metabolic activity. Late endocytic compartments accumulate around Plasmodium berghei liver-stage parasites during development, and whether this is a host defense strategy or active recruitment by the parasites is unknown. In support of the latter hypothesis, we observed that the recruitment of host late endosomes (LEs) and lysosomes is reduced in uis4- parasites, which lack a parasitophorous vacuole membrane protein and arrest during liver-stage development. Analysis of parasite development in host cells deficient for late endosomal or lysosomal proteins revealed that the Niemann-Pick type C (NPC) proteins, which are involved in cholesterol export from LEs, and the lysosome-associated membrane proteins (LAMP) 1 and 2 are important for robust liver-stage P. berghei growth. Using the compound U18666A, which leads to cholesterol sequestration in LEs similar to that seen in NPC- and LAMP-deficient cells, we show that the restriction of parasite growth depends on cholesterol sequestration and that targeting this process can reduce parasite burden in vivo. Taken together, these data reveal that proper LE and lysosome function positively contributes to liver-stage Plasmodium development.

Characterization of the complex formed by ß-glucocerebrosidase and the lysosomal integral membrane protein type-2.

The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of ß-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2-derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.

Parkinson's disease: acid-glucocerebrosidase activity and alpha-synuclein clearance.

The role of mutations in the gene GBA1 encoding the lysosomal hydrolase ß-glucocerebrosidase for the development of synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, was only very recently uncovered. The knowledge obtained from the study of carriers or patients suffering from Gaucher disease (a common lysosomal storage disorder because of GBA1 mutations) is of particular importance for understanding the role of the enzyme and its catabolic pathway in the development of synucleinopathies. Decreased activity of ß-glucocerebrosidase leads to lysosomal dysfunction and the accumulation of its substrate glucosylceramide and related lipid derivatives. Glucosylceramide is suggested to stabilize toxic oligomeric forms of α-synuclein that negatively influence the activity of ß-glucocerebrosidase and to partially block export of newly synthesized ß-glucocerebrosidase from the endoplasmic reticulum to late endocytic compartments, amplifying the pathological effects of α-synuclein and ultimately resulting in neuronal cell death. This pathogenic molecular feedback loop and most likely other factors (such as impaired endoplasmic reticulum-associated degradation, activation of the unfolded protein response and dysregulation of calcium homeostasis induced by misfolded GC mutants) are involved in shifting the cellular homeostasis from monomeric α-synuclein towards oligomeric neurotoxic and aggregated forms, which contribute to Parkinson's disease progression. From a therapeutic point of view, strategies aiming to increase either the expression, stability or delivery of the ß-glucocerebrosidase to lysosomes are likely to decrease the α-synuclein burden and may be useful for an in depth evaluation at the organismal level. Lysosomes are critical for protein and lipid homeostasis. Recent research revealed that dysfunction of this organelle contributes to the development of neurodegenerative diseases such as Parkinson's disease (PD). Mutations in the lysosomal hydrolase ß-glucocerebrosidase (GBA1) are a major risk factor for the development of PD and the molecular events linked to the reduced activity of GBA1 and the pathological accumulation of lipids and α-synuclein are just at the beginning to be understood. New therapeutic concepts in regards to how to increase the expression, stability, or delivery of ß-glucocerebrosidase to lysosomes are currently developed. This article is part of a special issue on Parkinson disease.

Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis.

BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis. METHODS: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP de-glycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. RESULTS: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger LAMPs' bulk de-glycosylation, but induces their degradation via CatB-mediated cleavage of LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, stimulates the basal and inhibits CCK-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. CONCLUSIONS: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis, and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction.

Mannose 6-phosphate-independent Lysosomal Sorting of LIMP-2.

The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for ß-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.

LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease.

The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with the myopathy, is also present in LAMP-2-deficient mice. Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain. LAMP-2A, one specific LAMP-2 isoform, was proposed to be important for the lysosomal degradation of selective proteins involved in neurodegenerative diseases such as Huntington's and Parkinson's disease. To elucidate the neuronal function of LAMP-2 we analyzed knockout mice for neuropathological changes, MA and steady-state levels of CMA substrates. The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning. The latter abnormality points to hippocampal dysfunction caused by altered lysosomal activity, distinct accumulation of p62-positive aggregates, autophagic vacuoles and lipid storage within hippocampal neurons and their presynaptic terminals. The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions. Our data contribute to the understanding of intellectual dysfunction observed in Danon disease patients and highlight the role of LAMP-2 within the central nervous system, particularly the hippocampus.

Lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) is a substrate of cathepsin-F, a cysteine protease mutated in type-B-Kufs-disease.

The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase ß-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

Chronic enzyme replacement therapy ameliorates neuropathology in alpha-mannosidosis mice.

OBJECTIVE: The lysosomal storage disease alpha-mannosidosis is caused by the deficiency of the lysosomal acid hydrolase alpha-mannosidase (LAMAN) leading to lysosomal accumulation of neutral mannose-linked oligosaccharides throughout the body, including the brain. Clinical findings in alpha-mannosidosis include skeletal malformations, intellectual disabilities and hearing impairment. To date, no curative treatment is available. We previously developed a beneficial enzyme replacement therapy (ERT) regimen for alpha-mannosidase knockout mice, a valid mouse model for the human disease. However, humoral immune responses against the injected recombinant human alpha-mannosidase (rhLAMAN) precluded long-term studies and chronic treatment. METHODS: Here, we describe the generation of an immune-tolerant alpha-mannosidosis mouse model that allowed chronic injection of rhLAMAN by transgenic expression of a catalytically inactive variant of human LAMAN in the knockout background. RESULTS: Chronic ERT of rhLAMAN revealed pronounced effects on primary substrate storage throughout the brain, normalization of lysosomal enzyme activities and morphology as well as a decrease in microglia activation. The positive effect of long-term ERT on neuronal lysosomal function was reflected by an improvement of cognitive deficits and exploratory activity. in vivo and in vitro uptake measurements indicate rapid clearance of rhLAMAN from circulation and a broad uptake into different cell types of the nervous system. INTERPRETATION: Our data contribute to the understanding of neurological disorders treatment by demonstrating that lysosomal enzymes such as rhLAMAN can penetrate into the brain and is able to ameliorate neuropathology.

LIMP-2 expression is critical for ß-glucocerebrosidase activity and α-synuclein clearance.

Mutations within the lysosomal enzyme ß-glucocerebrosidase (GC) result in Gaucher disease and represent a major risk factor for developing Parkinson disease (PD). Loss of GC activity leads to accumulation of its substrate glucosylceramide and α-synuclein. Since lysosomal activity of GC is tightly linked to expression of its trafficking receptor, the lysosomal integral membrane protein type-2 (LIMP-2), we studied α-synuclein metabolism in LIMP-2-deficient mice. These mice showed an α-synuclein dosage-dependent phenotype, including severe neurological impairments and premature death. In LIMP-2-deficient brains a significant reduction in GC activity led to lipid storage, disturbed autophagic/lysosomal function, and α-synuclein accumulation mediating neurotoxicity of dopaminergic (DA) neurons, apoptotic cell death, and inflammation. Heterologous expression of LIMP-2 accelerated clearance of overexpressed α-synuclein, possibly through increasing lysosomal GC activity. In surviving DA neurons of human PD midbrain, LIMP-2 levels were increased, probably to compensate for lysosomal GC deficiency. Therefore, we suggest that manipulating LIMP-2 expression to increase lysosomal GC activity is a promising strategy for the treatment of synucleinopathies.

The lysosomal polypeptide transporter TAPL is stabilized by interaction with LAMP-1 and LAMP-2.

TAPL (ABCB9) is a homodimeric polypeptide translocation machinery which transports cytosolic peptides into the lumen of lysosomes for degradation. Since the function of proteins is strongly dependent on the interaction network involved, we investigated the interactome of TAPL. A proteomic approach allowed identification of the lysosome-associated membrane proteins LAMP-1 and LAMP-2B as the most abundant interaction partners. Albeit with low frequency, major histocompatibility complex II subunits were also detected. The interaction interface with LAMP was mapped to the four-transmembrane helices constituting the N-terminal domain of TAPL (TMD0). The LAMP proteins bind independently to TAPL. This interaction has influence on neither subcellular localization nor peptide transport activity. However, in LAMP-deficient cells, the half-life of TAPL is decreased by a factor of five, whereas another lysosomal membrane protein, LIMP-2, is not affected. Reduced stability of TAPL is caused by increased lysosomal degradation, indicating that LAMP proteins retain TAPL on the limiting membrane of endosomes and prevent its sorting to intraluminal vesicles.

A critical histidine residue within LIMP-2 mediates pH sensitive binding to its ligand ß-glucocerebrosidase.

The lysosomal membrane protein type 2 is a novel identified lysosomal sorting receptor for ß-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.

Cerebellar alterations and gait defects as therapeutic outcome measures for enzyme replacement therapy in α-mannosidosis.

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.

Role for LAMP-2 in endosomal cholesterol transport.

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.

Lysosomal membrane proteins: life between acid and neutral conditions.

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 ß-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.

Disease-causing mutations within the lysosomal integral membrane protein type 2 (LIMP-2) reveal the nature of binding to its ligand beta-glucocerebrosidase.

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.