Proteomic Analysis of Lysosomal Acid Hydrolases Secreted by Osteoclasts
2005; Elsevier BV; Volume: 5; Issue: 1 Linguagem: Inglês
10.1074/mcp.m500291-mcp200
ISSN1535-9484
AutoresCornelia Czupalla, Hannu Mansukoski, Thilo Riedl, Dorothee Thiel, Eberhard Krause, Bernard Hoflack,
Tópico(s)Bone and Dental Protein Studies
ResumoOsteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-κB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna. Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-κB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna. Osteoclasts are the bone-resorbing cells involved in the tightly regulated process of bone remodeling. These multinucleated cells are formed by the fusion of mononuclear progenitors of monocyte/macrophage origin (1Boyle W.J. Simonet W.S. Lacey D.L. Osteoclast differentiation and activation.Nature. 2003; 423: 337-342Google Scholar). Proliferation of the precursor cells and their differentiation into mature osteoclasts are essentially driven by two hematopoietic factors, macrophage colony-stimulating factor and receptor activator of NF-κB ligand (RANKL), 1The abbreviations used are: RANKL, receptor activator of NF-κB ligand; Cap, capillary; Man-6-P, mannose 6-phosphate; MPR, mannose 6-phosphate receptor; TRAcP, tartrate-resistant acid phosphatase; Z, benzyloxycarbonyl. 1The abbreviations used are: RANKL, receptor activator of NF-κB ligand; Cap, capillary; Man-6-P, mannose 6-phosphate; MPR, mannose 6-phosphate receptor; TRAcP, tartrate-resistant acid phosphatase; Z, benzyloxycarbonyl. respectively (2Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Google Scholar). Activation of the corresponding receptors present at the cell surface of osteoclast precursors results in the regulated expression of a plethora of genes, including those encoding tartrate-resistant acid phosphatase (TRAcP), cathepsin K, and αvβ3 integrin that typify osteoclasts. Many of the corresponding gene products are required for reorganizing several key cellular functions such as cell adhesion, cytoskeleton dynamics, cell polarity, and membrane traffic along osteoclast differentiation. To digest bone, osteoclasts attach onto the bone surface, polarize, and create a specialized highly convoluted membrane domain, i.e. the ruffled border membrane that faces the bone surface thereby forming a sealed resorption lacuna (3Väänänen H.K. Zhao H. Mulari M. Halleen J.M. The cell biology of osteoclast function.J. Cell Sci. 2000; 113: 377-381Google Scholar). This resorption pit is acidified by vacuolar ATPases present on the ruffled border membrane leading to the breakdown of inorganic bone matrix mainly consisting of calcium hydroxyapatite (4Blair H.C. Teitelbaum S.L. Ghiselli R. Gluck S. Osteoclastic bone resorption by a polarized vacuolar proton pump.Science. 1989; 245: 855-857Google Scholar, 5Väänanen H.K. Karhukorpi E.K. Sundquist K. Wallmark B. Roininen I. Hentunen T. Tuukkanen J. Lakkakorpi P. Evidence for the presence of a proton pump of the vacuolar H+-ATPase type in the ruffled borders of osteoclasts.J. Cell Biol. 1990; 111: 1305-1311Google Scholar). In contrast, the digestion of the organic bone matrix, mostly made of collagens, proteoglycans, and other glycoproteins, requires the secretion of different acid hydrolases, in particular proteases, glycosidases, and sulfatases, most of them not identified yet.Acid hydrolases are mainly soluble glycoproteins that, after synthesis as preproproteins in the endoplasmic reticulum, acquire a mannose 6-phosphate (Man-6-P) sorting marker on their N-linked oligosaccharides. This marker is recognized by two types of mannose 6-phosphate receptors (MPRs) that sort the acid hydrolases within the trans-Golgi network, the last sorting station of the secretory pathway (6Ludwig T. Le Borgne R. Hoflack B. Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly.Trends Cell Biol. 1995; 5: 202-206Google Scholar). Resulting transport intermediates containing ligand-bound MPRs fuse with endosomes where the MPRs release their cargo. Acid hydrolases are then transported to and stored in lysosomes where they lose the Man-6-P marker and, in many cases, their prosequence to become mature, fully active enzymes. How acid hydrolases are secreted into the resorption lacuna of osteoclasts is not entirely clear yet. In early morphological studies a co-distribution of MPRs and some acid hydrolases along the secretory pathway from the site of biosynthesis to the ruffled border membrane has been shown, thus proposing a constitutive pathway for acid hydrolase secretion (7Baron R. Neff L. Brown W. Courtoy P.J. Louvard D. Farquhar M.G. Polarized secretion of lysosomal enzymes: co-distribution of cation-independent mannose-6-phosphate receptors and lysosomal enzymes along the osteoclast exocytic pathway.J. Cell Biol. 1988; 106: 1863-1872Google Scholar). Furthermore in more recent morphological studies lysosomal membrane glycoproteins, such as lgp110 and lamp2 as well as the vacuolar ATPase α3 subunit characteristic for late endosomes/lysosomes, were detected in the ruffled border membrane (8Baron R. Neff L. Louvard D. Courtoy P.J. Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.J. Cell Biol. 1985; 101: 2210-2222Google Scholar, 9Palokangas H. Mulari M. Väänänen H.K. Endocytic pathway from the basal plasma membrane to the ruffled border membrane in bone-resorbing osteoclasts.J. Cell Sci. 1997; 110: 1767-1780Google Scholar, 10Toyomura T. Murata Y. Yamamoto A. Oka T. Sun-Wada G.H. Wada Y. Futai M. From lysosomes to the plasma membrane: localization of vacuolar-type H+-ATPase with the a3 isoform during osteoclast differentiation.J. Biol. Chem. 2003; 278: 22023-22030Google Scholar). This led to the proposal that late endocytic compartments, namely late endosomes and lysosomes, can fuse with the membrane of the ruffled border thereby releasing their content of mature, fully active acid hydrolases into the resorption lacuna (11Mulari M.T.K. Zhao H. Lakkakorpi P.T. Väänänen H.K. Osteoclast ruffled border has distinct subdomains for secretion and degraded matrix uptake.Traffic. 2003; 4: 113-125Google Scholar).In the present study, we analyzed the secretion of acid hydrolases by osteoclasts in detail. We observed a strong increase in the secretion of different acid hydrolases, which still contained the Man-6-P targeting signal. Thus, we applied immobilized, cation-independent MPRs as highly specific affinity reagents for the purification of the complete set of Man-6-P-containing acid hydrolases and associated proteins secreted by osteoclasts. We present an exhaustive list of ≈60 proteins identified by MS, many of them being implicated in bone homeostasis. Our analysis further revealed that these Man-6-P-modified acid hydrolases are secreted as proforms thus suggesting a modification of MPR trafficking pathways during osteoclastogenesis.EXPERIMENTAL PROCEDURESCell Culture and Metabolic Labeling—Soluble recombinant RANKL was produced in Pichia yeast as described previously (12Czupalla C. Mansukoski H. Pursche T. Krause E. Hoflack B. Comparative study of protein and mRNA expression during osteoclastogenesis.Proteomics. 2005; 5: 3868-3875Google Scholar). Raw 264.7 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS (Hyclone Laboratories, Perbio Science, Erembodegem-Aalst, Belgium) and antibiotics. In vitro osteoclastogenesis was induced by addition of RANKL as described previously (12Czupalla C. Mansukoski H. Pursche T. Krause E. Hoflack B. Comparative study of protein and mRNA expression during osteoclastogenesis.Proteomics. 2005; 5: 3868-3875Google Scholar). Prior to collection of conditioned medium, cells were serum-starved for 6 h or overnight. For metabolic labeling, cells were incubated for 2 h with methionine-free Dulbecco's modified Eagle's medium containing 1 mCi/ml [35S]methionine, 10 mm Hepes, pH 7.0, and 10% dialyzed FCS followed by a 3-h chase in the presence of 10 mm Man-6-P.Immunoprecipitation of Cathepsin D—Conditioned medium from [35S]methionine-labeled cells was diluted in lysis buffer (PBS supplemented with 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and complete protease inhibitors (Roche Diagnostics)). Cells were washed twice with PBS, homogenized in lysis buffer, and centrifuged at 13,000 rpm for 15 min. Cell extracts and conditioned media were precleared with Protein A-Sepharose CL-4B (Amersham Biosciences), and cathepsin D was immunoprecipitated with a polyclonal antibody (13Ludwig T. Griffiths G. Hoflack B. Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.J. Cell Biol. 1991; 115: 1561-1572Google Scholar). Immunocomplexes were collected with Protein A-Sepharose and analyzed by SDS-PAGE followed by autoradiography.Measurement of Enzyme Activities—For measurement of glycosidase activities, conditioned media were incubated for 3 h at 37 °C in assay buffer I (100 mm sodium citrate, pH 4.6, 0.2% Triton X-100) supplemented with a 1 mm concentration of the appropriate 4-methylumbelliferyl substrates. Reactions were stopped by addition of 0.5 m Na2CO3. Fluorescent reaction products were measured using a fluorescence multiwell plate reader (Tecan, Grödig, Austria) with excitation at 360 nm and emission at 440 nm. Cathepsin K activity was measured using (Z-Leu-Arg)2-Rh110 bisamide (Calbiochem) in assay buffer II (100 mm sodium acetate, pH 5.5, 1 mm EDTA, 0.1 mm DTT). Reactions were terminated after 20 h at 37 °C by addition of 100 mm Tris, pH 8.0, 100 mm sodium iodoacetate, and fluorescence was measured at 485/530 nm. TRAcP activity was determined after dilution of conditioned media in HBSS buffer (118 mm NaCl, 4.6 mm KCl, 1 mm CaCl2, 10 mm glucose, 20 mm Hepes, pH 7.2). Reactions were started by addition of assay buffer III (100 mm sodium acetate, pH 4.6, 40 mm sodium tartrate, 20 mmp-nitrophenyl phosphate) and stopped after 30 min at 37 °C by addition of 0.1 m NaOH. Colored reaction products were measured at 405 nm. Protein concentrations of corresponding cell extracts were determined using the DC protein assay (Bio-Rad), and enzymatic activities were normalized to the protein amount of the cells secreting these enzymes.Purification of Man-6-P-containing Enzymes—Soluble bovine cation-independent MPR was purified from FCS by affinity chromatography on immobilized phosphomannan and coupled to Affi-Gel 15 (Bio-Rad) as described previously (14Hoflack B. Kornfeld S. Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver.J. Biol. Chem. 1985; 260: 12008-12014Google Scholar). Conditioned media were diluted in column buffer (50 mm imidazole, pH 6.5, 150 mm NaCl, 5 mm sodium β-glycerophosphate, 2 mm EDTA, 0.05% Triton X-100) and applied to an MPR Affi-Gel affinity column. The column was washed with 5 volumes of column buffer followed by 3 volumes of 5 mm glucose 6-phosphate in column buffer and 5 volumes of column buffer. Bound proteins were eluted with 5 volumes of 5 mm Man-6-P in column buffer and analyzed for their enzymatic activity or by SDS-PAGE.Protein Identification by Mass Spectrometry—Proteins were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Protein bands were excised, washed, in-gel reduced, S-alkylated, and in-gel digested with trypsin (Promega, Madison, WI) as described previously (15Czupalla C. Nürnberg B. Krause E. Analysis of class I phosphoinositide 3-kinase autophosphorylation sites by mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 17: 690-696Google Scholar). Peptides were extracted by addition of 0.3% trifluoroacetic acid in acetonitrile, the separated supernatant was dried under vacuum, and samples were redissolved in 0.1% (v/v) trifluoroacetic acid in water. MALDI-MS measurements were performed using an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in reflectron mode using α-cyano-4-hydroxycinnamic acid as matrix. All peptide mass fingerprint spectra were internally calibrated with trypsin autolysis peaks. MALDI-TOF/TOF fragment ion analysis was carried out in the LIFT mode of the instrument. Spectra were processed using FlexAnalysis software. Protein identification, both by peptide mass fingerprinting and fragment ion analysis, was performed using MASCOT (Matrix Science, London, UK). Search criteria were as follows: taxonomy, mouse; mass accuracy, 50 ppm for peptide mass fingerprinting and 0.5 Da for fragment analysis; modifications, carbamidomethylation and methionine oxidation; maximum one missed cleavage site. The National Center for Biotechnology Information (NCBI) non-redundant protein database (version 20041117; 2,171,938 sequences) and Swiss-Prot (version 45.5, 215,444 sequences) were searched. CapLC-MS/MS experiments were performed on the quadrupole orthogonal acceleration time-of-flight mass spectrometer Q-TOF Ultima (Micromass, Manchester, UK) equipped with a Z-spray nanoelectrospray source. A Micromass CapLC liquid chromatography system was used to deliver the peptide solution to the electrospray source. Peptides were separated using an analytical column (PepMap C18, 3 μm, 100 Å, 150 mm % 75-μm inner diameter; LC Packings, Sunnyvale, CA) and an eluent flow rate of 200 nl/min. Mobile phase A was 0.1% formic acid (v/v) in acetonitrile-water (5:95, v/v), and B was 0.1% formic acid in acetonitrile-water (8:2, v/v). Runs were performed using a gradient of 3–64% B in 60 min. To perform MS/MS experiments automatic function switching (survey scanning) was used. The MS survey range was m/z 300–1990, and the scan duration was 1.0 s. The collision gas was argon at a pressure of 6.0 % 10−5 millibar. The MS/MS ion search option of the MASCOT program (www.matrixscience.com) was used to search against the NCBI non-redundant protein database and Swiss-Prot. The mass tolerance of precursor and sequence ions was set to 0.1 and 0.2 Da, respectively (other search criteria were as above).Immunofluorescence and Confocal Laser Scanning Microscopy—Purified MPRs were fluorescently labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Raw 264.7 cells and osteoclasts grown on glass were fixed with 3% paraformaldehyde in PBS for 10 min and permeabilized in 0.05% saponin in PBS for 15 min. Samples were incubated with fluorescently labeled MPR fragments and primary anti-cathepsin D antibody in 1% bovine serum albumin in PBS for 1 h followed by incubation with Texas Red-conjugated goat anti-rabbit secondary antibody (Molecular Probes). Samples were viewed with a Zeiss LSM 510 Meta confocal laser scanning microscope (Zeiss, Jena, Germany).DNA Microarray and Real Time PCR—mRNA isolation, cDNA synthesis, and Affymetrix expression analysis were done as described previously (12Czupalla C. Mansukoski H. Pursche T. Krause E. Hoflack B. Comparative study of protein and mRNA expression during osteoclastogenesis.Proteomics. 2005; 5: 3868-3875Google Scholar). Real time PCR was performed with a Stratagene Mx4000 QPCR system and Brilliant SYBR Green QPCR kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions using the following primers: cation-dependent MPR, 5′-GGAATGGAGCAGTTTCCTCA-3′ and 5′-GGCAGGTTAGGGTCAAATCA-3′; cation-independent MPR, 5′-GCTGCTGCAGAAGAAGCTC-3′ and 5′-GTGATATGGCCATTTTCTTGC-3′; TRAcP, 5′-TTGTCAAGAACTTGCGACCA-3′ and 5′-TAGCGGACAAGCAGGACTCT-3′; cathepsin K, 5′-TGATGAAAATTGTGACCGTGA-3′ and 5′-CCTTCCAAAGCCACCAATATC-3′; cathepsin D, 5′-CCTGAAGCTAGGAGGCAAAA-3′ and 5′-AGGGTCCAGCAACACTAAGC-3′; legumain, 5′-TATGTGCTGGCCAATCTCTG-3′ and 5′-CCACCCAAACTGGCTTCTTA-3′; and β-glucuronidase, 5′-TGAATGGGATTCATGTGGTG-3′ and 5′-TGCTTGAAGCCTTTTTCTCC-3′.RESULTSOsteoclasts Secrete Acid Hydrolases Still Containing Mannose 6-Phosphate—As a model system of osteoclastogenesis, we have used the mouse myeloid cell line Raw 264.7. Treatment of Raw 264.7 cells with RANKL for 4 days results in cultures mainly consisting of multinucleated osteoclasts (12Czupalla C. Mansukoski H. Pursche T. Krause E. Hoflack B. Comparative study of protein and mRNA expression during osteoclastogenesis.Proteomics. 2005; 5: 3868-3875Google Scholar, 16Matsumoto M. Sudo T. Saito T. Osada H. Tsujimoto M. Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-κB ligand (RANKL).J. Biol. Chem. 2000; 275: 31155-31161Google Scholar). We first followed the secretion of enzymatic activities of known acid hydrolases such as β-galactosidase and β-hexosaminidase as well as of the well characterized osteoclastogenesis markers cathepsin K and TRAcP (Fig. 1A). As expected, no secretion of cathepsin K and TRAcP was observed in precursor Raw 264.7 cells or during the early phase of differentiation. In contrast, secretion of both markers dramatically increased during later phases of differentiation when multinucleated osteoclasts were mostly present in the culture. Similarly we observed an increased secretion of β-galactosidase and β-hexosaminidase during osteoclastogenesis, reaching a maximum after 4 days of RANKL treatment. Osteoclasts secreted ≈10-fold more of both glycosidases than non-induced Raw 264.7 cells. Secretion of other acid hydrolases tested, such as α- and β-glucosidase, β-glucuronidase, and β-mannosidase, was also increased in osteoclasts (Fig. 1B).Once in lysosomes, acid hydrolases lose the Man-6-P marker (17Neufeld E.F. Lysosomal storage diseases.Annu. Rev. Biochem. 1991; 60: 257-280Google Scholar). Because efficient processing of acid hydrolases cannot be expected in the resorption lacunae of osteoclasts grown on plastic, retention of the Man-6-P targeting signal on acid hydrolases might indicate that these enzymes do not reach lysosomes before secretion. To investigate this, the acid hydrolases secreted into culture media of osteoclasts were tested for their capability to interact with purified MPRs immobilized on an affinity gel matrix. Fig. 2 shows that ≈80% of active cathepsin K interacted with immobilized MPRs, whereas ≈40–55% of the secreted β-galactosidase, β-glucuronidase, β-hexosaminidase, and β-mannosidase bound to the receptor. Thus, a significant fraction of these secreted enzymes still contains the Man-6-P marker as a typical hallmark of newly synthesized acid hydrolases.Fig. 2Osteoclasts secrete Man-6-P-modified acid hydrolases. Raw 264.7 cells (1.3 % 104/cm2) were treated with RANKL for 5 days to generate osteoclasts. Conditioned media collected from osteoclasts grown for 6 h in serum-free medium were passed over an MPR affinity column. The following enzymatic activities of the conditioned media as well as of the bound material eluted with 5 mm Man-6-P were determined: β-galactosidase (β-gal), β-glucuronidase (β-gluc), β-hexosaminidase (β-hex), β-mannosidase (β-man), and cathepsin K (ctsK). Shown are results from one representative experiment of three performed in duplicate.View Large Image Figure ViewerDownload (PPT)Identification of Acid Hydrolases Secreted by Osteoclasts—Our results showing that several acid hydrolases are secreted as Man-6-P-containing proteins prompted us to identify the complete set of Man-6-P-modified proteins secreted by osteoclasts. Proteins were purified by their affinity to immobilized MPRs and further fractionated by SDS-PAGE as shown in Fig. 3. First the major Coomassie-stained bands were analyzed by peptide mass fingerprinting, and identified proteins were confirmed by MALDI-TOF/TOF peptide sequencing. This led to the identification of 14 proteins (see Fig. 3 and Table I). The major secreted Man-6-P-containing proteins are peptidases such as cathepsins A, B, D, S, and Z; legumain (asparaginyl endopeptidase); and tripeptidyl-peptidase I. Moreover glycosidases such as α-mannosidase, α-N-acetylglucosaminidase, β-hexosaminidase, and β-glucuronidase, enzymes involved in lipid metabolism, i.e. palmitoyl-protein thioesterase 1 and the epididymal secretory protein E1 (Niemann Pick C2 protein homolog), and another lysosomal protein, interferon γ-inducible protein 30 were found. To identify additional, less abundant Man-6-P-containing proteins, the gel was cut into 50 slices of equal size, and each of them was subjected to protein identification by tryptic in-gel digestion followed by CapLC-ESI tandem mass spectrometry. Analysis of 11,191 MS/MS spectra revealed a total number of 58 different proteins belonging to several families of acid hydrolases including the known osteoclast markers cathepsin K and TRAcP (see Table I). This includes 15 peptidases, 16 glycosidases, five sulfatases, 10 enzymes involved in lipid metabolism, and 12 others. Among those, 33 proteins have been described previously as lysosomal acid hydrolases carrying the typical Man-6-P recognition marker such as cathepsins A, B, and D or α- and β-mannosidase (18Sleat D.E. Lackland H. Wang Y. Sohar I. Xiao G. Li H. Lobel P. The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.Proteomics. 2005; 5: 1520-1532Google Scholar). Our analysis also revealed lysosomal enzymes such as dipeptidyl-peptidase II, angiotensinase C-like protein, γ-glutamyl hydrolase, cathepsins F and Z, legumain, di-N-acetylchitobiase, and lysosomal phospholipase 2A for which a Man-6-P modification has not been described yet, indicating that they might be transported to lysosomes by a Man-6-P-dependent pathway in normal cells. Moreover we identified a third group of proteins that have not been classified as lysosomal acid hydrolases so far, namely sphingomyelinase-like phosphodiesterase 3a, interleukin-4-induced protein 1, and dentin matrix protein 4. It is interesting to note that β-glucosidase (β-glucocerebrosidase) and lipoprotein lipase, which do not contain Man-6-P residues, were also detected, probably reflecting their interaction with other Man-6-P-containing ligands. This probably holds true for four other proteins that are clearly not lysosomal enzymes, i.e. lysozyme C and three glycosyltransferases (N-acetylgalactosaminyltransferase 6, β-1,4-galactosyltransferase 5, and CMP-N-acetyl-poly-α-2,8-sialyltransferase), which were counted as possible contaminants. These glycosyltransferases are type II transmembrane proteins, which could be released as a soluble form after proteolytic cleavage.Fig. 3Protein profile of Man-6-P-containing proteins secreted by osteoclasts. Raw 264.7 cells (1.0 % 107) were differentiated into osteoclasts in the presence of RANKL during 5 days and then grown overnight in serum-free medium. Conditioned medium was collected, and Man-6-P-containing proteins were purified on an MPR affinity column and separated by SDS-PAGE. Coomassie-stained bands were excised and analyzed by MALDI-TOF/TOF mass spectrometry. Molecular mass markers are indicated.View Large Image Figure ViewerDownload (PPT)Table IProteins secreted by osteoclasts captured on an MPR affinity columnProteinNCBI gene identifierPredicted molecular massNo. of sequenced peptidesMan-6-PPropeptide sequence (position, MS/MS score)Change in mRNA expressionaAverage -fold change ratio in Raw 264.7 cells vs. osteoclasts (p < 0.05).Bone phenotype (syndrome)DaPeptidases Dipeptidyl-peptidase I302345452,34310bPeptide sequencing by LC-ESI-Q-TOF.Yes149VNMNAAHLGGLQER162 (89), 167LYTHNHNFVK176 (42), 184SWTATAYK191 (35), 184SWTATAYKEYEK195 (35)bPeptide sequencing by LC-ESI-Q-TOF.−2.7Yes (Papillon-Lefevre, Haim-Munk) Dipeptidyl-peptidase II1362639056,2349bPeptide sequencing by LC-ESI-Q-TOF.1.3 Tripeptidyl-peptidase I1264408561,30412bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.Yes183QRPEPQQVGTVSLHLGVT-PSVLR205 (44)cPeptide sequencing by MALDI-TOF/TOF.1.3 Retinoid-inducible serine carboxypeptidase4847458650,93413bPeptide sequencing by LC-ESI-Q-TOF.−1.9 Angiotensinase C-like3346901555,7641bPeptide sequencing by LC-ESI-Q-TOF., dPrecursor ion, 537.27[M + 2H]2+; sequence, K113AMLVFAEHR121Y; score, 72. Cathepsin A13108253,80913bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.YesYes (Goldberg) γ-Glutamyl hydrolase1312425635,4159bPeptide sequencing by LC-ESI-Q-TOF.−2.3 Cathepsin Z1258520933,97410bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.31SGQTCYHPIRGDQLALL-GR49 (45), 51TYPRPHEYLSPADLP-K66 (45)cPeptide sequencing by MALDI-TOF/TOF. Cathepsin B11571237,25611bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.Yes72VAFGEDIDLPETFDAR87 (108)cPeptide sequencing by MALDI-TOF/TOF.−1.8 Cathepsin L11574237,52312bPeptide sequencing by LC-ESI-Q-TOF.Yes21FDQTFSAEWHQWK33 (81), 38RLYGTNEEEWR48 (32)cPeptide sequencing by MALDI-TOF/TOF.−1.8Yes Cathepsin S1264331838,4136bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.Yes29DPTLDYHWDLWK40 (48), 29DPTLDYHWDLWKK41 (54), 49DKNEEEVR56 (49)bPeptide sequencing by LC-ESI-Q-TOF.−2.2 Legumain2161782149,34112bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.324TNDVKESQNLIGQIQQFLD-AR344 (80), 329ESQNLIGQIQQFLDAR344 (51), 354IVSLLAGFGETAER367 (59)cPeptide sequencing by MALDI-TOF/TOF.−3.8 Cathepsin K1264432036,8658bPeptide sequencing by LC-ESI-Q-TOF.Yes36QYNSKVDEISR46 (30)bPeptide sequencing by LC-ESI-Q-TOF.8.4Yes Cathepsin F1264332151,6286bPeptide sequencing by LC-ESI-Q-TOF.47FALDMYNYGR56 (64), 244SINDLAPPEWDWR256 (72)bPeptide sequencing by LC-ESI-Q-TOF.1.6 Cathepsin D11571844,92512bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.Yes35TMTEVGGSVEDLILKGPITK54 (52)cPeptide sequencing by MALDI-TOF/TOF.−1.4Glycosidases Di-N-acetylchitobiase2722920441,5042bPeptide sequencing by LC-ESI-Q-TOF.−1.6 Sialidase 11736796744,5635bPeptide sequencing by LC-ESI-Q-TOF.YesYes Lysosomal α-glucosidase51338793106,18010bPeptide sequencing by LC-ESI-Q-TOF.Yes α-Galactosidase A170321047,6111bPeptide sequencing by LC-ESI-Q-TOF., ePrecursor ion, 545.30[M + 2H]2+; sequence, R407VNPSGTVLFR416L; score, 51.Yes β-Galactosidase11494473,07414bPeptide sequencing by LC-ESI-Q-TOF.Yes3.2Yes (Morquio) Lysosomal α-mannosidase17380364114,53234bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.Yes−1.6Yes Epididymis-specific α-mannosidase17367999115,55131bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.−1.8Yes β-Mannosidase13310141101,3202bPeptide sequencing by LC-ESI-Q-TOF.Yes1.6Yes β-Glucuronidase11496474,19223bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.YesYes β-Glucosidase12128457,5852bPeptide sequencing by LC-ESI-Q-TOF.NoYes (Gaucher) α-N-Acetylglucosaminidase730529982,1154bPeptide sequencing by LC-ESI-Q-TOF.Yes−1.3Yes (Sanfilippo) Tissue α-l-fucosidase3154178153,4522bPeptide sequencing by LC-ESI-Q-TOF.Yes−1.5 β-Hexosaminidase α chain23225560,56016bPeptide sequencing by LC-ESI-Q-TOF.Yes37YTLYPNNFQFR47 (47)cPeptide sequencing by MALDI-TOF/TOF. β-Hexosaminidase β chain134628061,07720bPeptide sequencing by LC-ESI-Q-TOF., cPeptide sequencing by MALDI-TOF/TOF.Yes31LQPALWPFPR40 (47)cPeptide sequencing by MALDI-TOF/TOF.1.8 α-l-Iduronidase135242471,13511bPeptide sequencing by LC-ESI-Q-TOF.YesYes (Hurler) N4-(β-N-Acetylglucosaminyl)-l-asparaginase249816336,9985bPeptide sequencing by LC-ESI-Q-TOF.YesSulfatases Galactosamine (N-acetyl)-6-su
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