N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells
2018; Elsevier BV; Volume: 17; Issue: 4 Linguagem: Inglês
10.1074/mcp.ra117.000129
ISSN1535-9484
AutoresMarko Košiček, Ivan Gudelj, Anita Horvatić, Tanja Jovic, Frano Vučković, Gordan Lauc, Silva Hečimović,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoIncreasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4–5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration. Increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4–5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration. Since their first description scientists have tried to fully characterize lysosomal composition and function. Today, many facts are known about lysosomal physiology. The acidic pH, ionic gradients and the membrane potential make lysosomes an ideal environment for activity of luminal lysosomal hydrolases and a cellular center for nutrient sensing and recycling (1.Xu H. Ren D. Lysosomal Physiology.Annu. Rev. Physiol. 2015; 77: 57-80Crossref PubMed Scopus (589) Google Scholar). Lysosome's primary role is to digest a cargo from endocytic, phagocytic or autophagocytic pathways. More than 50 lysosomal hydrolases have been characterized and dysfunction in their activity/levels leads to accumulation of the lysosomal cargo which causes lysosomal storage disorders (LSDs) 1The abbreviations used are: LSD, Lysosomal storage disorder; AD, Alzheimer's disease; APP, β-amyloid precursor protein; BACE1, β-secretase; CHO, Chinese hamster ovary; HCD, Higher energy collisional dissociation; HILIC, Hydrophilic interaction liquid chromatography; EEA1, Early Endosome Antigen 1 - early endosomal marker; ESI, Electrospray ionization; FASP, Filter aided sample preparation; GO, Gene ontology; LAMP1, Lysosomal Associated Membrane Protein 1 - lysosomal marker; MALDI, Matrix-assisted laser desorption/ionization; NAG, N-Acetyl-β-D-glucosaminidase; NPC, Niemann-Pick Type C; PD, Parkinson's disease; Rab7, RAS-related GTP-binding protein 7- late endosomal marker; SPE, Solid-phase extraction; TfR, Transferrin receptor – a marker of recycling endosomes; TOF, Time-of-flight; UPLC, Ultra-performance liquid chromatography. 1The abbreviations used are: LSD, Lysosomal storage disorder; AD, Alzheimer's disease; APP, β-amyloid precursor protein; BACE1, β-secretase; CHO, Chinese hamster ovary; HCD, Higher energy collisional dissociation; HILIC, Hydrophilic interaction liquid chromatography; EEA1, Early Endosome Antigen 1 - early endosomal marker; ESI, Electrospray ionization; FASP, Filter aided sample preparation; GO, Gene ontology; LAMP1, Lysosomal Associated Membrane Protein 1 - lysosomal marker; MALDI, Matrix-assisted laser desorption/ionization; NAG, N-Acetyl-β-D-glucosaminidase; NPC, Niemann-Pick Type C; PD, Parkinson's disease; Rab7, RAS-related GTP-binding protein 7- late endosomal marker; SPE, Solid-phase extraction; TfR, Transferrin receptor – a marker of recycling endosomes; TOF, Time-of-flight; UPLC, Ultra-performance liquid chromatography. (2.Futerman A.H. van Meer G. The cell biology of lysosomal storage disorders.Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (628) Google Scholar). Besides luminal hydrolases, integral lysosomal membrane proteins are also important for proper function of lysosomes. Mutations in genes encoding these proteins lead to defects in the transport of lysosomal cargo and/or ions across the lysosomal membrane also causing the LSDs (3.Saftig P. Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function.Nat. Rev. Mol. Cell Biol. 2009; 10: 623-635Crossref PubMed Scopus (1104) Google Scholar). Individuals with lysosomal storage disorders often develop symptoms early in life and in majority of LSDs the brain, especially neurons, are affected (4.Schultz M.L. Tecedor L. Chang M. Davidson B.L. Clarifying lysosomal storage diseases.Trends Neurosci. 2011; 34: 401-410Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Niemann-Pick Type C (NPC) disease is a rare, autosomal recessive, progressive and fatal disorder characterized by abnormal cholesterol trafficking and intracellular accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. NPC is caused by loss of function of either NPC1, a multi-transmembrane lysosomal protein, or NPC2, a small luminal lysosomal protein that mediate intracellular cholesterol transport (5.Pacheco C.D. Lieberman A.P. The pathogenesis of Niemann-Pick type C disease: a role for autophagy?.Expert. Rev. Mol. Med. 2008; : 1069-1075Google Scholar). Recently, NPC disease has shown to share several pathological features with the most common and complex Alzheimer's disease (AD) (6.Malnar M. Hecimovic S. Mattsson N. Zetterberg H. Bidirectional links between Alzheimer's disease and Niemann-Pick type C disease.Neurobiol. Dis. 2014; 72: 37-47Crossref PubMed Scopus (62) Google Scholar). Our previous work has suggested that dysfunction of the late endosomal/lysosomal compartments in NPC1-cellular model is, most likely, responsible for the AD-like features in NPC (7.Malnar M. Kosicek M. Lisica A. Posavec M. Krolo A. Njavro J. Omerbasic D. Tahirovic S. Hecimovic S. Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells.Biochim. Biophys. Acta - Mol. Basis Dis. 2012; 1822: 1270-1283Crossref PubMed Scopus (29) Google Scholar, 8.Malnar M. Kosicek M. Mitterreiter S. Omerbasic D. Lichtenthaler S.F. Goate A. Hecimovic S. Niemann-Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the β-secretase pathway.Biochim. Biophys. Acta - Mol. 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Sci. 2012; 13: 15801-15812Crossref PubMed Scopus (6) Google Scholar). Besides cholesterol, other lipids, especially phospholipids and sphingolipids are also involved in these processes (11.Kosicek M. Hecimovic S. Phospholipids and Alzheimer's disease: Alterations, mechanisms and potential biomarkers.Int. J. Mol. Sci. 2013; 14: 1310-1322Crossref PubMed Scopus (128) Google Scholar). Glycosylation is one of the most common co-translational and post-translational modification which regulates the structure, stability, localization and function of various proteins, and N-glycosylation has been the most studied type of it (12.Stanley, P., Schachter, H., and Taniguchi, N., (2009) Chapter 8. N-Glycans, Essentials of Glycobiology, 2nd Edition,Google Scholar). N-glycans are known to be versatile and responsive to environmental stimuli and undergo significant changes in numerous diseases including those of central nervous system (13.Vučković F. Krištić J. Gudelj I. Teruel M. Keser T. Pezer M. Pučić-Baković M. Štambuk J. Trbojević-Akmačić I. Barrios C. Pavić T. Menni C. Wang Y. Zhou Y. Cui L. Song H. Zeng Q. Guo X. Pons-Estel B.A. McKeigue P. Leslie Patrick A. Gornik O. Spector T.D. Harjaček M. Alarcon-Riquelme M. Molokhia M. Wang W. Lauc G. Association of systemic lupus erythematosus with decreased immunosuppressive potential of the IgG glycome.Arthritis Rheumatol. 2015; 67: 2978-2989Crossref PubMed Scopus (165) Google Scholar, 14.Gudelj I. Baciarello M. Ugrina I. De Gregori M. Napolioni V. Ingelmo P.M. Bugada D. De Gregori S. Ðerek L. Pučić-Baković M. Novokmet M. Gornik O. Saccani Jotti G. Meschi T. Lauc G. Allegri M. Changes in total plasma and serum N-glycome composition and patient-controlled analgesia after major abdominal surgery.Sci. Rep. 2016; 6: 31234Crossref PubMed Scopus (21) Google Scholar, 15.Barrios C. Zierer J. Gudelj I. Štambuk J. Ugrina I. Rodríguez E. Soler M.J. Pavić T. Šimurina M. Keser T. Pučić-Baković M. Mangino M. Pascual J. Spector T.D. Lauc G. Menni C. Glycosylation Profile of IgG in Moderate Kidney Dysfunction.J. Am. Soc. Nephrol. 2015; : 1-9Google Scholar, 16.Freidin M.B. Keser T. Gudelj I. Štambuk J. Vučenović D. Allegri M. Pavić T. Šimurina M. Fabiane S.M. Lauc G. Williams F.M.K. The Association Between Low Back Pain and Composition of IgG Glycome.Sci. Rep. 2016; 5: 26815Crossref Scopus (21) Google Scholar, 17.Bieberich E. Synthesis, Processing, and Function of N-glycans in N-glycoproteins.Adv. Neurobiol. 2014; 9: 47-70Crossref PubMed Google Scholar). Moreover, it has been recently shown that modulation of glycosylation of APP, a key protein in the pathogenesis of AD, may represent a potential target for AD therapy (18.Jacobsen K.T. Iverfeldt K. O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-β precursor protein (APP).Biochem. Biophys. Res. Commun. 2011; 404: 882-886Crossref PubMed Scopus (80) Google Scholar). It has been previously shown that glycoproteins accumulate in NPC model (19.Mbua N.E. Flanagan-Steet H. Johnson S. Wolfert M.A. Boons G.-J. Steet R. Abnormal accumulation and recycling of glycoproteins visualized in Niemann-Pick type C cells using the chemical reporter strategy.Proc. Natl. Acad. Sci. 2013; 110: 10207-10212Crossref PubMed Scopus (26) Google Scholar), and that blocking the O-linked glycosylation lowers cholesterol levels and increases the number of lysosomes (20.Li J. Deffieu M.S. Lee P.L. Saha P. Pfeffer S.R. Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells.Proc. Natl. Acad. Sci. 2015; 112: 14876-14881Crossref PubMed Scopus (37) Google Scholar), thus rescuing the NPC cellular defects. In the present work, we studied N-glycosylation profile of the lysosomal membrane proteins in NPC1-null cells versus wt-cells. We tested the hypothesis that alteration of the lysosomal glycocalyx is an additional feature of the lysosomal dysfunction in NPC disease, as well as in other LSDs. To the best of our knowledge, here we describe the first complete N-glycome of the lysosomal glycocalyx in NPC disease cellular model, which potentially could be useful for restoring lysosomal storage defects in NPC disease and other LSDs as well as for rescuing pathological processes occurring in AD. Chinese hamster ovary wild type cells (CHOwt), CHO cells lacking NPC1 protein (NPC1-null) and NPC1-null cells stably express human NPC1 protein (NPC1-null + NPC1) were kindly provided by Dr. Daniel Ory. The cells were grown in DMEM/F12 medium supplemented with 10% FBS, 2 mm l-glutamine and antibiotic/antimycotic mix, all from Sigma-Aldrich (MO). Lysosomes were purified according the Walker and Lloyde Evans protocol (21.Walker M.W. Lloyd-Evans E. A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles.Methods Cell Biol. 2015; 126: 21-43Crossref PubMed Scopus (22) Google Scholar). Briefly, the cells were grown in T75 flasks, they were incubated with 10% ferrofluid solution (superparamagnetic iron oxide nanoparticles, 10 mg/ml of 40 kDa dextran-stabilized magnetite, Liquids Research Ltd, UK) and 10 mm HEPES pH 7.2 in growth medium for 24 h. After washing and the chase period in the regular medium for 24 h, cells were trypsinized, harvested and resuspended in 2 ml of hypotonic buffer (15 mm KCL, 1.5 mm MgAc, 1 mm DTT, 10 mm HEPES and proteinase inhibitor (Roche, Switzerland)). After homogenization in dounce homogenizer (30 times) and passing through 23G needle (10 times), 0.5 ml of hypertonic buffer was added (220 mm HEPES pH 7.2, 0.1 mm sucrose, 375 mm KCl, 22.5 mm MgAc, 1 mm DTT, 50 ml of DNase 1 (Roche Applied Science)). Following incubation for 5 min, the cellular homogenate was centrifuged and the same amount of proteins in the supernatant of CHOwt and NPC1-null cells was subjected to MS column (MACS Miltenyi Biotec, Germany) activated with 0.5% BSA in PBS and attached to QuadroMACS magnetic separator. Flow-through was collected, and column was washed with DNase solution and Phosphate Buffer Saline (PBS) supplemented with 0.1 mm sucrose. The column was removed from magnetic separator and lysosomes were eluted with 0.5 ml of PBS supplemented with 0.1 mm sucrose and proteinase inhibitor mixture (Roche Applied Science). All collected fractions were stored at −80 °C before analysis. Isolated fractions were mixed with sample buffer (6 times concentrated: 60% glycerol, 12% SDS, 3% DTT, 1/8 v/v 0.5 m Tris pH 6.8, bromphenol blue) and 20 μl of each fraction including the cell lysate (the input) was subjected to SDS-PAGE on 8% Tris-glycine gel. Proteins were transferred to PVDF membrane (Roche Applied Science), and blocked with I-block (Tropix, Thermo Fisher Scientific, MA). LAMP1 (rabbit polyclonal, Sigma-Aldrich, MO) was used as a lysosomal marker and EEA1 (mouse monoclonal, BD Transduction Laboratories, CA) was used as an early endosomal marker. Actin (rabbit polyclonal, Sigma-Aldrich) was used as a loading control. For detection, HRP conjugated secondary antibodies mouse/rabbit (Bio-Rad, CA) were used. Proteins were visualized by chemiluminescence using POD chemiluminescence blotting substrate (Roche Applied Science) on UviTec (UVItec ltd. Cambridge, UK). Western blots were quantified using ImageJ software (National Institutes of Health). Statistical validation of the data was achieved by Student t test. Total protein concentration was measured using commercially available Pierce BCA Protein Assay Kit (Thermo Scientific) according to manufacturer's protocol on Multiskan EX (Thermo Scientific). Total cholesterol concentration was measured using commercially available AmplexRed Cholesterol Assay (Molecular Probes) according to manufacturer's protocol on Fluoroskan Ascent FL (Thermo Scientific). N-Acetyl-β-d-glucosaminidase (NAG) activity was measured using 4-Nitrophenyl N-acetyl-β-d-glucosaminide substrate (Alfa Aesar). Ten μl of lysate fraction and lysosomal fraction were mixed with 90 μl of substrate (1 mg/ml in 0.09 m Citrate buffer solution, pH 4.8). After 30 min of incubation at 37 °C the reaction was stopped my adding 200 μl of 0.4 m Na2CO3. The absorbance of p-nitrophenylate ion was measured at 405 nm. The hydrodynamic size of isolated lysosomes was measured using a Zetasizer Nano ZS instrument (Malvern, UK). The hydrodynamic size was measured from a dilute (1:20) suspension of the sample in PBS pH 7.4 in a disposable plastic cuvette at 25 °C. The results were analyzed using the Zetasizer software v.6.32 provided by the manufacturer. Cells were grown on 12 mm glass covered slips and treated with 40 kDa dextran labeled with Fluorescein (Molecular Probes, Invitrogen, CA) in growth medium for 24 h. Cells were washed with PBS and further grown in the fresh growth medium for additional 0, 2, 4, 6, 8, and 24 h. After that cells were washed again in PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich) and mounted with Polyvinyl alcohol mounting medium with DABCO antifading (Fluka). All samples were analyzed using inverted fluorescent confocal microscope Leica SP8 X FLIM. For measuring cellular and secreted fluorescein labeled dextran, cells were grown in 6-well plates, they were incubated with 40 kDa fluorescein-labeled dextran for 24 h, following washing and 24 h chase in the regular medium. As a control the cells which were not incubated with dextran were used. Cells were lysed (50 mm Tris pH 7.6, 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, proteinase inhibitor) and fluorescein levels in the cell lysate and in the medium were measured on Fluoroskan Ascent FL (Thermo Scientific). Lysosomal fractions were prepared in triplicate and processed using filter aided sample preparation (FASP) protocol (22.Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Wi J.R. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 377Crossref PubMed Scopus (5097) Google Scholar) with some modifications. For the analysis, 8 μg of total protein from lysosomal fraction were mixed with the FASP-urea buffer (8 m urea in 0.1 m Tris-HCl pH 8.5) to final volume of 150 μl. SDS (Sigma-Aldrich) was added to final concentration of 1% (v/v). Before FASP, samples were sonicated for 10 min using ultrasonic bath, reduced (10 mm DTT, 30 min, 55 °C) and mixed with 100 μl of FASP-urea buffer. After transferring to the 10-kDa membrane filter units (Microcon YM-10, Merck Millipore), samples were centrifuged (13 000 × g, 45 min, 22 °C) and washed subsequently with 200 μl of FASP-urea buffer followed by centrifugation. Before digestion, proteins were alkylated (50 mm IAA, 20 min at room temperature in the dark), washed twice with FASP-urea buffer and then twice with ammonium bicarbonate (50 mm NH4HCO3 pH 7.6) followed by centrifugation (13 000 × g, 30 min). Protein digestion was achieved by adding trypsin (Promega) (enzyme-to-protein ratio 1:30, v/v) and by incubation at 37 °C overnight. Tryptic peptides were collected from filter units by centrifugation (13,000 × g, 30 min), washing with 50 μl of ammonium bicarbonate (50 mm NH4HCO3 pH 7.6) and subsequent centrifugation, followed by vacuum drying. Peptides were dissolved in loading solvent (1% ACN, 0.1% formic acid) and separated using Ultimate 3000 RSLCnano system (Dionex, CA) before on-line ESI-MS/MS analysis by Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). A total of amount of 1.5 μg was injected onto the trapping column (C18 PepMap100, 5 μm, 100A, 300 μm × 5 mm). After washing for 15 min with loading solvent at a flow rate of 15 μl/min, peptides were eluted onto the analytical column (PepMap™ RSLC C18, 50 cm × 75 μm) using linear gradient 5–45% mobile phase B (0.1% formic acid in 80% ACN) over 150 min, 45% to 90% for 2 min, held at 80% for 2 min and re-equilibrated at 5% B for 20 min at a flow rate of 300 nL/min. Mobile phase A consisted of 0.1% formic acid in water. Eluate from the column was introduced into the mass spectrometer via Nanospray Flex ion source and SilicaTip emitter (New Objective). The ionization voltage was set at 1.9 kV and the ion transfer capillary temperature at 250 °C. MS was operating in positive ion mode using HCD MS2 in data dependent acquisition mode. Full scan FTMS spectra were acquired in range from m/z 350.0 to 1800.0 with a resolution of 70,000. The maximum injection time for FTMS full scan was set at 100 ms reaching an automatic gain control (AGC) target value of 1 × 106. Top 15 most intense precursor ions were chosen for further HCD fragmentation with a resolution of 17500 using injection time 60 ms and MS2 AGC target of 1 × 105. The collision energy was set as 28% NCE. A ± 1.7 Da isolation window was applied to isolate precursor ions with dynamic exclusion of 15 s. MS raw files were processed by Proteome Discoverer software (version 2.0.0.802., Thermo Fisher Scientific) and SEQUEST search against Homo sapiens FASTA files (42116 sequences, downloaded November 5, 2016 from SwissProt database, TaxID = 9609 and subtaxonomies). Static peptide modification included carbamidomethylation (C), and dynamic oxidation (M) and deamidation (N,Q). Maximum two trypsin missed cleavage sites were allowed. Precursor tolerance and ion fragment tolerance were set at 10 ppm and 0.05 Da, respectively. Percolator confidence levels were set at 1% false discovery rate (FDR) (high) and 5% FDR (middle), for both peptide and protein levels FDR is determined automatically by Percolator node based on targeted-decoy strategy (23.Spivak M. Weston J. Bottou L. Kall L. Noble W.S. Improvements to the percolator algorithm for peptide identification from shotgun proteomics data sets.J. Proteome Res. 2009; 8: 3737-3745Crossref PubMed Scopus (188) Google Scholar). For peptide confidence, validation was based on q-value (minimal FDR at which the identification is considered correct (24.Käll L. Storey J.D. MacCoss M.J. Noble W.S. Posterior error probabilities and false discovery rates: Two sides of the same coin.J. Proteome Res. 2008; 7: 40-44Crossref PubMed Scopus (212) Google Scholar)) which was set at 0.01 (high). At least two unique peptides and 5% FDR were required for reporting confidently identified proteins. Gene ontology analysis of identified proteins was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) (version 6.8) (25.Dennis G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H. Lempicki R.A. DAVID: Database for Annotation, Visualization, and Integrated Discovery.Genome Biol. 2003; 4: R60Crossref Google Scholar). The significance of enrichment (p value) and enrichment rate (fold enrichment) was calculated by DAVID tool. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (26.Vizcaíno J.A. Csordas A. Del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2780) Google Scholar) partner repository with the dataset identifier PXD008438. The whole procedure was performed as previously reported (27.Pavić T. Gudelj I. Keser T. Pučić-Baković M. Gornik O. Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N-glycosylation.Biochim. Biophys. Acta - Gen. Subj. 2016; 1860: 1710-1715Crossref Scopus (7) Google Scholar). Briefly, the most hydrophilic molecules from commercial Triton X-114 were eliminated by adding of 490 ml of 10 mm Tris-HCl pH 7.4, 150 mm NaCl and keeping the solution at 4 °C for Triton X-114 dissolution and at 37 °C for its condensation. The whole procedure was repeated three times. Isolated lysosomes were homogenized in Triton lysis buffer (10 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% (v/v) Triton X-114 in PBS and protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany)), using an ultrasonic processor (UP100H Hielscher, Teltow, Germany) (four cycles, 15–20 s). Samples were then incubated overnight at 4 °C and the lysosome lysate was clarified by centrifugation (30 min, 10,000 × g, 4 °C). The clear supernatant was overlaid on 200 μl of sucrose cushion (6% (w/v) sucrose, 10 mm Tris-HCl pH 7.4, 150 mm NaCl and 0.06% Triton X-114) and incubated at 37 °C for 20 min. Clouding of the solution occurred. Samples were centrifuged for 3 min, 400 × g, 37 °C and two phases (detergent-rich and detergent-poor) formed. The detergent-poor phase was transferred to a fresh tube and kept on ice. The detergent-rich phase was resuspended in 500 μl of cold PBS and the phase separation was repeated. This aqueous phase was pooled with initial one and re-extracted by adding 50 μl of Triton stock solution and the phase separation was performed as described previously. Proteins from each phase were isolated by adding four times the sample volume of methanol and twice the initial sample volume of chloroform (Merck, Darmstadt, Germany) and vortexed well. At the end, three times of the initial sample volume of water was added, samples were vortexed vigorously and centrifuged for 1 min, 9 000 × g, 4 °C. After centrifugation the proteins were in the liquid interphase. The aqueous top layer was removed, an additional three volumes of methanol were added after which the samples were vortexed and centrifuged again for 2 min, 9,000 × g, 4 °C to pellet the proteins. Supernatant was removed, as much as possible without disturbing the precipitate, and samples were left to air-dry. The isolated proteins were denatured with the addition of SDS (Invitrogen) and by incubation at 65 °C. The excess of SDS was neutralized with Igepal-CA630 (Sigma-Aldrich) and N-glycans were released following the addition of PNGase F (Promega, WI) in PBS. The released N-glycans were labeled with 2-AB. Free label and reducing agent were removed from the samples using hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE). Glycans were eluted with ultrapure water and stored at −20 °C until use. The cell lysate proteins were prepared using the same protocol except there was not membrane protein isolation. Fluorescently labeled N-glycans were separated by HILIC on an Acquity UPLC instrument (Waters) consisting of a quaternary solvent manager, sample manager, and an FLR fluorescence detector set with excitation and emission wavelengths of 250 and 428 nm, respectively. The instrument was under the control of Empower 3 software, build 3471 (Waters Corp., MA). Labeled N-glycans were separated on a Waters BEH Glycan chromatography column, 150 × 2.1 mm i.d., 1.7 μm BEH particles, with 100 mm ammonium formate, pH 4.4, as solvent A and ACN as solvent B. The separation method used a linear gradient of 30–47% solvent A at flow rate of 0.56 ml/min in a 23 min analytical run. Samples were maintained at 10 °C before injection, and the separation temperature was 25 °C. Data processing was performed using an automatic processing method with a traditional integration algorithm, after which each chromatogram was manually corrected to maintain the same intervals of integration for all the samples. The chromatograms were all separated in the same manner into 37 and 25 chromatography peaks for the lysosomal and cell lysate N-glycome, respectively. HILIC-UPLC chromatograms were used for quantification, and abundance of each glycan was expressed as percentage of total integrated area. The identity of N-glycans separated by HILIC-UPLC was determined by MALDI-TOF/TOF-MS. Prior to MS analysis, fractions of each N-glycan chromatography peak were collected, dried down in a vacuum concentrator and resuspended in 10 μl of ultrapure water. To stabilize and distinguish α2,3-linked from α2,6-linked sialic acids ethyl esterification was performed as previously described (28
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