Eukaryotic formylglycine-generating enzyme catalyses a monooxygenase type of reaction.

C α-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 µM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor.

Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins.

Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies.

Differential involvement of the extracellular 6-O-endosulfatases Sulf1 and Sulf2 in brain development and neuronal and behavioural plasticity.

The extracellular sulfatases Sulf1 and Sulf2 remove specific 6-O-sulfate groups from heparan sulfate, thereby modulating numerous signalling pathways underlying development and homeostasis. In vitro data have suggested that the two enzymes show functional redundancy. To elucidate their in vivo functions and to further address the question of a putative redundancy, we have generated Sulf1- and Sulf2-deficient mice. Phenotypic analysis of these animals revealed higher embryonic lethality of Sulf2 knockout mice, which can be associated with neuroanatomical malformations during embryogenesis. Sulf1 seems not to be essential for developmental or postnatal viability, as mice deficient in this sulfatase show no overt phenotype. However, neurite outgrowth deficits were observed in hippocampal and cerebellar neurons of both mutant mouse lines, suggesting that not only Sulf2 but also Sulf1 function plays a role in the developing nervous system. Behavioural analysis revealed differential deficits with regard to cage activity and spatial learning for Sulf1- and Sulf2-deficient mouse lines. In addition, Sulf1-specific deficits were shown for synaptic plasticity in the CA1 region of the hippocampus, associated with a reduced spine density. These results reveal that Sulf1 and Sulf2 fulfil non-redundant functions in vivo in the development and maintenance of the murine nervous system.

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways.

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19 degrees C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro. The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.

Paralog of the formylglycine-generating enzyme--retention in the endoplasmic reticulum by canonical and noncanonical signals.

Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the oxidation of a specific cysteine residue to formylglycine, which is the catalytic residue required for sulfate ester hydrolysis. This post-translational modification occurs in the endoplasmic reticulum (ER), and is an essential step in the biogenesis of this enzyme family. A paralog of FGE (pFGE) also localizes to the ER. It shares many properties with FGE, but lacks formylglycine-generating activity. There is evidence that FGE and pFGE act in concert, possibly by forming complexes with sulfatases and one another. Here we show that human pFGE, but not FGE, is retained in the ER through its C-terminal tetrapeptide PGEL, a noncanonical variant of the classic KDEL ER-retention signal. Surprisingly, PGEL, although having two nonconsensus residues (PG), confers efficient ER retention when fused to a secretory protein. Inducible coexpression of pFGE at different levels in FGE-expressing cells did not significantly influence the kinetics of FGE secretion, suggesting that pFGE is not a retention factor for FGE in vivo. PGEL is accessible at the surface of the pFGE structure. It is found in 21 mammalian species with available pFGE sequences. Other species carry either canonical signals (eight mammals and 26 nonmammals) or different noncanonical variants (six mammals and six nonmammals). Among the latter, SGEL was tested and found to also confer ER retention. Although evolutionarily conserved for mammalian pFGE, the PGEL signal is found only in one further human protein entering the ER. Its consequences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved.

The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum.

Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.

ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum.

Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.

Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency.

Multiple Sulfatase Deficiency (MSD) is a rare inborn autosomal-recessive disorder, which mainly combines clinical features of metachromatic leukodystrophy, mucopolysaccharidosis and X-linked ichthyosis. The clinical course ranges from neonatal severe to mild juvenile cases. MSD is caused by mutations in the SUMF1 gene encoding the formylglycine-generating enzyme (FGE). FGE posttranslationally activates sulfatases by generating formylglycine in their catalytic sites. We analyzed the functional consequences of missense mutations p.A177P, p.W179S, p.A279V and p.R349W with regard to FGE's subcellular localization, enzymatic activity, protein stability, intracellular retention and resulting sulfatase activities. All four mutations did not affect localization of FGE in the endoplasmic reticulum of MSD fibroblasts. However, they decreased its specific enzymatic activity to less than 1% (p.A177P and p.R349W), 3% (p.W179S) or 23% (p.A279V). Protein stability was severely decreased for p.A279V and p.R349W, and almost comparable to wild type for p.A177P and p.W179S. The patient with the mildest clinical phenotype carries the mutation p.A279V leading to decreased FGE protein stability, but high residual enzymatic activity and only slightly reduced sulfatase activities. In contrast, the most severely affected patient carries the mutation p.R349W leading to drastically decreased protein stability, very low residual enzymatic activity and considerably reduced sulfatase activities. Our functional studies provide novel insight into the molecular defect underlying MSD and reveal that both residual enzyme activity and protein stability of FGE contribute to the clinical phenotype. The application of improved functional assays to determine these two molecular parameters of FGE mutants may enable the prediction of the clinical outcome in the future.

Golgi GDP-fucose transporter-deficient mice mimic congenital disorder of glycosylation IIc/leukocyte adhesion deficiency II.

Modification of glycoproteins by the attachment of fucose residues is widely distributed in nature. The importance of fucosylation has recently been underlined by identification of the monogenetic inherited human disease "congenital disorder of glycosylation IIc," also termed "leukocyte adhesion deficiency II." Due to defective Golgi GDP-fucose transporter (SLC35C1) activity, patients show a hypofucosylation of glycoproteins and present clinically with mental and growth retardation, persistent leukocytosis, and severe infections. To investigate effects induced by the loss of fucosylated structures in different organs, we generated a mouse model for the disease by inactivating the Golgi GDP-transporter gene (Slc35c1). Lectin binding studies revealed a tremendous reduction of fucosylated glycoconjugates in tissues and isolated cells from Slc35c1(-/-) mice. Fucose treatment of cells from different organs led to partial normalization of the fucosylation state of glycoproteins, thereby indicating an alternative GDP-fucose transport mechanism. Slc35c1-deficient mice presented with severe growth retardation, elevated postnatal mortality rate, dilatation of lung alveoles, and hypocellular lymph nodes. In vitro and in vivo leukocyte adhesion and rolling assays revealed a severe impairment of P-, E-, and L-selectin ligand function. The diversity of these phenotypic aspects demonstrates the broad general impact of fucosylation in the mammalian organism.

Molecular characterization of the hypothetical 66.3-kDa protein in mouse: lysosomal targeting, glycosylation, processing and tissue distribution.

Recently, we and others identified the 66.3-kDa protein as one of several putative novel lysosomal matrix proteins by analyzing mannose 6-phosphate receptors binding proteins [Kollmann K., Mutenda K.E., Balleininger M., Eckermann E., von Figura K., Schmidt B., Lübke T. (2005) Identification of novel lysosomal matrix proteins by proteome analysis. Proteomics 5(15), 3966-3678, Sleat D.E., Lackland H., Wang Y., Sohar I., Xiao G., Li H., Lobel P. (2005) The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins. Proteomics. 5(6), 1520-1532]. Here, we describe the expression of the mouse 66.3-kDa protein in HT1080 cells in which it is synthesized as a precursor of about 75kDa and subsequently processed by limited proteolysis to mature polypeptides accumulating in the lysosomal compartment. The lysosomal localisation of the endogenous 66.3-kDa protein was verified by indirect immunofluorescence in mouse embryonic fibroblasts and by subcellular fractionation of tyloxapol-filled mouse liver lysosomes. Northern blot analysis reveals high transcriptional levels in testis, liver and kidney, whereas Western blot analysis shows high protein levels in brain, heart, lung and spleen. Interestingly, in mouse the endogenous 66.3-kDa protein is processed in a highly tissue-dependent manner to mature forms.

Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity.

HS (heparan sulfate) is essential for normal embryonic development. This requirement is due to the obligatory role for HS in the signalling pathways of many growth factors and morphogens that bind to sulfated domains in the HS polymer chain. The sulfation patterning of HS is determined by a complex interplay of Golgi-located N- and O-sulfotransferases which sulfate the heparan precursor and cell surface endosulfatases that selectively remove 6-O-sulfates from mature HS chains. In the present study we generated single or double knock-out mice for the two murine endosulfatases mSulf1 and mSulf2. Detailed structural analysis of HS from mSulf1-/- fibroblasts showed a striking increase in 6-O-sulfation, which was not seen in mSulf2-/- HS. Intriguingly, the level of 6-O-sulfation in the double mSulf1-/-/2-/- HS was significantly higher than that observed in the mSulf1-/- counterpart. These data imply that mSulf1 and mSulf2 are functionally co-operative. Unlike their avian orthologues, mammalian Sulf activities are not restricted to the highly sulfated S-domains of HS. Mitogenesis assays with FGF2 (fibroblast growth factor 2) revealed that Sulf activity decreases the activating potential of newly-synthesized HS, suggesting an important role for these enzymes in cell growth regulation in embryonic and adult tissues.

Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality.

Mutations in the cytosolic enzyme phosphomannomutase 2 (PMM2), which catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, cause the most common form of congenital disorders of glycosylation, termed CDG-Ia. It is an inherited multisystemic disease with severe neurological impairment. To study the pathophysiology of CDG-Ia and to investigate possible therapeutic approaches, we generated a mouse model for CDG-Ia by targeted disruption of the Pmm2 gene. Heterozygous mutant mice appeared normal in development, gross anatomy, and fertility. In contrast, embryos homozygous for the Pmm2-null allele were recovered in embryonic development at days 2.5 to 3.5. These results indicate that Pmm2 is essential for early development of mice. Mating experiments of heterozygous mice with wild-type mice could further show that transmission of the female Pmm2-null allele is impaired.

LAMP-2 deficient mice show depressed cardiac contractile function without significant changes in calcium handling.

OBJECTIVE: Mutations in the highly glycosylated lysosome associated membrane protein-2 (LAMP-2) cause, as recently shown, familial Danon disease with mental retardation, mild myopathy and fatal cardiomyopathy. Extent and basis of the contractile dysfunction is not completely understood. METHODS: In LAMP-2 deficient mice, we investigated cardiac function in vivo using Doppler-echocardiography and contractile function in vitro in isolated myocardial trabeculae. RESULTS: LAMP-2 deficient mice displayed reduced ejection fraction (EF) (58.9+/-3.4 vs. 80.7+/-5.1%, P<0.05) and reduced cardiac output (8.3+/-3.1 vs. 14.7+/-3.6 ml/min, P<0.05) as compared to wild-type controls. Isolated multicellular muscle preparations from LAMP-2 deficient mice confirmed depressed force development (3.2+/-0.6 vs. 8.4+/-0.9 mN/mm2, P<0.01). All groups showed similar force-frequency behaviour when normalised to baseline force. Post-rest potentiation was significantly depressed at intervals>15 s in LAMP-2 deficient mice (P<0.05). Although attenuated in absolute force development, the normalised inotropic response to increased calcium and beta-adrenoreceptor stimulation was unaltered. Electron microscopic analysis revealed autophagic vacuoles in LAMP-2 deficient cardiomyocytes. Protein analysis showed unaltered levels of SERCA2a, calsequestrin and phospholamban. CONCLUSIONS: Cardiac contractile function in LAMP-2 deficient mice as a model for Danon disease is significantly attenuated. The occurrence of autophagic vacuoles in LAMP-2 deficient myocytes is likely to be causal for the depressed contractile function resulting in an attenuated cardiac pump reserve.

The early vertebrate Danio rerio Mr 46000 mannose-6-phosphate receptor: biochemical and functional characterisation.

Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, gamma-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish.

A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme.

The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism.

Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease).

In cathepsin D-deficient (CD-/-) and cathepsins B and L double-deficient (CB-/-CL-/-) mice, abnormal vacuolar structures accumulate in neurons of the brains. Many of these structures resemble autophagosomes in which part of the cytoplasm is retained but their precise nature and biogenesis remain unknown. We show here how autophagy contributes to the accumulation of these vacuolar structures in neurons deficient in cathepsin D or both cathepsins B and L by demonstrating an increased conversion of the molecular form of MAP1-LC3 for autophagosome formation from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In both CD-/- and CB-/-CL-/- mouse brains, the membrane-bound LC3-II form predominated whereas MAP1-LC3 signals accumulated in granular structures located in neuronal perikarya and axons of these mutant brains and were localized to the membranes of autophagosomes, evidenced by immunofluorescence microscopy and freeze-fracture-replica immunoelectron microscopy. Moreover, as in CD-/- neurons, autofluorescence and subunit c of mitochondrial ATP synthase accumulated in CB-/-CL-/- neurons. This suggests that not only CD-/- but also CB-/-CL-/- mice could be useful animal models for neuronal ceroid-lipofuscinosis/Batten disease. These data strongly argue for a major involvement of autophagy in the pathogenesis of Batten disease/lysosomal storage disorders.

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation.

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.

Identification of novel lysosomal matrix proteins by proteome analysis.

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.

Neurocognitive and psychotiform behavioral alterations and enhanced hippocampal long-term potentiation in transgenic mice displaying neuropathological features of human alpha-mannosidosis.

Mice with alpha-mannosidase gene inactivation provide an experimental model for alpha-mannosidosis, a lysosomal storage disease with severe neuropsychological and psychopathological complications. Neurohistological alterations in these mice were similar to those in patients and included vacuolations and axonal spheroids in the CNS and peripheral nervous system. Vacuolation was most prominent and evenly distributed in neuronal perikarya of the hippocampal CA2 and CA3 regions, whereas CA1 and dentate gyrus were weakly or not affected. Field potential recordings from CA1 region in hippocampal slices showed enhanced theta burst-induced long-term potentiation (LTP) in alpha-mannosidase-deficient mice. Longitudinal assessment in age-matched alpha-mannosidase-deficient and wild-type littermates, using an extended test battery, demonstrated a neurocognitive and psychotiform profile that may relate to the psychopathological alterations in clinical alpha-mannosidosis. Brainstem auditory-evoked potentials and basic neuromotor abilities were not impaired and did not deteriorate with age. Exploratory and conflict tests revealed consistent decreases in exploratory activity and emotional blunting in the knock-out group. alpha-Mannosidosis mice were also impaired in aversively motivated learning and acquisition of signal-shock associations. Acquisition and reversal learning in the water maze task, passive avoidance learning in the step-through procedure, as well as emotional response conditioning in an operant procedure were all impaired. Acquisition or shaping of an appetitive instrumental conditioning task was unchanged. Appetitive odor discrimination learning was only marginally impaired during shaping, whereas both the discrimination and reversal subtasks were normal. We propose that prominent storage and enhanced LTP in hippocampus have contributed to these specific behavioral alterations in alpha-mannosidase-deficient mice.