Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II*

The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver.

Soluble lysosomal proteins undergo a specific modification on high-mannose type N-linked oligosaccharides for an efficient transport from the Golgi apparatus to late endosomes and lysosomes by phosphorylation of distinct mannose residues in the C6 position. This modification is catalyzed by the subsequent action of two enzymes: the first enzyme is an N-acetylglucosamine (GlcNAc) 1 -1-phosphotransferase (PT), which mediates the transfer of GlcNAc-1-phosphate from uridine diphosphate (UDP)-GlcNAc to mannose acceptors forming GlcNAc-phosphomannose diester (1)(2)(3). The second so called 'uncovering enzyme' removes GlcNAc to expose mannose 6-phosphate (M6P) residues (4). Two M6P-specific receptors (MPRs), MPR46 and MPR300, can bind M6P-modified lysosomal proteins in the trans Golgi network (TGN) and mediate their segregation from the secretory pathway (5). Lysosomal enzymes which escape the binding to MPRs are secreted but can be reinternalized by MPR300 localized at the plasma membrane and delivered to lysosomes via the endocytic pathway. MPR-ligand complexes reach late endosomal compartments where the acidic pH induces the dissociation of lysosomal cargo. MPRs are then transported back to the TGN or the plasma membrane for further rounds of transport (6).
PT is a heterohexameric enzyme complex consisting of two ␣, two ␤, and two ␥ subunits (7). The ␣and ␤-subunits are encoded by the GNPTAB gene (8,9), and the ␥-subunits are coded by GNPTG (10). The ␣and ␤-subunits contain the catalytic center for PT activity and the recognition sites for lysosomal protein substrates and UDP-GlcNAc (11) whereas the role of the ␥-subunits are less defined, but might be involved in the folding and stabilization of the PT complex, prerequisite for optimum catalytic activity, and selective recognition of subsets of lysosomal proteins (12,13). Mutations in GNPTAB and GNPTG lead to lysosomal storage diseases, mucolipidosis type II (ML II also called I-cell disease) and III (ML III) (14). The failure or partial formation of M6P residues on soluble lysosomal proteins in ML II and ML III, respectively, impair their proper delivery to lysosomes and result in their hypersecretion into the extracellular space and bloodstream. The defect in mannose phosphorylation, however, allows processing of high mannose-type oligosaccharides to negatively charged complex-type units and the formation of abnormal proteolytically processed intracellular and secreted forms of lysosomal enzymes (15)(16)(17). Although all cells and tissues in ML II are deficient in PT activity, fibroblast and mesenchymal cells are most strikingly affected (18), indicating that these cells particularly rely on M6P-dependent lysosomal protein delivery pathways. In contrast, many cell types (such as hepatocytes, Kupffer cells and leukocytes) and several organs (liver, spleen, kidney and brain) have nearly normal level of lysosomal enzymes (18 -21). These observations suggest the existence of lysosomal transport pathways that do not require M6P recognition marker on lysosomal enzymes.
We have generated previously a Gnptab knock-in (PT ki ) mouse model for ML II by insertion a mutation found in ML II patients which led to a complete loss of PT activity (17). These PT ki mice show all biochemical and clinical symptoms of the human disease (17,22,23). Here we present a comprehensive quantitative proteomic approach of purified lysosomes ("tritosomes") from liver of wild-type (wt) and PT ki mice. This allowed us to identify proteins for which M6P is indispensable for their proper delivery to lysosomes. We found proteins which are significantly decreased, as well as sets of proteins of similar or even increased expression in lysosomes from PT ki mice in comparison with wild-type controls. The analytic data were biochemically evaluated and compared with primary cultured hepatocytes and sinusoid endothelial cells (LSECs). Further, pulse-chase experiments with cultured hepatic cells indicated that hypersecretion of nonphosphorylated lysosomal enzymes followed by M6P-independent recapture mechanisms are important for maintenance of lysosomal functions in ML II liver.

EXPERIMENTAL PROCEDURES
Antibodies and Materials-The Npc2 antiserum was a kind gift from Dr. Shutish Patel and described previously (24), Neu1 and Ppca antibodies were a kind gift from Dr. Allessandra D'Azzo (St. Jude Children's Research Hospital; Memphis), Mpr300 antibodies were a kind gift from Dr. Kurt von Figura (University of Gö ttingen) and ␤-glucuronidase antibody was kindly provided by Dr. William S. Sly (St. Louis University). Recombinant human arylsulfatase B (ARSB) was kindly provided by Dr. Michel Vellard (Biomarin/Genzyme, San Rafael, CA). The monoclonal Lamp1 (clone 1D4B) and Lamp2 (clone Abl93) antibodies were purchased from the Developmental Studies Hybridoma Bank. Ctsd antiserum used for immunofluorescence analysis and immunoprecipitation was described previously (25). Antibodies against Ctsd for Western blotting, Ctsh, Ctsa, and Ctss were obtained from Santa Cruz (Dallas, TX). Ctsb was purchased from Neuromics, Ctsz, and Ctsl from R&D Systems (Minneapolis, MN), Lrp1 and Ldlr from Abcam. Chemicals were, if not stated otherwise, purchased from Sigma Aldrich (Cambridge, United Kingdom).
Mice-The PT ki mouse model was generated by the insertion of a cytosine in position 3082 of the murine Gnptab gene (c.3082insC) (17). Mice were housed in a pathogen-free animal facility at the University Medical Center Hamburg-Eppendorf, and experimental procedures were performed according to the institutional guidelines and approved by the Amt fü r Gesundheit und Verbraucherschutz and the Ministerium fü r Landwirtschaft, Umwelt und lä ndliche Rä ume des Landes Schleswig-Holstein.
Isolation of Primary Hepatocytes and Liver Sinusoidal Endothelial Cells (LSECs)-Primary mouse hepatocytes were isolated by liver perfusion, EDTA dissociation and centrifugation on a Percoll gradient as described previously (26). LSECs were isolated as described previously (27).
Isolation of Lysosomes from Liver (Tritosomes)-Mice were injected with 4 l/g bodyweight of a 17% (w/v in 0.9% NaCl) Triton WR1339 solution (Sigma Aldrich) 4 days prior to sacrifice. The livers were removed and homogenized in five volumes of ice-cold 0.25 M sucrose with three strokes with a Potter-Elvejhem homogenizer. The homogenates were centrifuged at 1000 ϫ g for 10 min at 4°C. After removal of the supernatant and resuspension of the pellet followed by another centrifugation step at 1000 ϫ g for 10 min at 4°C, the supernatants were pooled (post nuclear supernatant, PNS) and centrifuged in an ultracentrifuge at 56,000 ϫ g (70.1 Ti rotor, Beckmann, Coulter, Indianapolis, IN) for 7 min at 4°C. After rehomogenization of the pellet with 0.25 M sucrose followed by another ultracentrifugation step, the supernatant was discarded and the pellet was resuspended in sucrose solution with a density of 1.21 (resulting in the ML-fraction, representing the mitochondrial-lysosomal fraction). The ML fraction (ϳ3.5 ml) was overlaid each with 2.5 ml sucrose solutions of 1.15, 1.14, and finally 1.06 density yielding a discontinuous sucrose gradient. After centrifugation at 110,000 ϫ g for 150 min in a swinging bucket rotor (SW41 Ti rotor, Beckmann Coulter), lysosomes/tritosomes were collected at the interphase between 1.14 and 1.06 sucrose (F2-fraction).
Western Blotting-Five micrograms (tritosome protein), 100 g (total liver protein), or 25 g hepatocytes or LSECs protein were separated by SDS-PAGE and blotted on nitrocellulose membranes. After blocking with low-fat milk powder for 30 min at room temperature, membranes were incubated with the primary antibodies over night at 4°C. After further incubation with Horseradish peroxidasecoupled secondary antibodies, blots were developed with enhanced chemiluminescence solution. Densitometric analyses were performed with the ImageJ software (NIH) to quantify band intensities.
Two-dimensional Gel Electrophoresis-Two-dimensional gel electrophoresis was carried out essentially as described previously (28) with Immobiline DryStrip gels (IPG strip) (GE Healthcare Life Sciences) in a pH range of 4 -7 and 400 g of total tritosome protein.
Metabolic Labeling and Immunoprecipitation of Cathepsin D and Z-Cells grown on 35 mm dishes were metabolically labeled with 150 Ci/ml [ 35 S] methionine (Hartmann Analytik; 37 Bq/mmol)in methionine-free DMEM for 45 min. After removal of the radioactive medium, cells were either harvested or washed with PBS and chased for 5 h in DMEM containing 5% dialyzed fetal bovine serum (FBS). Cell extracts and media were analyzed by immunoprecipitation of cathepsin D and cathepsin Z followed by SDS-PAGE and fluorography (29).
Endocytosis of Arylsulfatase B (ARSB)-Human ARSB was iodinated with sodium [ 125 I] (Hartmann Analytik, Braunschweig, Germany, 74 TBq/mmol) and IODO-GEN ® (Pierce, Waltham, MA) as described (30) to a specific activity of 22 Ci/g. Hepatocytes grown in 35 mm dishes were preincubated with DMEM containing 0.1% bovine serum albumin (BSA) (DMEM/BSA) for 30 min, followed by incubation with [ 125 I]-labeled ARSB (ϳ600,000 cpm/ml in DMEM/BSA) in the absence or presence of 10 mM M6P or 10 M receptor associated protein (RAP) for 20 min at 37°C as indicated. After washing, the cells were either harvested in 10 mM Tris/HCl, pH 7.4 containing 0.25 M sucrose or chased for 6 h in DMEM/BSA. The cells were solubilized in PBS containing 0.2% Triton X-100 and protease inhibitors, separated by SDS-PAGE and visualized by autoradiography.
Immunofluorescence Staining of Hepatocytes-Double immunofluorescence staining of hepatocytes was performed as described previously (31). For filipin staining the cells were stained with 1 g/l filipin in PBS.
Immunofluorescence Staining of Liver Sections-Formalin-fixed liver was equilibrated in 30% (w/v) sucrose in 0.1 M phosphate buffer (PB), pH 7.4 overnight and sectioned using a sliding microtome (SM2000R, Leica) into 30 m thick free floating sections and collected in PB. After blocking in PB containing 0.5% Triton X-100 and 4% normal goat serum for 2 h at room temperature, sections were incubated with the primary antibodies in the blocking solution overnight at 4°C. After washing three times in PB containing 0.25% Triton X-100 (washing solution), sections were incubated with fluorophore-coupled secondary antibodies (AlexaFluor 488 and 594; Molecular Probes, Waltham, MA) for one hour at room temperature. After additional three washing steps in washing solution, sections were coverslipped in Mowiol/DABCO. Confocal microscopy was performed on an Olympus FV1000 fluorescence microscope.
Measurement of iduronate-2-sulfatase activity was performed as described previously (32). Using 100 and 5 g protein of liver extracts and tritosomes, respectively, as enzyme source. The liberated 4-methylumbelliferone was measured fluorometrically (excitation at 355 nm and emission 460 nm) related to 4-MU standard solutions.
Protein Digestion-Each sample (20 g of protein) was dissolved in 0.1% (w/v) RapiGest (Waters Corporation, Milford, MA) in 50 mM ammonium bicarbonate (Sigma Aldrich, St. Louis, MO) and heated at 80°C for 45 min. The samples were reduced in the presence of 5 mM dithiotreitol (Sigma Aldrich) at 60°C for 30 min and alkylated in the presence of 10 mM iodoacetamide (Sigma Aldrich) at ambient temperature in the dark for another 30 min. Proteolytic digestion was initiated by adding sequencing grade TMPK-treated trypsin (Promega, Madison, MI) at a 1:50 (w/w) ratio and incubation conducted for 4 h at 37°C. A second aliquot of trypsin was added at a 1:50 (w/w) ratio and followed by incubation overnight at 37°C. TFA was added to a final concentration of 0.5% (v/v) in order to hydrolyze RapiGest and the solutions incubated at 37°C for 20 min before being vortexed and centrifuged for 30 min at 13,000 rpm.
LC-MS Configuration-LC separations were performed with an ACQUITY M Class system (Waters Corporation), equipped with a Symmetry C18 5 m, 2 cm ϫ 180 m precolumn and an HSS T3 C18 1.7 m, 25 cm ϫ 75 m analytical column. The samples were transferred with aqueous 0.1% (v/v) formic acid to the precolumn at a flow rate of 5 l/min. Mobile phase A was water containing 0.1% (v/v) formic acid, whereas mobile phase B was acetonitrile containing 0.1% (v/v) formic acid. The peptides were eluted from the precolumn to the analytical column and separated with a gradient of 3-40% mobile phase B over 90 min at a flow rate of 300 nl/min. The analytical column temperature was maintained at 35°C. The lock mass compound, [Glu1]-Fibrinopeptide B (200 fmol/l), was delivered at 600 nl/min to the reference sprayer of the mass spectrometer.
Mass spectrometric analysis of tryptic peptides was performed using a Synapt G2-Si mass spectrometer (Waters Corporation, Wilmslow, United Kingdom). The mass spectrometer was operated in v-mode with nominal resolution of 20,000 FWHM. All analyses were performed in positive mode ESI. The ion source block temperature and capillary voltage were set to 100°C and 3.2 kV, respectively. The time of flight analyzer (ToF) of the mass spectrometer was externally calibrated with a NaCsI mixture from m/z 50 to 1990. LC-MS data were collected in mobility assisted data independent (UDMSE) mode of acquisition (33). The spectral acquisition time in each mode was 0.5 s with a 0.02 s interscan delay. In low energy mode, data were collected at constant Trap collision energy of 4 eV (per unit charge) and a constant Transfer collision energy of 2 eV (per unit charge). In elevated energy mode, the Trap collision energy was ramped from 4 eV to 5 eV (per unit charge) in 0.5 s, and the Transfer collision energy was set to ramp during each 14 ms ion mobility separation, with the ramp applying the optimum collision energy for species at a particular drift time. The ion mobility separations were combined in a 0.5 s total elevated energy integration time. The reference sprayer sampled every 60 s and the data post-acquisition lock mass corrected.
Data Processing and Database Searching-ProteinLynx Global-SERVER v3.0.2 (Waters Corporation) was used to process the acquired data. Protein identifications were obtained by searching a Mus Musculus UniProt database (16,674 reviewed entries, release 2014_10). To assess protein and peptide FDR values, decoy database entries were created by reversing the amino acid sequences and concatenating them with the original database entries. The principle of the search algorithm has been described (34). Peptide and fragment ion tolerances were determined automatically, equaling 5 and 10 ppm respectively, one missed cleavage allowed, as well as fixed carbamidomethylation modification of cysteine and variable oxidation modification of methionine. ISOQuant was applied for the integrated quantitative analysis of data derived from multiple LC-MS runs (http:// www.isoquant.net). The results have been deposited to the Proteome Xchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (35) with the data set identifier PXD001556.
Experimental Design and Statistical Rationale, Statistics, Hierarchical Clustering, Enrichment Analysis, and Principal Component Analysis-The experimental design comprised four biological and three technical replicates of PT ki and WT each, a number adequate enough to detect small changes in the protein abundance in the tritosome samples with sufficient statistical power. Two-tailed unpaired Student's t-tests were calculated using Excel (tables) or Graphpad Prism (graphs). The protein abundance data from WT and PT ki mice followed a normal distribution. Errors bars represent the means Ϯ S.E. of mean (S.E.). For annotation of proteomics data as heat maps, GENE-E software was used (http://www.broadinstitute.org/cancer/ software/GENE-E). Both columns and rows were hierarchically clustered and the "one minus Pearson correlation" was chosen as the column distance metric. Principal component analysis was performed by ClustVis (36). For functional annotation of the proteomics data, the "Database for Annotation, Visualization and Integrated Discovery" (DAVID) was used and the "Functional Annotation Chart" was chosen for enrichment-analysis of KEGG pathways (37).

Proteomic Analysis of Isolated Liver Lysosomes-Early
studies measuring the activities of few lysosomal hydrolases in liver tissue of ML II patients have revealed nearly normal levels (19,20). In order to determine the impact of the loss of M6P targeting signals on the lysosomal proteome in the liver, Triton WR1339-filled lysosomes (tritosomes) were purified from the liver of wt and PT ki mice by differential and subsequent discontinuous sucrose gradient centrifugation ( Fig. 1A; (38)). Western blotting analysis of liver lysates and different fractions obtained showed highest enrichment of lysosomes in the F2-fraction characterized by the lysosomal membrane protein Lamp1. The enrichment and yield of tritosomes were comparable between wt and PT ki preparations ( Fig. 1B and  1C). One-dimensional SDS-PAGE and Coomassie Blue staining of wt and PT ki lysosomes, however, revealed significant differences in intensity and band pattern (Fig. 1D). Separation of the lysosomal F2 fractions by 2D gel electrophoresis followed by Coomassie staining indicated additional differences in the composition of the lysosomal proteome between wt and PT ki mouse liver, such as the amounts and isoform pattern for lysosomal proteases tripeptidyl peptidase 1 (Tpp1), cathepsin H (Ctsh), Ctsz, Ctsb, and Ctsd (supplemental Fig.   S1). The altered charges and molecular masses of proteins in PT ki lysosomes are most likely because of differences in complex type oligosaccharides and proteolytic modifications (15,39). Because 2D gel electrophoresis resolve only the most abundant lysosomal proteins representing a small part of the proteome (28), quantitative label-free LC-/MS (ion mobilityassisted data independent analysis (UDMSE (33)) was applied to tritosome samples purified from four different animals of each genotype from distinct littermates. The protein amounts were estimated by expressing summed peptide intensity responses versus that of an exogenous protein spike (40), and normalized, to account for experimental differences, to the total tritosome protein content and expressed as g/g tritosome protein values. About 900 proteins have been identified in the lysosomal F2 fractions of wt and PT ki animals, but among them lysosomal proteins represent half of the most abundant 150 proteins (supplemental Table S1). The numerous nonlysosomal proteins identified here represent mostly mitochondrial, cytosolic as well as intermediate filament proteins that we considered as contaminants (supplemental Fig.  S2). However, we cannot exclude that several of these proteins may reach lysosomes via the degradative autophagy pathway. Hierarchical clustering of the list of selected soluble lysosomal proteins revealed clear differences in the levels of most proteins between wt and PT ki mice ( Fig. 2A). Principal component analysis (PCA) of the soluble lysosomal proteins showed a clear separation of the two genotypes (Fig. 2B). Furthermore, sigmoidal plot of all identified proteins demonstrated differential protein levels for a number of proteins including soluble lysosomal enzymes like Neu1 and Ctsf which are decreased in PT ki mice. Others such as the lysosomal membrane proteins Lamp1 and Lamp2 proteins were unchanged, whereas a number of soluble proteins like Ctsk and Mpo were found to be increased in PT ki mice (Fig. 2C).
The levels of only five soluble lysosomal proteins in PT ki tritosomes were found to be lower than 50% in PT ki in comparison with wt tritosomes. This subset includes neuraminidase 1 (Neu1), cathepsin F (Ctsf), Niemann Pick type C 2 protein (Npc2), cathepsin L1 (Ctsl), and sphingomyelin phosphodiesterase 1 (Smpd1). A number of additional proteins showed significant, but less pronounced decrease in PT ki lysosomes. The majority of lysosomal proteins showed no or only marginal differences in their concentration in tritosomes between wt and PT ki mice. Surprisingly, the level of 13 lysosomal proteins was increased in tritosomes of PT ki mice. This was most striking for cathepsin K (Ctsk) (1400%, p ϭ 4.35 ϫ 10 Ϫ4 ). In addition to these established lysosomal proteins, we identified a number of soluble proteins, for which a lysosomal residency was suggested (42,44). These include e.g. biotinidase, plasma ␣-L-fucosidase, serum amyloid P-component, or ribonuclease UK114 (supplemental Table S2). However, the levels of none of them were significantly affected in PT ki mice.
Abundant lysosomal membrane proteins like Lamp1, Lamp2, Limp2, and Npc1 showed no differences in their total levels in tritosomes between wt and PT ki mice (Table I, supplemental Table S3). A total of 45 known and 33 putative lysosomal membrane proteins (supplemental Table S3 and S4), identified previously in tritosomes (45), were detected and quantified. Additional 64 proteins known to function in endosomal and lysosomal trafficking processes were covered by our proteomic analysis (supplemental Table S5).
Expression Levels of Lysosomal Proteins-In order to validate our lysosomal proteomic dataset, we examined the expression of soluble lysosomal proteins and enzymes by Western blotting and enzyme activity assays. As shown in Fig. 3 the amount of immunoreactive polypeptides Neu1, Npc2, Ctsl, Ctsh, and Ctsa were significantly reduced in tritosomes of PT ki mice. The expression of Ctsb, Ctsd, Ctsz, and ␤-glucuronidase (Gusb), as well as the lysosomal membrane protein Lamp1, were not or only marginally affected. Of note, for Ctsb and Ctsd changes in the proportion of single chain (sc), and their cleaved heavy (hc) and light chain (lc) forms were observed. The relative amounts of Ctss was elevated in PT ki tritosomes in comparison to wt fractions (Fig. 3). The data for these selected lysosomal proteins were in complete agreement with the MS data analysis (Table I). For a number of lysosomal enzymes we additionally determined the specific activity in extracts of both liver tissue and tritosomes. Both iduronate 2-sulfatase (Ids) and Fuca1 activities were 2-to 3-fold higher in liver tissue as well as tritosomes of Pt ki mice than of wt animals (Fig. 4A) corresponding to the increased protein level of these enzymes. No significant changes where observed for Gusb and Idua (Fig. 4A). No or only modest changes in the mRNA levels of Npc2, Idua, Fuca1, Ctsk, Neu1, Ctsf, and Ids were observed in liver tissue (supplemental Fig. S3A) whereas the respective lysosomal proteins were increased or decreased, respectively, in tritosomes of PT ki mice (Table I). These data excluded transcriptional regulation rather than posttranslational sorting events to explain the different concentrations of these lysosomal proteins in lysosomal fractions, which might be representative for the other lysosomal components. Western blotting analyses of whole liver lysates generally resembled the protein expression level in purified lysosomes, indicating that the bulk of these proteins are found in lysosomes (supplemental Fig. S3B).
Distribution of Lysosomal Proteases in Liver Tissue-To evaluate whether the almost unchanged concentrations of the lysosomal proteases Ctsd, Ctsb, and Ctsz in liver tissue or tritosomes between wt and PT ki mice are caused by differential distribution in parenchymal cells (hepatocytes) and nonparenchymal cells (Kupffer cells, stellate cells, liver sinusoid endothelial cells (LSECs)), we performed immunofluorescence staining on liver sections. Ctsd, Ctsz, and Ctsb were found to be colocalized with the lysosomal marker protein Lamp1 both in wt and PT ki hepatocytes representing the major cell type in liver tissue (Fig. 4B). Ctsl, however, that was reduced in PT ki tritosomes to 40% of wt fractions, was almost absent in PT ki hepatocytes but expressed in nonparenchymal cells (Fig. 4B).
Cultured Hepatic Cells-To examine differences in the composition of lysosomes between wt and PT ki on the cellular level in more detail, we analyzed the steady state concentration, transport and sorting efficiency, and lysosomal localization of selected soluble lysosomal proteins in cultured hepatocytes and LSECs. Western blotting analysis revealed the almost complete loss of mature Npc2 from PT ki hepatocytes, and the concomitant accumulation of Npc2 precursor polypeptides in the medium (Fig. 5A). Surprisingly, no accumulation of filipin-positive nonesterified cholesterol was observed in PT ki hepatocytes (supplemental Fig. S4A) suggesting that the residual amounts of Npc2 are sufficient to mediate the egress of lipoprotein-derived cholesterol from lysosomes. Similarly, to the tritosome data, the steady state concentration of Neu1 was strongly reduced in PT ki hepatocytes, whereas comparable amounts of Ctsb and Ctsz were found in hepatocytes of both genotypes. Different proteolytically processed forms of Ctsl and Ctsd were observed in wt and PT ki hepatocytes. The latter lack the 20 kDa mature Ctsl form and exhibit a distinct proteolytic processing of single chain and heavy chain Ctsd forms. For both Ctsl (short exposure)

TABLE I List of soluble and selected lysosomal membrane proteins identified in wt and PT ki liver lysosomes
Proteins known to be components of lysosomes (41,44) are given. The total levels for each protein in g/g tritosome protein is the average of n ϭ 4. The fold change is given as the ratio between the mean PT ki /wt. ns ϭ not significant, p Ͻ 0.05 ϭ *; p Ͻ 0.01 ϭ **; p Ͻ 0.001 ϭ ***. and Ctsd increased amounts of missorted precursor forms were detected in the medium (Fig. 5A).
To analyze targeting, sorting and processing of Ctsd and Ctsz in hepatocytes in more detail, [ 35 S] methionine pulsechase experiments were performed. The rate of synthesis of Ctsd and Ctsz was comparable in wt and PT ki hepatocytes (Fig. 5B, lane 1 and 3). During the chase period proteolytic maturation of newly synthesized Ctsd was impaired in PT ki hepatocytes, and only a small portion was retained intracel-lularly accompanied by hypersecretion of the precursor form (Fig. 5B, lane 4 and 6). During the chase Ctsz was completely missorted by PT ki hepatocytes into the medium (Fig. 5B, lane  4 and 6). The presence of other lysosomal enzymes, ␣-Lfucosidase, ␤-glucuronidase, and ␣-L-iduronidase, were detected by measurement of their enzymatic activity in hepatocyte extracts and conditioned medium. Similar activities of these lysosomal hydrolases were detected in wt and PT ki hepatocytes with the exception of ␣-L-fucosidase that was FIG. 3. Analysis of lysosomal proteins that are specifically changed in their expression in PT ki lysosomes. Western blotting analysis for Neu1, Npc2, Ctsl, Ctsh, Ctsa, Ctsb, Ctsd, Ctsz, Gusb, Ctss, and Lamp1 in lysosome-enriched fractions from wt and PT ki mice. Densitometric evaluation of the corresponding immunoreactive bands confirms the significant differences between wt and PT ki samples (n ϭ 4; sc, single chain; hc, heavy chain; lc, light chain; ns, not significant; p Ͻ 0.05, *; p Ͻ 0.01, **; p Ͻ 0.001, ***).
increased both in cells and the medium (supplemental Fig.  S4B). Double immunofluorescence microscopic imaging revealed the prominent expression of Ctsd, Ctsb and Neu1 in Lamp 1-positive structures of wt and PT ki hepatocytes (Fig.  5C). These data were supported by Western blotting analyses of lysosomal proteins in cultured LSECs. The expression pattern of Neu1, Npc2, Ctsb, and Ctsz appear to be similar of those in cultured hepatocytes (supplemental Fig. S5). In contrast, the amounts and the proteolytic processing of Ctsl and Ctsd in PT ki LSECs differed from cultured PT ki hepatocytes, Cargo Receptors-The majority of soluble lysosomal proteins in PT ki tritosomes were found in comparable amounts as in wt controls suggesting their M6P-independent transport. Because it has been shown that in PT ki fibroblasts Lrp1 and LDL receptors (Ldlr) are involved in M6P-independent targeting of nonphosphorylated lysosomal proteins (31), we have analyzed the expression of known and potential cargo receptors. No differences in the expression of Lrp1 protein have been observed in tritosome fractions, liver lysate or cultured hepatocytes between PT ki and wt controls (supplemental Fig.  S6A and S6B). Of note, there is an accumulation of Lrp1 fragments in PT ki tritosomes. In cultured hepatocytes the amount of the Ldlr was increased most likely because of increased transcriptional activation (supplemental Fig. S6B and S6C). The total amounts of Mpr300 in PT ki hepatocytes are not altered and corresponded with the relative mRNA expression level (supplemental Fig. S6B and S6C). However, the 1.8-fold higher amount of the internalized recombinant M6P-containing Mpr300 ligand, [ 125 I] arylsulfatase B (ARSB), indicated that the number of Mpr300 localized at the surface of PT ki hepatocytes is increased (supplemental Fig. S6D). DISCUSSION This study reports on the first quantitative analysis of the lysosomal proteome of mouse liver recovering almost all known soluble lysosomal enzymes and accessory proteins. These data were compared with the lysosomal proteome from mice lacking the lysosomal targeting signal mannose 6-phos-  5-6). The immunocomplexes were solubilized, separated by SDS-PAGE and visualized by fluorography. Secretion of newly synthesized Ctsd and Ctsz is increased in PT ki hepatocytes, accompanied by a significant reduction or complete loss of processed intracellular mature forms, respectively, during the chase. C, Isolated hepatocytes from wt and PT ki mice were fixed, and immunostained for localization of endogenous cathepsin D (Ctsd), cathepsin B (Ctsb) and neuraminidase 1 (Neu1) in red and the lysosomal marker protein Lamp1 (green). Nuclei were stained with DAPI (blue). Only merged images are shown in which yellow indicates colocalization of the antigens. Magnified images of the indicated white rectangles are shown in the insets. Scale bars: 7.5 m.
phate (M6P), and provided valuable insights into i) the efficiency of alternate transport systems in liver cells to prevent lysosomal dysfunctions, and ii) peripheral proteins attached transiently to the cytosolic surface of lysosomes playing an important role in lysosomal positioning, fusion, and signaling. Our approach also extended previous studies combining affinity purification of M6P-containing proteins with mass spectrometry of several tissues and lysosomal disease models (46 -48) which neither allow quantitative analyses of lysosomal proteomes nor its application to cells or tissues of mucolipidosis II patients or corresponding mouse models, respectively.
Furthermore, our approach allowed the quantification of steady-state concentrations of peripheral membrane proteins on the cytoplasmic side of lysosomes involved in vesicular targeting, positioning, and signaling. Among the numerous proteins playing a role in the vesicular transport machinery (supplemental Table S5), the adaptor protein AP3 complex subunits comprised the most abundant proteins (17.4 -31.7 nmoles/g lysosomal protein). In addition, five of the eight subunits of the BLOC-one-related complex (BORC) essential for lysosome positioning (49) have been identified in our preparations. Their expression ranges from 1.1 nmoles (Mef2bnb) to 6.2 nmoles/g lysosomal protein (myrlysin). In addition, rab7a and Arl8b, which have been recently reported to be involved in redistribution of lysosomes (50), belong to the more abundant peripheral proteins (11.7 and 24.2 nmoles/g lysosomal protein). Finally, we found all five Ragulator subunits LAMTOR 1 to 5 (Lysosomal Adaptor and Mitogen-activated protein kinase and mTOR activator/regulator) which interact with and recruit Rag GTPases to the lysosomal surface and mediate the translocation and activation of mTOR complex 1 (51). mTORC1 is a central component of fine tuned signaling pathways coupling internal and external stimuli (52).
The primary biochemical defect in ML II is the lack in GlcNAc-1-phosphotransferase (PT) activity that causes the inability to generate the M6P recognition marker for efficient targeting of soluble lysosomal enzymes. Therefore, fibroblasts and mesenchymal cells are deficient for a large number of lysosomal enzymes because of their hypersecretion (53). However, in certain liver cells of ML II patients such as hepa-tocytes or Kupffer cells, and in liver tissue of patients and PT ki mice nearly normal levels of few selected lysosomal enzymes were reported (17,19,20). In agreement with these observations, our quantitative analysis of the lysosomal proteome revealed that the concentration of only 5 out of 67 soluble proteins (Neu1, Ctsf, Npc2, Ctsl, and Smpd1) were more than 50% reduced in comparison with wild-type proteins (Table I) independent of transcriptional regulation (supplemental Fig.  S3A). The proteome data were validated by Western blotting analyses of 11 soluble lysosomal enzymes in tritosome fractions (Fig. 3) and by activity measurements of four additional lysosomal enzymes (Fig. 4A). The low amounts of neuraminidase 1 (Neu1, sialidase) might lead to increased excretion of sialic acid-containing oligosaccharides in the urine as described in ML II patients (54). The loss of neuraminidase 1 from lysosomes is, however, far more severe in fibroblasts, where no activity of neuraminidase 1 in fibroblasts of three unrelated ML II patients was found (55). This has been confirmed by SILAC-based lysosomal proteome analysis of PT ki mouse fibroblasts that found Ͻ5% neuraminidase 1 protein compared with wild-type controls (31). It is obvious, however, that the partial loss of a few select lysosomal enzymes is not limiting for lysosome functions as demonstrated by the absence of storage material in PT ki liver sections by electron microscopy (supplemental Fig. S7) that is also supported by our results in cultured hepatocytes ( Fig. 5 and supplemental Fig. S4) and liver sinusoid endothelial cells (supplemental Fig.  S5) prepared from wild-type and PT ki mice. The majority (49 of 67) of luminal (Table I), integral and peripheral lysosomal proteins were not or only moderately changed in their concentration in PT ki lysosomal fractions (supplemental Tables  S3 and S5).
The nearly normal composition of luminal proteins in liver lysosomes in the absence of M6P residues suggests that there are alternate M6P-independent targeting systems to lysosomes. Several M6P-independent receptors for the transport of lysosomal enzymes have been identified such as the LDL receptor-related protein 1 (Lrp1; (31,43,56,57)), Lrp2 (megalin;(58)), LIMP-2 (59), Sortilin (60 -62), SEZ6L2 (63), and mannose receptors (64). For the majority of these alternate M6P-independent receptors one or only a few lysosomal enzyme ligands have been tested or verified. Therefore it is unclear how many different receptor routes are used in liver cells of PT ki mice in vivo to allow the targeting of the ϳ60 lysosomal enzymes. At least in part, the acid hydrolases secreted by all cells of PT ki mice are cleared from the plasma by mannose receptors localized on macrophage-like Kupffer cells and hepatic endothelial cells (64) and exhibiting specificities for terminal mannose, GlcNAc and fucose residues (65). Additionally, the high number of LDL receptor and Lrp1 on the surface of hepatocytes most likely contribute to the re-internalization of missorted, nonphosphorylated lysosomal enzymes (31).
In summary, our lysosomal proteome analyses reveal striking protein-specific differences in the abundance of soluble and membrane-bound proteins as well as in peripheral proteins localized on the cytosolic side of mouse liver lysosomes. The lysosomal composition and the lack of storage material in liver unable to form the major lysosomal targeting signal, mannose 6-phosphate, suggests that alternate cargo receptor routes guarantee the transport of nonphosphorylated lysosomal enzymes in sufficient amounts to maintain almost normal functions of liver cells. These observations emphasize that the lysosomal proteome must be quantified systematically in various organs and cell types in order to further our understanding of the physiological role of individual and subsets of lysosomal enzymes in cellular homeostasis.