Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria.

BACKGROUND: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms. METHODS: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis. RESULTS: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis. CONCLUSIONS: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.

Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes.

Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions.

Antigen processing and presentation and cytotoxic targeting depend on the activities of several lysosomal enzymes that require mannose 6-phosphate (M6P) sorting signals for efficient intracellular transport and localization. In this paper, we show that mice deficient in the formation of M6P residues exhibit significant loss of cathepsin proteases in B cells, leading to lysosomal dysfunction with accumulation of storage material, impaired antigen processing and presentation, and subsequent defects in B cell maturation and antibody production. The targeting of lysosomal and granular enzymes lacking M6P residues is less affected in dendritic cells and T cells and sufficient for maintenance of degradative and lytic functions. M6P deficiency also impairs serum immunoglobulin levels and antibody responses to vaccination in patients. Our data demonstrate the critical role of M6P-dependent transport routes for B cell functions in vivo and humoral immunity in mice and human.

Molecular characterization of arylsulfatase G: expression, processing, glycosylation, transport, and activity.

Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor protein localizes to pre-lysosomal compartments and tightly associates with organelle membranes, most likely the endoplasmic reticulum. In contrast, proteolytically processed ARSG fragments of 34-, 18-, and 10-kDa were found in lysosomal fractions and lost their membrane association. The processing sites and a disulfide bridge between the 18- and 10-kDa chains could be roughly mapped. Proteases participating in the processing were identified as cathepsins B and L. Proteolytic processing is dispensable for hydrolytic sulfatase activity in vitro. Lysosomal transport of ARSG in the liver is independent of mannose 6-phosphate, sortilin, and Limp2. However, mutation of glycosylation site N-497 abrogates transport of ARSG to lysosomes in human fibrosarcoma cells, due to impaired mannose 6-phosphate modification.

Serine carboxypeptidase SCPEP1 and Cathepsin A play complementary roles in regulation of vasoconstriction via inactivation of endothelin-1.

The potent vasoconstrictor peptides, endothelin 1 (ET-1) and angiotensin II control adaptation of blood vessels to fluctuations of blood pressure. Previously we have shown that the circulating level of ET-1 is regulated through its proteolytic cleavage by secreted serine carboxypeptidase, cathepsin A (CathA). However, genetically-modified mouse expressing catalytically inactive CathA S190A mutant retained about 10-15% of the carboxypeptidase activity against ET-1 in its tissues suggesting a presence of parallel/redundant catabolic pathway(s). In the current work we provide direct evidence that the enzyme, which complements CathA action towards ET-1 is a retinoid-inducible lysosomal serine carboxypeptidase 1 (Scpep1), a CathA homolog with previously unknown biological function. We generated a mouse strain devoid of both CathA and Scpep1 activities (DD mice) and found that in response to high-salt diet and systemic injections of ET-1 these animals showed significantly increased blood pressure as compared to wild type mice or those with single deficiencies of CathA or Scpep1. We also found that the reactivity of mesenteric arteries from DD mice towards ET-1 was significantly higher than that for all other groups of mice. The DD mice had a reduced degradation rate of ET-1 in the blood whereas their cultured arterial vascular smooth muscle cells showed increased ET-1-dependent phosphorylation of myosin light chain 2. Together, our results define the biological role of mammalian serine carboxypeptidase Scpep1 and suggest that Scpep1 and CathA together participate in the control of ET-1 regulation of vascular tone and hemodynamics.

A hereditary spastic paraplegia mouse model supports a role of ZFYVE26/SPASTIZIN for the endolysosomal system.

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.

Identification of the ubiquitin-like domain of midnolin as a new glucokinase interaction partner.

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.

Decreased bone formation and increased osteoclastogenesis cause bone loss in mucolipidosis II.

Mucolipidosis type II (MLII) is a severe multi-systemic genetic disorder caused by missorting of lysosomal proteins and the subsequent lysosomal storage of undegraded macromolecules. Although affected children develop disabling skeletal abnormalities, their pathogenesis is not understood. Here we report that MLII knock-in mice, recapitulating the human storage disease, are runted with accompanying growth plate widening, low trabecular bone mass and cortical porosity. Intralysosomal deficiency of numerous acid hydrolases results in accumulation of storage material in chondrocytes and osteoblasts, and impaired bone formation. In osteoclasts, no morphological or functional abnormalities are detected whereas osteoclastogenesis is dramatically increased in MLII mice. The high number of osteoclasts in MLII is associated with enhanced osteoblastic expression of the pro-osteoclastogenic cytokine interleukin-6, and pharmacological inhibition of bone resorption prevented the osteoporotic phenotype of MLII mice. Our findings show that progressive bone loss in MLII is due to the presence of dysfunctional osteoblasts combined with excessive osteoclastogenesis. They further underscore the importance of a deep skeletal phenotyping approach for other lysosomal diseases in which bone loss is a prominent feature.

Ultrastructural analysis of neuronal and non-neuronal lysosomal storage in mucolipidosis type II knock-in mice.

The GlcNAc-1-phosphotransferase catalyzes the first step in the formation of mannose 6-phosphate (M6P) residues on lysosomal acid hydrolases that is essential for the efficient transport of newly synthesized lysosomal enzymes to lysosomes and the maintenance of lysosomal functions. Mutations in the GlcNAc-1-phosphotransferase cause the lysosomal storage disease mucolipidosis type II (MLII), resulting in mistargeting and hypersecretion of multiple lysosomal hydrolases and subsequent lysosomal accumulation of nondegraded material in several tissues. To describe cell-type specificity, compositional differences, and subcellular distribution of the stored material we performed an in-depth ultrastructural analysis of lysosomal storage in brain and retina of MLII knock-in mice using electron microscopy. Massive vacuoles filled with heterogeneous storage material have been found in the soma, swollen axons, and dendrites of Purkinje, and granular cells in 9-month-old MLII mice. In addition, non-neuronal cells, such as microglial, astroglial, and endothelial cells, exhibit storage material. Fucose-specific lectin histochemistry demonstrated the accumulation of fucose-containing oligosaccharides, indicating that targeting of the lysosomal α-fucosidase is strongly impaired in all cerebellar cell types. The data suggest that the accumulation of storage material might affect neuronal function and survival in a direct cell-autonomous manner, as well as indirectly by disturbed metabolic homeostasis between glial and neuronal cells or by cerebrovascular complications.

Cell biology and function of neuronal ceroid lipofuscinosis-related proteins.

Neuronal ceroid lipofuscinoses (NCL) comprise a group of inherited lysosomal disorders with variable age of onset, characterized by lysosomal accumulation of autofluorescent ceroid lipopigments, neuroinflammation, photoreceptor- and neurodegeneration. Most of the NCL-related genes encode soluble and transmembrane proteins which localize to the endoplasmic reticulum or to the endosomal/lysosomal compartment and directly or indirectly regulate lysosomal function. Recently, exome sequencing led to the identification of four novel gene defects in NCL patients and a new NCL nomenclature currently comprising CLN1 through CLN14. Although the precise function of most of the NCL proteins remains elusive, comprehensive analyses of model organisms, particularly mouse models, provided new insight into pathogenic mechanisms of NCL diseases and roles of mutant NCL proteins in cellular/subcellular protein and lipid homeostasis, as well as their adaptive/compensatorial regulation at the transcriptional level. This review summarizes the current knowledge on the expression, function and regulation of NCL proteins and their impact on lysosomal integrity. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.

A key enzyme in the biogenesis of lysosomes is a protease that regulates cholesterol metabolism.

Mucolipidosis II is a severe lysosomal storage disorder caused by defects in the α and ß subunits of the hexameric N-acetylglucosamine-1-phosphotransferase complex essential for the formation of the mannose 6-phosphate targeting signal on lysosomal enzymes. Cleavage of the membrane-bound α/ß-subunit precursor by an unknown protease is required for catalytic activity. Here we found that the α/ß-subunit precursor is cleaved by the site-1 protease (S1P) that activates sterol regulatory element-binding proteins in response to cholesterol deprivation. S1P-deficient cells failed to activate the α/ß-subunit precursor and exhibited a mucolipidosis II-like phenotype. Thus, S1P functions in the biogenesis of lysosomes, and lipid-independent phenotypes of S1P deficiency may be caused by lysosomal dysfunction.

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)ß(2)γ(2)). The α- and ß-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.

Proteolytic processing of the gamma-subunit is associated with the failure to form GlcNAc-1-phosphotransferase complexes and mannose 6-phosphate residues on lysosomal enzymes in human macrophages.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

A novel single-chain antibody fragment for detection of mannose 6-phosphate-containing proteins: application in mucolipidosis type II patients and mice.

Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III.

Mannose phosphorylation in health and disease.

Lysosomal hydrolases catalyze the degradation of a variety of macromolecules including proteins, carbohydrates, nucleic acids and lipids. The biogenesis of lysosomes or lysosome-related organelles requires a continuous substitution of soluble acid hydrolases and lysosomal membrane proteins. The targeting of lysosomal hydrolases depends on mannose 6-phosphate residues (M6P) that are recognized by specific receptors mediating their transport to an endosomal/prelysosomal compartment. The key role in the formation of M6P residues plays the GlcNAc-1-phosphotransferase localized in the Golgi apparatus. Two genes have been identified recently encoding the type III alpha/beta-subunit precursor membrane protein and the soluble gamma-subunit of GlcNAc-1-phosphotransferase. Mutations in these genes result in two severe diseases, mucolipidosis type II (MLII) and III (MLIII), biochemically characterized by the missorting of multiple lysosomal hydrolases due to impaired formation of the M6P recognition marker, and general lysosomal dysfunction. This review gives an update on structural properties, localization and functions of the GlcNAc-1-phosphotransferase subunits and improvements of pre- and postnatal diagnosis of ML patients. Further, the generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysosomal disorders.

Molecular characterization and gene disruption of mouse lysosomal putative serine carboxypeptidase 1.

The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix protein that was initially identified in a screen for genes induced by retinoic acid. Recently, it has been spotlighted by several proteome analyses of the lysosomal compartment, but its cellular function and properties remain unknown to date. In this study, Scpep1 from mice was analysed with regard to its intracellular processing into a mature dimer consisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDa C-terminal fragment and the glycosylation status of the mature Scpep1 fragment. Although Scpep1 shares notable homology and a number of structural hallmarks with the well-described lysosomal carboxypeptidase protective protein/cathepsin A, the purified recombinant 55 kDa precursor and the homogenates of Scpep1-overexpressing cells do not show proteolytic activity or increased serine carboxypeptidase activity towards artificial serine carboxypeptidase substrates. Hence, we disrupted the Scpep1 gene in mice by a gene trap cassette, resulting in a Scpep1/beta-galactosidase/neomycin phosphotransferase fusion protein. The fusion protein is devoid of the C-terminal half of Scpep1, including two amino acids of the assumed catalytic triad which is indispensable for its predicted serine carboxypeptidase activity. However, Scpep1-deficient mice were viable and fertile, and did not exhibit either lysosomal storage or reduced lysosomal SC activity under any tested condition.

De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse.

The 66.3 kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75 kDa preproprotein which is further processed into 28 and 40 kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3 kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3 kDa variant and the double-chain form consisting of a 28 kDa fragment and a 40 kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3 kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9 A on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4 A using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an R(free) factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks.

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.

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.