“Subcellular Proteomics” of Neuromelanin Granules Isolated from the Human Brain
2005; Elsevier BV; Volume: 4; Issue: 7 Linguagem: Inglês
10.1074/mcp.m400117-mcp200
ISSN1535-9484
AutoresFlorian Tribl, Manfred Gerlach, Katrin Marcus, Esther Asan, Thomas Tatschner, Thomas Arzberger, Helmut E. Meyer, Gerhard Bringmann, Peter Riederer,
Tópico(s)Connexins and lens biology
Resumo"Subcellular proteomics" is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate's substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosome-related organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone. "Subcellular proteomics" is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate's substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosome-related organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone. Melanins are widely distributed throughout the plant and animal kingdoms. In humans, these heterogeneous, complex polymer pigments occur naturally in the hair, the skin, the inner ear, the iris, and the choroid of the eye (1Prota G. Melanins and Melanogenesis. Academic Press Inc., San Diego, CA1992Google Scholar). Melanin in the brain has an appearance and structure similar to cutaneous melanins and has thus been named neuromelanin (NM) 1The abbreviations used are: NM, neuromelanin; SN, substantia nigra pars compacta; 1-D, one-dimensional; LAMP, lysosome-associated membrane glycoprotein; GNA, Galanthus nivalis agglutinin; LIMP, lysosome membrane protein; ER, endoplasmic reticulum; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; EEA1, early endosomal antigen 1; SNAP, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; AP, adaptor-related protein complex; BiP, polypeptide-binding protein. 1The abbreviations used are: NM, neuromelanin; SN, substantia nigra pars compacta; 1-D, one-dimensional; LAMP, lysosome-associated membrane glycoprotein; GNA, Galanthus nivalis agglutinin; LIMP, lysosome membrane protein; ER, endoplasmic reticulum; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; EEA1, early endosomal antigen 1; SNAP, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; AP, adaptor-related protein complex; BiP, polypeptide-binding protein. (2Lillie R.D. Metal reduction reactions of the melanins: histochemical studies.J. Histochem. Cytochem. 1957; 5: 325-333Google Scholar). NM is found inter alia in dopaminergic neurons of a small area in the human midbrain important for the control of movement that is known as the substantia nigra pars compacta (SN; from the Latin meaning "black body"). The loss of this dark pigment and the resulting pallor of the SN is one of the most striking features of the common movement disorder Parkinson disease. A relationship between the loss of the dopaminergic SN cells and their NM content (3Hirsch E. Graybiel A.M. Agid Y.A. Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson's disease.Nature. 1988; 334: 345-348Google Scholar), a specific affinity to iron (4Jellinger K. Kienzl E. Rumpelmair G. Riederer P. Stachelberger H. Ben-Shachar D. Youdim M.B. Iron-melanin complex in substantia nigra of parkinsonian brains: an x-ray microanalysis.J. Neurochem. 1992; 59: 1168-1171Google Scholar), and a significant binding of α-synuclein to NM in the diseased state (5Fasano M. Giraudo S. Coha S. Bergamasco B. Lopiano L. Residual substantia nigra neuromelanin in Parkinson's disease is cross-linked to α-synuclein.Neurochem. Int. 2003; 42: 603-606Google Scholar) suggest a functional role for NM in neurodegeneration in Parkinson disease (6Double K.L. Ben-Shachar D. Youdim M.B. Zecca L. Riederer P. Gerlach M. Influence of neuromelanin on oxidative pathways within the human substantia nigra.Neurotoxicol. Teratol. 2002; 24: 621-628Google Scholar).Although much is known about the peripheral melanins to which NM is thought to be related, many basic questions remain to be answered about NM in the brain. Thus it is unclear why only some human dopamine neurons produce NM within their cytoplasm (7McRitchie D.A. Halliday G.M. Cartwright H. Quantitative analysis of the variability of substantia nigra pigmented cell clusters in the human.Neuroscience. 1995; 68: 539-551Google Scholar). Little is also known about the structure of NM, and the understanding of its genesis and function within the cell remains speculative.Nuclear magnetic resonance spectroscopic studies have shown that NM resembles synthetic cysteinyldopamine melanin more closely than the more simple dopamine melanin; however, human NM appears to be a structurally more complex chemical structure than any of the synthetic models (8Double K.L. Zecca L. Costi P. Mauer M. Griesinger C. Ito S. Ben-Shachar D. Bringmann G. Fariello R.G. Riederer P. Gerlach M. Structural characteristics of human substantia nigra neuromelanin and synthetic dopamine melanins.J. Neurochem. 2000; 75: 2583-2589Google Scholar). In addition to the melanin backbone, nuclear magnetic resonance spectroscopic studies have demonstrated that cholesterol and other uncharacterized high molecular mass lipid components are closely associated with NM (8Double K.L. Zecca L. Costi P. Mauer M. Griesinger C. Ito S. Ben-Shachar D. Bringmann G. Fariello R.G. Riederer P. Gerlach M. Structural characteristics of human substantia nigra neuromelanin and synthetic dopamine melanins.J. Neurochem. 2000; 75: 2583-2589Google Scholar, 9Dzierzega-Lecznar A. Kurkiewicz S. Stepien K. Chodurek E. Wilczok T. Arzberger T. Riederer P. Gerlach M. GC/MS analysis of thermally degraded neuromelanin from the human substantia nigra.J. Am. Soc. Mass Spectrom. 2004; 15: 920-926Google Scholar, 10Zecca L. Costi P. Mecacci C. Ito S. Terreni M. Sonnino S. Interaction of human substantia nigra neuromelanin with lipids and peptides.J. Neurochem. 2000; 74: 1758-1765Google Scholar). Dolichol was identified as the major lipid component of NM (11Fedorow H. Pickford R. Hook J.M. Double K.L. Halliday G.M. Gerlach M. Riederer P. Garner B. Dolichol is the major lipid component of human substantia nigra neuromelanin.J. Neurochem. 2005; 92: 990-995Google Scholar). A proteinaceous component making up ∼5–15% of the isolated molecule is also present that has been suggested to represent an integral component of the polymer (8Double K.L. Zecca L. Costi P. Mauer M. Griesinger C. Ito S. Ben-Shachar D. Bringmann G. Fariello R.G. Riederer P. Gerlach M. Structural characteristics of human substantia nigra neuromelanin and synthetic dopamine melanins.J. Neurochem. 2000; 75: 2583-2589Google Scholar, 10Zecca L. Costi P. Mecacci C. Ito S. Terreni M. Sonnino S. Interaction of human substantia nigra neuromelanin with lipids and peptides.J. Neurochem. 2000; 74: 1758-1765Google Scholar).A general understanding of neuromelanogenesis could be provided by investigation of the synthetic pathway of peripheral melanins and comparison with what is known about NM. Genetic and enzymatic regulation of melanin production in the periphery has been primarily characterized by the study of fur pigmentation in the mouse. Similar experiments, however, cannot be used to elucidate the pathway of NM synthesis as NM does not occur in rodents. It has long been debated whether the NM synthesis is enzymatically controlled, like all melanins in the periphery, or whether NM arises from a simple autoxidation process (for reviews, see Refs. 12Fedorow H. Tribl F. Halliday G. Gerlach M. Riederer P. Double K.L. Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson's disease.Prog. Neurobiol. 2005; 75: 109-124Google Scholar and 13Zecca L. Tampellini D. Gerlach M. Riederer P. Fariello R.G. Sulzer D. Substantia nigra neuromelanin: structure, synthesis, and molecular behaviour.Mol. Pathol. 2001; 54: 414-418Google Scholar). In the apparent absence of a role for tyrosinase in neuromelanogenesis, the search for an enzyme associated with NM production has yielded no likely candidates to date (12Fedorow H. Tribl F. Halliday G. Gerlach M. Riederer P. Double K.L. Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson's disease.Prog. Neurobiol. 2005; 75: 109-124Google Scholar, 13Zecca L. Tampellini D. Gerlach M. Riederer P. Fariello R.G. Sulzer D. Substantia nigra neuromelanin: structure, synthesis, and molecular behaviour.Mol. Pathol. 2001; 54: 414-418Google Scholar). It is noteworthy that Parkinson disease patients treated with large quantities of the dopamine precursor l-3,4-dihydroxyphenylalanine (l-DOPA, levodopa) do not exhibit increased quantities of NM within their surviving SN neurons as might be expected to be the case if NM represents a product of pure dopamine autoxidation. The time course of NM appearance also supports the hypothesis that NM synthesis is a regulated process. In the human, NM is not present in functional dopaminergic neurons at birth but first appears at around 3–5 years of age.Although containing a melanin, neuronal NM granules are organelles different from melanosomes, which are mainly localized in melanocytes. In contrast to NM, skin- and hair-based melanins are contained within discrete regularly sized membrane-bound organelles called melanosomes, which differ structurally depending on which type of melanin they contain. It has been suggested that melanosomes are closely related to lysosomes (14Basrur V. Yang F. Kushimoto T. Higashimoto Y. Yasumoto K. Valencia J. Muller J. Vieira W.D. Watabe H. Shabanowitz J. Hearing V.J. Hunt D.F. Appella E. Proteomic analysis of early melanosomes: identification of novel melanosomal proteins.J. Proteome Res. 2003; 2: 69-79Google Scholar). In contrast, NM is bordered indistinctly, and the NM granules, as they are called, exhibit a wider size range than that of melanosomes (0.5- 2.5 μm) (15Duffy P.E. Tennyson V.M. Phase and electron microscopic observations of Lewy bodies and melanin granules in the substantia nigra and locus caeruleus in Parkinson's disease.J. Neuropathol. Exp. Neurol. 1965; 24: 398-414Google Scholar, 16Moses H.L. Ganote C.E. Beaver D.L. Schuffman S.S. Light and electron microscopic studies of pigment in human and rhesus monkey substantia nigra and locus coeruleus.Anat. Rec. 1966; 155: 167-183Google Scholar). The structurally segregated lipid component of NM that is not found in peripheral melanin pigments has been suggested to originate from the lipid-containing pigment lipofuscin, which also accumulates intracellularly with age (17Barden H. The histochemical relationship of neuromelanin and lipofuscin.J. Neuropathol. Exp. Neurol. 1969; 28: 419-441Google Scholar).Here we report a method to isolate intact, highly pure NM granules from human SN for subcellular proteomics. Proteomics is the large scale study of gene expression at the protein level that ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins (18Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Google Scholar, 19Binz P.A. Hochstrasser D.F. Appel R.D. Mass spectrometry-based proteomics: current status and potential use in clinical chemistry.Clin. Chem. Lab. Med. 2003; 41: 1540-1551Google Scholar, 20Jung E. Heller M. Sanchez J.C. Hochstrasser D.F. Proteomics meets cell biology: the establishment of subcellular proteomes.Electrophoresis. 2000; 21: 3369-3377Google Scholar). Subcellular proteomics thus is a powerful approach to gain new insight into functional properties of isolated organelles. We applied one-dimensional (1-D) SDS-PAGE, tryptic in-gel digestion, and nano-LC separation followed by ESI-MS/MS for protein analysis. Following this approach, we identified numerous proteins specific for organelles originating from the endosome-lysosome lineage.EXPERIMENTAL PROCEDURESIsolation of NM Granules Using Subcellular Fractionation—We used a sequential top-down approach that simultaneously allows the reduction of the complexity of the sample and the enrichment of the target structures at each isolation stage. Fig. 1 schematically summarizes the approach chosen. Brains were provided from the Austro-German Brain Bank in Würzburg. The use of postmortem human brain tissue was approved by the Ethics Committee of the University Clinics of Würzburg. The SN was dissected from postmortem brains of subjects with no history of neurological or neurodegenerative diseases within 36 h of death on a cool plate (−15 °C). 1.0 g of frozen SN tissue was thawed in "Separation Buffer" (10 mm HEPES, 10% glucose, pH 6) on ice and carefully passed through a polypropylene mesh into a Petri dish. The whole procedure was performed on a plate cooler set at 4 °C. The resulting cell suspension was layered on top of a discontinuous sucrose gradient (1, 1.2, 1.4, and 1.6 m) and separated by centrifugation at 4000 × g at 4 °C for 15 min. The pelleted dark cell bodies were recovered and washed with "Isolation Buffer" (10 mm HEPES, 1 mm EDTA, 100 mm KCl, 10% sucrose, pH 7.5) containing a protease inhibitor mixture (0.01% (v/v), Sigma). Subsequently the cell disruption was carried out by 10 passages through a 26-gauge needle to yield a suspension of cellular organelles that was layered on top of an 80% (v/v) Percoll cushion (Fluka, Buchs, Switzerland) and centrifuged at 4000 × g at 4 °C for 10 min. The pelleted dark granules were washed once with Isolation Buffer and twice with "Washing Buffer" (10 mm HEPES, 250 mm NaCl, 0.01% (v/v) Triton X-100, pH 7.5) to remove unspecifically associated proteins. The isolated NM granules were stored at −80 °C until analyzed.Transmission Electron Microscopy—The quality of the granule isolation and the preservation of the ultrastructural features were monitored by transmission electron microscopy. The aspect of cell homogenates and isolated NM granules were compared as a control to monitor the enrichment of the granules. The samples were fixed overnight in 2% (v/v) glutardialdehyde in 0.1 m phosphate-buffered saline, pH 7.4, at 4 °C and incubated in 2% OsO4, 1,5% (v/v) glutardialdehyde followed by dehydration with increasing concentrations of ethanol. After incubation in 1,2-epoxypropane (Sigma) (2 × 15 min) the samples were embedded in EPON™ epoxy resin (Sigma). Following polymerization at 65 °C (48 h) thin sections were prepared that were contrasted with lead citrate and uranyl acetate (21Reynolds E.S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.J. Cell Biol. 1963; 17: 208-212Google Scholar) before being monitored under the transmission electron microscope (LEO 912 AB, LEO Elektronenmikroskopie, Oberkochen, Germany).Sample Preparation for 1-D SDS-PAGE—Proteins of isolated NM granules were extracted with 16 mm 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate and SDS, mixed with reducing sample buffer containing dl-dithiothreitol, and heated for 10 min at 95 °C.1-D SDS-PAGE—The protein samples were separated electrophoretically on 10–20% Tricine gels (Novex, San Diego, CA) in an XCell II™ Mini-Cell (Invitrogen) using Tricine-SDS running buffer. Following electrophoresis the gel was either stained with colloidal Coomassie Brilliant Blue G-250 (22Neuhoff V. Arold N. Taube D. Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.Electrophoresis. 1988; 9: 255-262Google Scholar) or further processed for Western blotting.Antibodies and Materials—For Western blot analysis anti-human monoclonal antibodies to cis-Golgi matrix protein (GM130), induced myeloid leukemia cell differentiation protein (Mcl-1), early endosomal antigen 1 (EEA1), integrin α2 (VLA-2α), nucleoporin p62, lysosome-associated membrane glycoprotein 1 (LAMP-1), clathrin, and 78-kDa glucose-regulated protein (BiP/grp78) were used (BD Biosciences). Anti-human monoclonal antibody to dynamin was purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Anti-human antibodies to calnexin and cathepsin B were purchased from Calbiochem. Biotinylated Galanthus nivalis agglutinin (GNA) and horseradish peroxidase-linked streptavidin were obtained from Vector Laboratories Inc. (Burlingame, CA).Tissue Homogenate—To provide a positive control for the Western blot analysis, 0.5 g of SN tissue was disrupted using a Potter-Elvehjem homogenizer in Lysis Buffer containing protease inhibitor mixture (0.01%, v/v), and proteins were extracted with 16 mm 3-[(3-cholamidopropyl)dimethylamino-1-propanesulfonate (Calbiochem).Western Blot Analysis—The separated proteins were transferred electrophoretically onto nitrocellulose membranes (Invitrogen) using the XCell II blot module. Nonspecific binding was blocked with 5% (w/v) nonfat dried milk, 0.5% (v/v) Tween 20 in Tris-buffered saline, pH 7.3, for 1 h at 20 °C. Immunoblots were probed with primary antibodies at the appropriate dilutions at 4 °C overnight or at room temperature for 1 h. Membranes were washed in Tris-buffered saline containing 0.1% (v/v) Tween 20 (3 × 10 min) followed by incubation with the secondary antibody at 20 °C for 1 h. Additional washing was performed with Tris-buffered saline containing 0.1% (v/v) Tween 20 (3 × 10 min), and the immunocomplexes were visualized by enhanced chemiluminescence (ECL™ system, Boehringer Ingelheim). Stripping of immunoblots for repeated probing was performed by incubating the membranes at 50 °C for 15 min in 100 mm 2-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7.Detection of Mannosylated Proteins by GNA Lectin—The biotinylated GNA lectin was applied to visualize mannosylated proteins (23Liao Y.F. Lal A. Moremen K.W. Cloning, expression, purification, and characterization of the human broad specificity lysosomal acid α-mannosidase.J. Biol. Chem. 1996; 271: 28348-28358Google Scholar, 24Shibuya N. Goldstein I.J. Van Damme E.J. Peumans W.J. Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb.J. Biol. Chem. 1988; 263: 728-734Google Scholar) of isolated NM granules and total SN tissue homogenate. The proteins were transferred electrophoretically onto polyvinylidene difluoride membranes (Invitrogen) using the XCell II blot module. The membranes were blocked with Tris-buffered saline containing 0.1% (v/v) Tween 20 at 20 °C for 1 h and incubated with biotinylated GNA lectin overnight at 4 °C followed by incubation with horseradish peroxidase-linked streptavidin at 20 °C for 1 h. The bands of mannosylated proteins were visualized by enhanced chemiluminescence (ECL system).In-gel Digestion with Trypsin—An entire lane of a gel previously stained with colloidal Coomassie Brilliant Blue G-250 was sliced into 4-mm cubes, and each of these was placed into a separate quartz reaction tube (Sigma) (25Schafer H. Nau K. Sickmann A. Erdmann R. Meyer H.E. Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry.Electrophoresis. 2001; 22: 2955-2968Google Scholar). The gel cubes were washed three times with 10 mm NH4HCO3, pH 7.8, and 10 mm NH4HCO3, pH 7.8, acetonitrile (1:1, v/v) each for 10 min. The gel cubes were subsequently reswollen by addition of 2 μl of modified trypsin (Promega, Madison, WI; 0.05 μg/μl in 10 mm NH4HCO3, pH 7.8). The digestion was performed overnight at 37 °C.Sample Preparation for LC Separation—10 μl of 0.1% (v/v) trifluoroacetic acid/acetonitrile (1:1, v/v) were added to each gel slice followed by sonication for 10 min. This step was repeated twice, and the supernatants containing the extracted peptides were combined in separate quartz tubes.Mass Spectrometric Analysis—Nano-HPLC-ESI-MS/MS analysis of the tryptically generated peptides was carried out as described previously (26Marcus K. Moebius J. Meyer H.E. Differential analysis of phosphorylated proteins in resting and thrombin-stimulated human platelets.Anal. Bioanal. Chem. 2003; 376: 973-993Google Scholar). Spectra were recorded on a Finnigan LCQ™ Classic (Thermo Electron, San Jose, CA) ion trap mass spectrometer equipped with a nanoelectrospray ion source (Pico View™ 100, New Objective Inc., Woburn, MA). The peptides were preconcentrated by loading onto a μ-precolumn (0.3-mm inner diameter × 5 mm, PepMap™, LC Packings Dionex, Idstein, Germany) before being separated by reverse-phase nano-LC (75-μm inner diameter × 250 mm, PepMap, LC Packings Dionex) using a precolumn split.Mass Spectrometry Data Analysis—The analysis of the raw MS/MS data occurred automatically based on the Sequest™ algorithm (27Eng J.K. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Google Scholar, 28Yates III, J.R. Eng J.K. McCormack A.L. Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.Anal. Chem. 1995; 67: 1426-1436Google Scholar). The data were searched against the human NCBInr data base (www.ncbi.nlm.nih.gov) using the following parameters: average masses, partial oxidation of methionine (+16 Da), a mass tolerance of ± 1.5 Da, trypsin was used as a specific protease, and a maximum of two missed cleavage sites was tolerated. Furthermore the analysis was restricted to ions in the mass range of 500–5000 Da and a total ion current greater than 3 × 105. In general, a cross correlation value (Xcorr) of greater than 2.0 and a Δ correlation score (ΔCn) greater than 0.1 was accepted for confident identification; inspection of the spectra was performed to confirm the Sequest results.RESULTSSubcellular Fractionation to Isolate NM-containing Granules—We developed a mild procedure for the isolation of intact and pure NM granules from human SN to enable subcellular protein analysis. Fig. 1 shows a schematic summary of the approach used. As a first step, the tissue was disaggregated by mechanical sieving into a cell suspension that was subsequently fractionated by centrifugation through a discontinuous sucrose gradient. This step allowed the enrichment of dark cell bodies as a pellet at the bottom sucrose layer. In the second step, these dark cell bodies were disrupted and subjected to an additional centrifugation step for finally isolating the NM granules by subcellular fractionation.Quality Control of Isolated Specimens by Transmission Electron Microscopy—The purity and quality of the granule isolation were monitored by transmission electron microscopy to evaluate the level of enrichment achieved by this approach as well as the structural and morphological appearance of the isolated granules (Fig. 2). Compared with the homogenates of pigmented cell bodies prior to the NM granule isolation, the isolated NM granules were virtually free from contaminating organelles; this was attributed to the exceptional density of NM granules. Up to now the essential density to penetrate an 80% (v/v) Percoll cushion has only been reported for highly melanized stage IV melanosomes isolated from Xenopus laevis melanophores (29Rogers S.L. Tint I.S. Fanapour P.C. Gelfand V.I. Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3720-3725Google Scholar, 30Testorf M.F. Roback K. Lundstrom I. Svensson S.P. Volume changes of individual melanosomes measured by scanning force microscopy.Pigm. Cell Res. 2001; 14: 445-449Google Scholar).Fig. 2.Comparative morphological analysis of the organelle preparations to monitor the enrichment of the NM granules. The organelle preparations were fixed in glutardialdehyde and OsO4 and were counterstained with lead citrate and uranyl acetate. Compared with the homogenate of pigmented cell bodies (A and B), according to the second step of the isolation strategy (Fig. 1), NM granules are highly enriched after isolation (C and D).View Large Image Figure ViewerDownload (PPT)Purified granules (Fig. 3) displayed all the morphological structures described previously for primate brain tissue by electron microscopy studies (16Moses H.L. Ganote C.E. Beaver D.L. Schuffman S.S. Light and electron microscopic studies of pigment in human and rhesus monkey substantia nigra and locus coeruleus.Anat. Rec. 1966; 155: 167-183Google Scholar, 31Hirosawa K. Electron microscopic studies on pigment granules in the substantia nigra and locus coeruleus of the Japanese monkey (Macaca fuscata yakui).Z. Zellforsch. Mikrosk. Anat. 1968; 88: 187-203Google Scholar) showing 1) the highly electron-dense patches attributable to the iron-rich NM, 2) the medium electron-dense protein matrix, and 3) vacuolar lipid bulbs. These characteristics were well preserved during isolation underscoring the potential of this strategy for isolation of NM granules.Fig. 3.The ultrastructural features of the NM granules are well preserved, showing the lobulated form of the granules. Electron-dense regions (arrows) are regarded as the pigment NM; lipid bulbs (arrowheads) are still attached to the granule.View Large Image Figure ViewerDownload (PPT)Quality Control by Western Immunoblotting—The level of enrichment was additionally assessed by Western immunoblotting applying antibodies against "marker proteins" specific for cell organelles (Fig. 4A) such as the Golgi network (GM130), mitochondria (Mcl-1), early endosomal compartments (EEA1), the plasma membrane (VLA-2α), the nucleus (nucleoporin p62), lysosomes (cathepsin B and LAMP-1), and the endoplasmic reticulum (BiP/grp78). The proteins extracted from NM granules were compared with control tissue homogenate and show the presence of lysosomal markers, whereas the other "organelle marker proteins" are absent (Fig. 4A, a–e and i).Fig. 4.Quality control by Western immunoblot analysis of proteins extracted from NM granules isolated from human SN compared with SN tissue homogenate.A, the Western immunoblot analysis shows the absence of organelle marker proteins of the Golgi complex (a, cis-Golgi matrix protein 130 (GM130)), mitochondria (a, induced myeloid leukemia cell differentiation protein (Mcl-1)), early endosomal compartments (b, early endosomal antigen 1 (EEA1)), plasma membrane (b, integrin α 2 (VLA-2α)), and nucleus (c, d; nucleoporin p62 (np62)). The protein extract of isolated NM granules compared with SN homogenate shows the presence of cathepsin B (d), a lysosomal proteinase, and LAMP-1 (e), a marker for late endosomes, lysosomes, and lysosome-related organelles. Dynamin (f) and clathrin (g), which are involved in vesicular traffic and are suggested to be associated to endosomal compartments, are detected. The melanogenic chaperone calnexin (h) is present in NM granules, although the marker for the endoplasmic reticulum (i, 78-kDa glucose-regulated protein (BiP/GRP78)) is absent after stripping. B, blotted proteins were probed with GNA lectin, which specifically binds to mannosylated proteins found in lysosomes or lysosome-related organelles.View Large Image Figure ViewerDownload (PPT)Protein Identification by Mass Spectrometry and Western Immunoblotting—As depicted in Fig. 5, the proteins extracted from NM granules were further fractionated by 1-D SDS-PAGE into a relatively large number of protein bands, although many of those were poorly resolved. Thus, we cut the entire gel lane into 16 slices and generated peptides from each slice by in-gel digestion with trypsin. These peptides were consequently analyzed in a second dimension by nano-LC coupled to ESI-MS/MS, which gave positive identification of 72 proteins (this experiment was repeated five times with comparable results). The proteins identified by mass spectrometry and the protein characteristics are compiled in Table I. Examples of mass spectral analyses depic
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