Characterization of Vesicles Secreted from Insulinoma NIT-1 Cells

Characterization of Vesicles Secreted from Insulinoma NIT-1 Cells Hyo Sun Lee, Jaeho Jeong, and Kong-Joo Lee* Center for Cell Signaling & Drug Discovery Research, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea 120-750 Received January 5, 2009

Insulinoma NIT-1, an insulin-secreting mouse cell line, secretes vesicles in response to glucose or calcium. These vesicles, like exosomes, are relatively homogeneous (30-100 nm). We analyzed their protein profiles employing one-dimensional SDS gel electrophoresis combined with nanoLC-ESI-qTOF tandem mass spectrometry, and searched for post-translational modifications (PTMs) using MODi algorithm. We identified 270 proteins which matched at least two peptides reproducibly in duplicate runs. These proteins included metabolic proteins, endocytosis/exocytosis related proteins, chaperones, cytoskeletal proteins, membrane transporters/ion channels, signaling molecules, and nucleic acid binding proteins. Over 200 of these are newly identified proteins for the first time in secreted vesicles, and included RNA- and translation-related proteins, ubiquitin- and protein-degradation related proteins and post-translationally modified proteins. The rest of the proteins identified in this study were similar to those reported by others to be present in exosomes of various origins. The present study demonstrates that vesicles secreted from insulinoma NIT-1 cells have some properties, common to exosomes from lymphocytes and cancer cells, and some differing from those of other types of exosomes. We believe that the modified and newly identified proteins we identified in secreted vesicles from insulinoma NIT-1 cells have the potential to provide insights into mechanisms of biogenesis and function of secreted vesicles and may help explain the impairment of insulin secretion in islets from type-2 diabetes. Keywords: proteomic analysis • pancreatic β cells • insulinoma • secreted vesicles • calcium • tandem mass spectrometry • post-translational modifications

Introduction Small vesicles are secreted from cultured hematopoietic cells as well as tumor and epithelial cells, among others.1,2 Although there is some evidence that these vesicles may play roles in immune response,3-5 as biomarkers of tumors6 and in neurodegenerative diseases,7 there is no clear understanding of these roles and of the mechanism of their secretion from various cells in vivo. Two types of vesicles have been described: small vesicles (30-100 nm diameter) that are secreted from the endosomal membrane compartment after the fusion of secretory granules with the plasma membrane, and large vesicles (100-1000 nm) that are secreted during plasma membrane blebbing by various stimuli such as calcium and cell death.2 Exosomes are minute, natural membrane vesicles released extracellularly by various types of cells concerned with the immune system. They are released from large multivesicular endosomes, present inside cells, fused with plasma membrane. Exosomes are characterized by endocytic marker proteins, surface proteins, tetraspanins and hsp70, among others. Many studies are underway directed at the biogenesis, functional * To whom correspondence should be addressed: College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea. Tel: 82-2-3277-3038. Fax: 82-2-3277-3760. E-mail: [email protected]. 10.1021/pr900009y CCC: $40.75

 2009 American Chemical Society

roles, and compositions of exosomes from lymphocytes, dendritic cells, cancer cells, neural stem cells and other cell types.8-16 Pancreatic β-cells are known to secrete insulin in response to glucose and calcium, by granule docking and cargo release. Long-term exposure of β-cells to high glucose or lipid leads to large reduction of insulin secretion. These effects were specific to β-cells. The impairment of insulin secretion in type-2 diabetes was suggested to relate to the reduction of key exocytotic proteins, including synaptotagmin, syntaxin 1, VAMP-2. These results suggest that defective secretory machinery may be involved in some types of type-2 diabetes.17 In this study, we attempted to elucidate the molecular mechanisms underlying these processes which are not well-understood.18,19 We found that insulinoma NIT-1 cells secrete vesicles. We identified the proteins in the secreted vesicles and semiquantitated their abundance by proteomic analysis, combined with 1D gel separation, western analysis and tandem mass spectrometry. Our results demonstrate the heterogeneity and complexity of vesicles naturally secreted from insulinoma cells and show that vesicles from insulinoma NIT-1 cells share some, but not all, of the characteristics of other exosomes and secretory vesicles.

Materials and Methods Cell Culture. Insulinoma NIT-1 cells were grown and maintained in a high glucose Dulbecco’s Modified Eagle Journal of Proteome Research 2009, 8, 2851–2862 2851 Published on Web 04/07/2009

research articles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 units/mL penicillin G, 3.75 µg/mL sodium bicarbonate, and 0.11 µg/mL sodium pyruvate at 37 °C in an atmosphere of 5% CO2-95% air. Preparation of NIT-1 Cells and the Secreted Vesicles. Insulinoma NIT-1 cells were seeded, and the medium was replaced with fresh media 1 day later and let stand for 48 h. The medium was then separated from the cells. The supernatant was centrifuged first at 100g for 5 min to remove cell debris and then at 22 300g for 30 min to obtain the secreted vesicles. The pellet was washed twice with PBS, and the secreted vesicles were isolated by a modified procedure described previously.11,12 The medium (200 µL) was loaded onto 4 mL of 4-40% sucrose gradient in PBS, ultracentrifuged at 200,000g for 7 h and each 200 µL fraction was collected at the bottom. The harvested cells and isolated vesicles were dissolved in a gel sample buffer for SDS-PAGE and Western analysis. The protein concentrations were measured using DC protein assay kit (Bio-Rad) following manufacturer’s instruction. Cell Survival Assay. Cell viability was monitored by MTT assay and FACS analysis. For MTT conversion assay, NIT-1 cells were seeded in 35 mm dishes at a density of 2.5 × 105 cells/ dish, and then incubated for indicated times (0, 24, and 48 h), 10 µL of MTT (5 mg/mL, Sigma) was added, and the cells again were incubated for 2 h to allow metabolization of MTT to 3-[4,5-dimethyldiazol-2-yl]-2,5-diphenylformazan at 37 °C. The purple precipitate was solubilized with 100 µL of acidified isopropanol and absorbance was measured at 570 nm in a microplate reader (Softmax Pro5). For FACS analysis, NIT-1 cells were seeded in 100 mm dishes at a density of 1.5 × 106 cells, incubated for indicated times (0 and 48 h), and then harvested. The cells were fixed in 70% ethanol overnight at 4 °C and stained with PI solution containing 50 µg/mL propidium iodide (Sigma) and 200 µg/mL RNase (Sigma) for 3 h at 37 °C. The stained cells were filtered in a Spectra nylon mesh (Spectrum, 100 µm pore size) and 10,000 cells were measured with a FACS flow cytometer (Beckton & Dickinson). The fractions of apoptotic cells were determined using Mod-fit (Cellquest Software). All experiments were conducted at least in triplicates. Calcium/EDTA Treatment of Insulinoma NIT-1 Cells. Confluent cells were incubated with a new medium containing 1 mM calcium chloride or 1 mM EDTA with 1 µM ionophore for 1 h. The secreted vesicles were isolated by centrifugation, as described above. Antibodies. Polyclonal anti-GAPDH, UCH-L1, Prx1, Prx2 and SOD II antibodies were purchased from Ab Frontier, Inc. (Seoul, Korea). Monoclonal anti-β-actin antibody was obtained from Cell Signaling Technology (Beverly, MA); monoclonal anti-Rtubulin antibody was from Santa Cruz Co. (CA). The sources of other antibodies were as follows: monoclonal anti-14-3-3 β, 14-3-3 θ, 14-3-3 ε, and polyclonal anti-14-3-3 δ protein antibodies were from Santa Cruz Co. (CA); polyclonal anti-Vsp 26, Vsp 29 and Vsp 35 antibodies were from Abcam (Cambridge, U.K.); monoclonal anti-ubiquitin antibody were form Chemicon (CA); monoclonal anti-PDI and Hsp90β antibodies were from Stressgen (MI). Western Analysis. Proteins separated by 10% SDS-PAGE were immediately transferred onto a semidry PVDF membrane for 45 min with 50 mA (Amersham, TE 77PWR). The membrane was blocked with 3% bovine serum albumin (BSA)-containing phosphate buffered saline for 2 h at a room temperature and sequentially incubated with each antibody diluted in PBS 2852

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Lee et al. containing 0.1% of Tween 20 for 2 h at room temperature. After washing three times with PBS containing 0.1% of Tween 20 (10 min each wash), the membrane was incubated with a peroxidase-conjugated secondary antibody, diluted in PBS containing 0.1% Tween 20 for 1 h at room temperature and washed three times with PBS containing 0.1% Tween 20 for 10 min. Detection of the immunocomplexes was performed using Intronbio appliances WEST-one (Intron, Co., Korea) and LAS-3000s (Fuji Photo Film, Co., Japan). Transmission Electron Microscopy (TEM) of Secreted Vesicles. One volume of each sample (cell fragment or vesicle) was fixed in 2 vol of 1% glutaraldehyde at room temperature for at least 2 h. One drop of each sample was applied to a copper grid, 400 mesh, and negative stained with 2% uranylacetate solution. Carbon was vacuum evaporated to prevent the accumulation of electrons during TEM observation. Transmission electron microscopy (TEM; TEOL, JEM 2100F) was used to clarify the sample with acceleration voltage of 200 kV. Bioinformatic Analysis. The proteins identified in the secreted vesicles were classified by gene ontology (www. geneontology.org) and their biological processes and functions inferred employing PANTHER database (www.pantherdb.org). Liquid Chromatography and Mass Spectrometry. For mass spectrometry analysis, proteins were separated on 1D gel electrophoresis, stained with silver and analyzed by tandem MS as described previously.20,21 The gel bands were excised with a scalpel, destained by 30 mM K4FeCN6/100 mM sodium thiosulfate and washed to remove destaining reagent. The pH was adjusted to 8.0 by 200 mM NH4HCO3 to facilitate trypsin digestion. The gels were dehydrated by adding acetonitrile (ACN), rehydrated by adding 10-20 µL of 25 mM NH4HCO3 containing 20 ng/mL of sequencing grade trypsin (Promega Co.), and incubated at 37 °C for 15-17 h. Peptides were extracted with 30 µL of solution containing 60% ACN/0.1% TFA. The extracts were pooled and evaporated to dryness in SpeedVac. Formic acid was added to the peptide extract so that the final concentration of formic acid in solvent was 0.1% to facilitate electrospray. Peptides were analyzed by nanoflow reversed-phased LC ESI-MS/MS with a mass spectrometer (Q-tof Ultima global, Waters Co., U.K.) comprising a three-pumping Waters nanoLC system with an autosampler, a stream selection module configured for precolumn plus analytical capillary column, and operated under MassLynx v.4.0 control (Waters Co., U.K.).20,21 Peptides were separated using a C18 reversed-phase 75 µm i.d. × 150 mm analytical column (3 µm particle size, Atlantis dC18, Waters Co., U.K.) with an integrated electrospray ionization SilicaTip ((10 µm, New Objective). Five microliters of peptide mixtures was dissolved in a buffer C (water/ACN/formic acid; 95:5:0.2, v/v), injected on a column and eluted by a linear gradient of 5-80% buffer B (ACN/water/formic acid; 95:5:0.2, v/v) over 120 min. Samples were desalted on-line prior to separation using a trap column (5 µm particle size, NanoEase dC18, Waters Co., U.K.) cartridge. Initially, the flow rate was set to 200 nL/min by CapLC (Waters Co., U.K.) and a capillary voltage of 3.0 keV was applied to the LC mobile phase before spray. Chromatography was performed on-line employing an MS spectrometer. MS parameters for efficient data-dependent acquisition were switch from MS scan to MS/MS acquisition

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Characterization of Vesicles Secreted from Insulinoma NIT-1 Cells Table 1. Identified Proteins in Secreted Vesicles Derived from Insulinoma NIT-1 Cells

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Table 1. Continued


research articles when intensity of individual ions rises above 10 counts/s and return to MS scan when intensity falls below 4 counts/s or after 14 s acquisition. The three most abundant precursors obtained from the first run were selected for MS/MS analysis. Database Search. The individual MS/MS spectra acquired for each of the precursors within a single LC run were smoothened (type of Mean, window ( 3 channels and iteration 2), deisotoped (threshold 3%), and centroided (top 80%, tof resolution 8000, and NP multiplier 0.7) using the Micromass ProteinLynx Global Server (PLGS) 2.1 data processing software and output, as a single MASCOT-searchable peak list (.pkl) file. The peak list files were used to query the Swiss-Prot v.50.8 (234 112 sequences; 85 963 701 residues) database using the MASCOT v.2.1.03 (global search engine) and MODi (Korea, http://modi.uos.ac.kr/modi/),22,23 with the following parameters: peptide mass tolerance, 0.5 Da; MS/MS ion mass tolerance, 0.2 Da; allowing up to 2 missed trypsin cleavage sites; considering variable modifications, such as acetylation (K), deamidation (N, Q), methylation (K), dimethylation (K), pyroglu (N-term E, Q), oxidation (M), phosphorylation (S, T, Y), ubiquitination (K) and propionamide (C) but not fixed modifications; enzyme limited to trypsin; and taxonomy limited to mouse. Only significant hits as defined by MASCOT probability analysis (probability based Mowse score p < 0.05) were considered. In addition, a minimum total score of 50 comprising at least a peptide match of ion score more than 20 was arbitrarily set as threshold for acceptance. All reported assignments were verified by automatic and manual interpretation of spectra from Mascot and MODi in a blind mode. Quantitative Analysis. We analyzed the abundance of identified proteins in the secreted vesicles by exponentially modified protein abundance index (emPAI).24,25 Protein abundance index (PAI) was calculated as the number of observed peptides per observable peptides on each proteins. The emPAI is an exponential form of PAI minus 1 and could be showed roughly proportional to protein abundance. The emPAI based protein contents in weight fraction percentages were calculated in Table 1.

Results and Discussion Purification of Vesicles Secreted from Insulinoma NIT-1 Cells. Insulinoma NIT-1 cells secrete vesicles at a high rate during normal culture conditions. This secretion was confirmed by metabolic labeling with [35S]-methionine (data not shown). Also, this secretion takes place in normal media, from healthy cells and no cell death was detected by MTT assay and FACS analysis (Supporting Information Figure 1). Because of the low abundance and heterogeneity of secreted vesicles, identification of secreted vesicles, an essential prerequisite for understanding their biological function, was difficult (Figure 1A). We therefore tried to obtain pellets of purified and concentrated secreted vesicles (SV) by centrifugation since the pellet obtained was equivalent to the vesicles at density 1.06 g/mL sucrose gradient centrifugation (see Materials and Methods) (Supporting Information Figure 2). The homogeneity and size of purified secreted vesicles were demonstrated by transmission electron microscopy (Figure 1C). The purified vesicles which contained no cell debris or protein aggregate had ‘saucer-like’ morphology and were 30-100 nm in diameter. This confirms that the purified secreted vesicles from insulinoma NIT-1 cells are exosome-like, and not plasma membrane sheddings which are derived from plasma membranes of apoptotic cell bodies2 and whose size is around 100-1000 nm. To identify the protein profiles of 2856


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Figure 1. One-dimensional gel profiles of secreted vesicles from insulinoma NIT-1 cells. Secreted vesicles in medium were obtained for 48 h from confluent insulinoma NIT-1 cells; 1.5 × 106 cells were seeded and changed with fresh media at 1 day after seeding. (A) Cells after 48 h were harvested and washed three times with cold PBS. The washed cells were lysed in 200 µL of gel sample buffer and 10 µL of these were applied onto 10% SDS-PAGE (WCL). To obtain the secreted vesicles, the supernatant obtained from the media was first centrifuged at 100g for 5 min to remove the cell debris, and then at 22,400g for 30 min. The secreted vesicles (SV) were obtained as a pellet and washed twice with cold PBS and dissolved in 200 µL of gel sample buffer and 10 µL of these were applied on 10% SDS-PAGE (SV) without concentration. Proteins were detected with silver staining. (B) Same amounts of proteins in WCL (10-fold dilution of panel A) and SV (10-fold concentration of panel A) were loaded and separated on 10% SDS-PAGE, and detected with silver staining. (C) Transmission electron microscopic images of purified secreted vesicles were obtained by negative staining. The diameter range of secreted vesicle sizes was 30-100 nm.

secreted vesicles, we concentrated the secreted vesicles from 1.5 × 106 NIT-1 cells and loaded onto 10% SDS polyacrylamide gel electrophoresis as shown in Figure 1B. The protein pattern of secreted vesicles was found to be completely different from that of whole cell lysate. This suggests that secreted vesicles are composed of unique protein complexes. Identification of Proteins in Vesicles Secreted by Insulinoma NIT-1 Cells by NanoLC-ESI-q-TOF Tandem Mass Spectrometry and Western Analysis. The proteins of vesicles secreted from insulinoma NIT-1 cells were separated on 10%


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lipid metabolism; as well as 22 proteins acting as transporters or channels; 30 proteins acting as structural (cytoskeletal) molecules; 23 proteins functioning in exocytosis, endocytosis and trafficking; 19 proteins acting as chaperones/mediators of protein folding; 33 proteins acting as nucleic acid binding molecules; 10 proteins acting in signal transduction; 8 proteins functioning in cell cycle and motility; 24 proteins relating protein degradation and modification; 13 proteins functioning in oxido-reduction; and 2 proteins with unknown functions.

Figure 2. Validation of insulinoma-derived SV proteins. To validate SV proteins identified by MS/MS, western analysis was performed for both whole cell lysate (WCL) and secreted vesicles (SV). (A) Proteins in WCL and SV were separated on 10% 1D SDSPAGE and detected by silver staining. (B) Western analyses of the same sample (A) using various antibodies were detected by chemiluminescence.

1D SDS gel electrophoresis, detected by silver staining, and the gel was dissected into 21 fractions based on relative molecular masses (Figure 1B). We performed proteomic analysis of each gel fraction, using a sensitive and accurate detection protocol developed in this laboratory.20,21 Each gel piece was digested with trypsin and the extracted peptides were analyzed by nanoLC-ESI-q-TOF tandem mass spectrometry. Total MS and MS/MS spectra were thus obtained. We identified 405 proteins from the 21 fractions including single hit matched proteins. Two hundred seventy of these proteins matched at least two peptides reproducibly in duplicate runs (Table 1). The functions, biological processes and localizations of these proteins were inferred using gene ontology (www.godatabase.org) and PANTHER (www.pantherdb.org). We identified, in all, 86 proteins involved with metabolism; 25 proteins involved in carbohydrate metabolism; 43 proteins involved in amino acid metabolism; 24 proteins in nucleic acid metabolism; and 4 in

To confirm the identities of proteins inferred by proteomics/ mass spectrometry, we performed Western analysis of secreted vesicles. The gels of whole cell lysate (WCL) and secreted vesicles (SV) visualized by silver staining are shown in Figure 2A. Those detected by immunostaining with various antibodies and ECL plus chemiluminescence are shown in Figure 2B. These data, in addition to showing that GAPDH and SOD are relatively enriched in secreted vesicles, and that some proteins identified are components of exosome including 14-3-3 isoforms, actin and tubulin, vacuolar protein sorting (Vps) isoforms, Hsp90 and peroxiredonxin (PRX) isoforms, also identify a new protein, ubiquitin C-terminal hydrolase L1 (UCH-L1). Thus Western analysis validated the identification of proteins by proteomics using mass spectrometry. Also, our list of identified proteins is in accord with the previous reports on proteins of exosomes from various cells. However, further confirmation of these identifications is needed for venturing an exact definition of the characteristics of the exosome from insulinoma NIT-1 cells. Identification of Proteins in Vesicles Secreted by Insulinoma NIT-1 Cells by Gene Ontology and PANTHER Databases. We further classified the proteins identified in vesicles secreted from NIT-1 cells by their biological role as gleaned from Gene ontology and PANTHER databases. Table 1 and Figure 3 show that the most abundant proteins in secreted vesicles are proteins functioning in metabolism (32%), followed by nucleic acid binding proteins (13%), structural (cytoskeletal) proteins (11%), proteins functioning in exocytosis, endocytosis and trafficking (9%), proteins involved in protein

Figure 3. Classification of proteins identified in secreted vesicles by gene ontology and biological function. Identified proteins (270 proteins) of SV in Table 1 were classified based on functions, biological processes and localizations inferred by using gene ontology and PANTHER database. Journal of Proteome Research • Vol. 8, No. 6, 2009 2857

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Table 2. Identified Proteins in Secreted Vesicles, Not Found in Exosomes accession no.

2858

protein name

peptide matched

MW

pI

P25444 P62908 P97351 P62702 P62754 P62242 Q6ZWN5 P14206 P14869 Q9CXW4 P47963 Q9CR57 Q9CPR4 Q9CPR4 P35980 P62717 P84099 P62751 Q8BP67 P47911 P14148 P12970 O70133 Q8CGC7 P10126 O70251 P57776 P58252 P60843 P23116 Q6NZJ6 Q99020 P49312 Q8BG05 P61979 Q9D0E1 O88569 Q8BMJ2 Q99K48 P62960 P17225 Q99PV0 Q8VH51 Q99NB9 Q8VIJ6 P10711 Q9Z1Q9

Proteins Related to RNA Processing and Translation 40S ribosomal protein S2 2 40S ribosomal protein S3 6 40S ribosomal protein S3a 5 40S ribosomal protein S4, X isoform 4 40S ribosomal protein S6 3 40S ribosomal protein S8 4 40S ribosomal protein S9 3 40S ribosomal protein SA 5 60S acidic ribosomal protein P0 2 60S ribosomal protein L11 6 60S ribosomal protein L13 5 60S ribosomal protein L14 2 60S ribosomal protein L17 6 60S ribosomal protein L17 6 60S ribosomal protein L18 3 60S ribosomal protein L18a 3 60S ribosomal protein L19 2 60S ribosomal protein L23a 6 60S ribosomal protein L24 5 60S ribosomal protein L6 2 60S ribosomal protein L7 4 60S ribosomal protein L7a 4 ATP-dependent RNA helicase A 6 Bifunctional aminoacyl-tRNA synthetase 4 Elongation factor 1-alpha 1 8 Elongation factor 1-beta 4 Elongation factor 1-delta 2 Elongation factor 2 8 Eukaryotic initiation factor 4A-I 2 Eukaryotic translation initiation factor 3 subunit 10 5 Eukaryotic translation initiation factor 4 gamma 1 7 Heterogeneous nuclear ribonucleoprotein A/B 5 Heterogeneous nuclear ribonucleoprotein A1 3 Heterogeneous nuclear ribonucleoprotein A3 3 Heterogeneous nuclear ribonucleoprotein K 5 Heterogeneous nuclear ribonucleoprotein M 2 Heterogeneous nuclear ribonucleoproteins A2/B1 7 Leucyl-tRNA synthetase, cytoplasmic 3 Non-POU domain-containing octamer-binding protein 3 Nuclease sensitive element-binding protein 1 3 Polypyrimidine tract-binding protein 1 2 Pre-mRNA-processing-splicing factor 8 3 RNA-binding region-containing protein 2 2 Splicing factor 3B subunit 1 3 Splicing factor, proline- and glutamine-rich 3 Transcription elongation factor A protein 1 3 Valyl-tRNA synthetase 4

31212 26657 29735 29448 28663 24059 22447 32567 34195 20109 24159 23418 21278 21278 21500 20719 23451 17684 17768 33358 31400 29827 149381 169830 50082 24547 31143 95122 46125 161852 175967 30812 34044 39628 50944 77466 35971 134106 54506 35578 56443 273427 59457 145724 75394 33859 140127

10.25 9.68 9.75 10.16 10.85 10.32 10.66 4.80 5.91 9.64 11.54 11.03 10.20 10.20 11.79 10.72 11.48 10.44 11.26 10.69 10.89 10.56 6.39 7.75 9.10 4.53 4.91 6.42 5.32 6.39 5.30 7.69 9.27 9.10 5.39 8.81 8.97 6.64 9.01 9.87 8.47 8.97 10.16 6.65 9.45 8.65 7.90

P62334 P16675 P61082 P70398 O70435 Q9R1P0 Q9Z2U1 Q9QUM9 Q9Z2U0 O09061 P99026 O55234 Q60692 P62991 P56399 Q9R0P9 Q02053

Proteins Related to Ubiquitin and Degradation Related 26S protease regulatory subunit S10B 2 Lysosomal protective protein precursor 2 NEDD8-conjugating enzyme Ubc12 3 Probable ubiquitin carboxyl-terminal hydrolase FAF-X 3 Proteasome subunit alpha type 3 2 Proteasome subunit alpha type 4 3 Proteasome subunit alpha type 5 5 Proteasome subunit alpha type 6 3 Proteasome subunit alpha type 7 3 Proteasome subunit beta type 1 4 Proteasome subunit beta type 4 precursor 2 Proteasome subunit beta type 5 precursor 8 Proteasome subunit beta type 6 precursor 11 Ubiquitin 6 Ubiquitin carboxyl-terminal hydrolase 5 2 Ubiquitin carboxyl-terminal hydrolase isozyme L1 2 Ubiquitin-activating enzyme E1 1 18

44145 53809 20887 290360 28256 29452 26394 27355 27838 26355 29097 22952 25362 8560 95772 24822 117734

7.25 5.55 7.57 5.65 5.29 7.58 4.74 6.35 8.59 8.32 5.46 8.64 4.99 6.56 4.89 5.14 5.43



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Table 3. List of Post-Translationally Modified Proteins in Secreted Vesicles

degradation and modification (8%), proteins acting as a transporter and channel (8%), chaperons in protein folding (7%), proteins acting in oxido-reduction (5%), signaling molecules (4%), cell cycle and motility related proteins (3%) and unknown (1%), respectively. Also, the abundance of each protein identified in secreted vesicles was presented as the number of peptides identified by MS/MS analysis, to semiquantitatively reflect the abundance of each protein. The proteins found in the cytosol, in endocytotic compartments, or in the plasma membrane in secreted vesicles are clearly quite distinct from those of whole cells. The proteins identified in secreted vesicles of NIT-1 cells can be sorted into two groups, based on previous studies of exosomes of melanoma, T cells, B cells, dentritic cells, colorectal cancer8-16 and other cells. One group of proteins identified in the exosomes of insulinoma NIT-1 cells, identified in this study, are common to other cell-type specific exosomes. Many cytosolic proteins found in our study are in the endocytic pathway. These include cytoskeletal proteins (actin, tubulin, myosin, ezrin, etc.), proteins functioning in endocytosis and trafficking (small G-protein Rab isoforms, Rab GDP dissociation inhibitor (GDI), annexin isoforms, ADP ribosylation factors, vacuolar protein sorting (VPS) isoforms), chaperone (Hsp70 and Hsp90), signaling molecules (14-3-3 isoforms), metabolic enzymes (GAPDH, pyruvate kinase, enolase, thioredoxin peroxidase, etc.) and cell surface amino peptidases. Previous studies showed that adhesion molecules (e.g., integrin, tetraspanin) and antigen presenting proteins (MHC class I and II), shared by exosomes from lymphocytes (B cell, T cell and dendritic cells), were not found in secreted vesicles from insulinoma. This may be because either insulinoma cell surface lacks these molecules, because vesicles secreted from insulinoma have specific ways of producing them, or because they escaped detection because of their low abundance. Clearly, vesicles secreted from insulinoma share some properties with exosomes from various other cells.

The second group of exosomal proteins from NIT-1 cells includes proteins that are newly identified in secreted vesicles and have not been previously reported as secreted proteins. These proteins could be insulinoma cell-type specific or have been newly found, because more sensitive proteomic methodology has become available. This group includes ribosomeand RNA-processing related proteins, and ubiquitin and proteasome related proteins as shown in Table 2. Forty-three proteins related to ribosomal and RNA processing were identified, but their functions in secreted vesicles are not yet clear. Recent studies show that ubiquitinated proteins targeting to membranes induce endosomal vesicles combined with endosomal sorting complexes, required for transport (ESCART), and for vesicle formation.26 As shown in Table 1, vacuolar protein sorting (Vps) isoforms, VAMP and ADP/ATP translocase, components of ESCART and various ubiquitin related proteins were identified in secreted vesicles from insulinoma NIT-1 cells, which supports this concept.26 Ribosomal proteins were detected in secretory vesicles from various cells.27 This suggests that vesicles secreted from insulinoma contain the origins of secretory vesicles, a suggestion that might provide insights into the mechanisms for biogenesis of sorting vesicles with further studies. Post-Translational Modification of Proteins Identified in Secreted Vesicles. During the identification of proteins in the gel by nanoLC-ESI-q-TOF tandem MS, many proteins identified in secreted vesicles were not detected at the corresponding molecular weight positions on the gel. As shown in Table 3, some proteins were detected in multiple molecular weight positions (EF1R1, Rab 1A and 1B, ADP/ATP translocase 1 and 2, GAPDH, fructose-1,6-diphosphatase); some were detected at much higher molecular weight positions (ubiquitin activation enzyme E1, hsp90β, tubulin R2 and β5, histon, ATPases, hnRNP A1, etc.), and some were found at much lower molecular weight positions (KIF13A, keratin, lysosomal protection protein precursor, etc.). To confirm these findings from mass spectromJournal of Proteome Research • Vol. 8, No. 6, 2009 2859

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Figure 4. Validation of modified proteins in secreted vesicles. Proteins in secreted vesicles were moved to different positions compared to theoretical molecular weights. These proteins were validated by western analysis of WCL and SV on 10% 1D SDSPAGE. Arrows indicate the positions of variously modified ubiquitin and hsp90β by tandem MS analysis in Table 1 and arrowhead indicates the right molecular weight of hsp90β without any modifications.

etry, Western analysis was conducted using antibodies against ubiquitin, hsp90β and GAPDH (data not shown). As shown in Figure 4, the proteins in secreted vesicles exist in multiple populations, distinct from those in cell lysate. This suggests that some uniquely modified proteins can be located in secreted vesicles. However, based on these findings, it is difficult to explain the relationship between protein modifications in secreted vesicles and biogenesis of vesicles or their functions. Post-translational modifications (PTMs) play key roles in determining the biological functions of proteins. Identification of the PTMs using mass spectrometry is a challenging problem because of the dynamic complexities of PTMs in vivo and their low abundance. We employed a strategy for rapid, efficient and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during LCESI-q-TOF MS/MS through replicated runs of a protein.20 A list of precursor ions of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. Exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. By examining the modification sites and modification status of the modified peptides of new bands in secreted vesicles using SEMSA technology and MODi algorithm (Figure 5), we detected post-translational modifications including ubiquitination and phosphorylations in some proteins in the vesicles.22,23 Many polyubiquitinated proteins were identified in various bands and phosphorylations including hsp90β were clearly identified with MS/MS, which were noted previously.27,28 However, the relationship between protein modifications de2860


Figure 5. Identification of post-translational protein modifications and the sites of modifications in secreted vesicles. Polyubiquitinated proteins in fraction 1 in Figure 1B and phosphorhorylated peptides of Hsp90β and endoplasmin precursor in fraction 5 and modification sites were determined by sequencing using nanoLCESI-q-TOF tandem MS combining PTM enriched method SEMSA and algorithm MODi.

tected in secreted vesicles and the biogenesis of vesicles or their functions should be further studied. Effect of Calcium on Vesicle Secretion from Insulinoma Cells. We investigated whether the secretion of vesicles from insulinoma is affected by calcium concentration by measuring the amount of secreted vesicles from cells treated with EDTA or calcium. Confluent insulinoma NIT-1 cells were incubated with 1 mM calcium chloride or 1 mM EDTA in PBS for 1 h, and the secreted vesicles were isolated. As shown in Figure 6A, calcium treatment increased the amount of vesicle secretion, without changing the protein profiles. Relatively homogeneous secreted vesicles were detected from both calcium and EDTA treated cells by transmission electron microscopy, and even


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Acknowledgment. We thank Professor Jin-Ho Choy and Myung Wook Jang in Center for Intelligent NanoBio Materials (CINBM, Ewha Womans University, Korea) for the technical support of TEM. This work was supported by KOSEF through the Center for Cell Signaling & Drug Discovery Research (CCS & DDR, R15-2006-020) and WCU (R31-2008-000-10010-0) at Ewha Womans University. H. S. Lee was supported by the Brain Korea 21 project. Supporting Information Available: Supplementary Table 1, identified proteins in secreted vesicles derived from insulinoma NIT cells; Supplementary Table 2, identified proteins in Figure 6A; Supplementary Figure 1, cell viability; Supplementary Figure 2, secreted vesicles separated by centrifugation and sucrose gradient centrifugation. This material is available free of charge via the Internet at http://pubs.acs.org. References Figure 6. Vesicle secretion from insulinoma cells was increased by raising intracellular calcium concentrations. (A) Insulinoma cells were incubated with 1 mM EDTA and CaCl2 in PBS for 1 h, vesicles secreted from cells for 1 h were isolated, and proteins were analyzed on 1D gel and detected with silver staining (upper panel) and Western analysis (lower panel). Identified proteins of each arrow were listed in Supporting Information Table 2. (B) Secreted vesicles from EDTA- and calcium-treated cells were visualized with TEM after glutaraldehyde fixation and uranyl acetate staining.

the population density of vesicles from calcium treated cells was higher than that from EDTA treated cells (Figure 6B). This demonstrates that vesicles from insulinoma NIT-1 cells are constitutively secreted under normal conditions and this secretion is enhanced by intracellular calcium concentration increase.

Conclusion In this study, we examined the protein composition and the biological properties of 270 proteins present in the vesicles secreted from insulinoma NIT-1 cells, with proteomic, bioinformatic and Western analyses and TEM approaches. Secreted vesicles from insulinoma were relatively homogeneous (30-100 nm). Protein profiles in the secreted vesicles were analyzed by combining one-dimensional SDS gel electrophoresis with nanoLC-ESI-q-TOF tandem mass spectrometry. We detected post-translational modifications (PTMs) including ubiquitination and phosphorylation in proteins using MODi algorithm combining the SEMSA technology. The 270 proteins detected, included metabolic proteins, endocytosis/exocytosis related proteins, chaperones, cytoskeletal proteins, membrane transporter/ion channels, signaling molecules, nucleic acid binding proteins. They also include >200 newly identified proteins in addition to the others previously shown in exosomes from various origins as reported in previous studies. Of note, RNAand translation-related proteins, ubiquitin- and proteindegradation related proteins and modified proteins were found in the secreted vesicles. These results demonstrate that secreted vesicles from insulinoma NIT-1 cells share some common properties with exosomes from lymphocyte and cancer cells and also differ from them in some properties. Understanding the nature of the newly and previously identified proteins in secreted vesicles might hold the key to understanding the biogenesis and function of secreted vesicles.

(1) The´ry, C.; Zitvogel, L.; Amigorena, S. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2002, 2, 569–579. (2) Fe´vrier, B.; Raposo, G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell. Biol. 2004, 16, 415–421. (3) Raposo, G.; Nijman, H. W.; Stoorvogel, W.; Liejendekker, R.; Harding, C. V.; Melief, C. J.; Geuze, H. J. B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 1996, 183, 1161–1172. (4) Saunderson, S. C.; Schuberth, P. C.; Dunn, A. C.; Miller, L.; Hock, B. D.; MacKay, P. A.; Koch, N.; Jack, R. W.; McLellan, A. D. Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor. J. Immunol. 2008, 180, 8146–8152. (5) Admyre, C.; Telemo, E.; Almqvist, N.; Lo¨tvall, J.; Lahesmaa, R.; Scheynius, A.; Gabrielsson, S. Exosomessnanovesicles with possible roles in allergic inflammation. Allergy 2008, 63, 404–408. (6) Schorey, J. S.; Bhatnagar, S. Exosome function: from tumor immunology to pathogen biology. Traffic 2008, 871–881. (7) Vella, L. J.; Sharples, R. A.; Nisbet, R. M.; Cappai, R.; Hill, A. F. The role of exosomes in the processing of proteins associated with neurodegenerative diseases. Eur. Biophys. J. 2008, 37, 323–332. (8) The´ry, C.; Boussac, M.; Ve´ron, P.; Ricciardi-Castagnoli, P.; Raposo, G.; Garin, J.; Amigorena, S. Proteomic analysis of dendritic cellderived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol 2001, 166, 7309–7318. (9) Aoki, N.; Jin-no, S.; Nakagawa, Y.; Asai, N.; Arakawa, E.; Tamura, N.; Tamura, T.; Matsuda, T. Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and hormonedependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology 2007, 148, 3850–3862. (10) Casey, T. M.; Meade, J. L.; Hewitt, E. W. Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome. Mol. Cell. Proteomics 2007, 6, 767– 780. (11) Choi, D. S.; Lee, J. M.; Park, G. W.; Lim, H. W.; Bang, J. Y.; Kim, Y. K.; Kwon, K. H.; Kwon, H.; Kim, K. P.; Gho, Y. S. Proteomic analysis of microvesicles derived from human colorectal cancer cells. J. Proteome Res. 2007, 6, 4646–4655. (12) Kang, D.; Oh, S.; Ahn, S. M.; Lee, B. H.; Moon, M. H. Proteomic analysis of exosomes from human neural stem cells by flow fieldflow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J. Proteome Res. 2008, 7, 3475–3480. (13) Mears, R.; Craven, A.; Hanrahan, S.; Totty, N.; Upton, C.; Young, S. L.; Patel, P.; Selby, P. J.; Banks, R. E. Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 2004, 4, 4019–4031. (14) Hegmans, J. P.; Bard, M. P.; Hemmes, A.; Kleijmeer, M. J.; Prins, J. B.; Zitvogel, L.; Burgers, S. A.; Hoogsteden, H. C.; Lambrecht, B. N. Proteomic analysis of exosomes secreted by human mesothelioma cells. Am. J. Pathol. 2004, 164, 1807–1815. (15) Fernandez, C.; Fransson, U.; Hallgard, E.; Spegel, P.; Holm, C.; Krogh, M.; Warell, K.; James, P.; Mulder, H. Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13). J. Proteome Res. 2008, 7, 400–411. (16) Wegrzyn, J.; Lee, J.; Neveu, J. M.; Lane, W. S.; Hook, V. Proteomics of neuroendocrine secretory vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters. J. Proteome Res. 2007, 6, 1652–1665.


research articles (17) Eliasson, L.; Abdulkader, F.; Braun, M.; Galvanovskis, J.; Hoppa, M. B.; Rorsman, P. Novel aspects of the molecular mechanisms controlling insulin secretion. J. Physiol. 2008, 586, 3313–3324. (18) Donelan, M. J.; Morfini, G.; Julyan, R.; Sommers, S.; Hays, L.; Kajio, H.; Briaud, I.; Easom, R. A.; Molkentin, J. D.; Brady, S. T.; Rhodes, C. J. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis. J. Biol. Chem. 2002, 277, 24232–24242. (19) Ma, L.; Bindokas, V. P.; Kuznetsov, A.; Rhodes, C.; Hays, L.; Edwardson, J. M.; Ueda, K.; Steiner, D. F.; Philipson, L. H. Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9266–9271. (20) Seo, J.; Jeong, J.; Kim, Y. M.; Hwang, N.; Paek, E.; Lee, K. J. Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase. J. Proteome Res. 2008, 7, 587–602. (21) Kim, Y. M.; Seo, J.; Kim, Y. H.; Jeong, J.; Joo, H. J.; Lee, D. H.; Koh, G. Y.; Lee, K. J. Systemic analysis of tyrosine phosphorylated proteins in angiopoietin-1 induced signaling pathway of endothelial cells. J. Proteome Res. 2007, 6, 3278–3290. (22) Kim, S.; Na, S.; Sim, J. W.; Park, H.; Jeong, J.; Kim, H.; Seo, Y.; Seo, J.; Lee, K. J.; Paek, E. MODi: a powerful and convenient web server for identifying multiple post-translational peptide modifications from tandem mass spectra. Nucleic Acids Res. 2006, 34, W25863.

2862


Lee et al. (23) Na, S.; Jeong, J.; Park, H.; Lee, K. J.; Paek, E. Unrestrictive identification of multiple post-translational modifications from tandem mass spectrometry using an error-tolerant algorithm based on an extended sequence tag approach. Mol. Cell. Proteomics 2008, 7, 2452–2463. (24) Rappsilber, J.; Ryder, U.; Lamond, A. I.; Mann, M. Large-scale proteomic analysis of the human spliceosome. Genome Res. 2002, 12, 1231–1245. (25) Ishihama, Y.; Oda, Y.; Tabata, T.; Sato, T.; Nagasu, T.; Rappsilber, J.; Mann, M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteomics 2005, 4, 1265–1272. (26) Nickerson, D. P.; Russell, M. R.; Odorizzi, G. A concentric circle model of multivesicular body cargo sorting. EMBO Rep. 2007, 8, 644–650. (27) Uriarte, S. M.; Powell, D. W.; Luerman, G. C.; Merchant, M. L.; Cummins, T. D.; Jog, N. R.; Ward, R. A.; McLeish, K. R. Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils. J. Immunol. 2008, 180, 5575– 5581. (28) Ogiso, H.; Kagi, N.; Matsumoto, E.; Nishimoto, M.; Arai, R.; Shirouzu, M.; Mimura, J.; Fujii-Kuriyama, Y.; Yokoyama, S. Phosphorylation analysis of 90 kDa heat shock protein within the cytosolic arylhydrocarbon receptor complex. Biochemistry 2004, 43, 15510–15519.

PR900009Y

Characterization of Vesicles Secreted from Insulinoma NIT-1 Cells

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