Exosomes Secreted by HeLa Cells Shuttle on Their Surface the

Oct 17, 2017 - ... the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on the exosomal surface could represent a useful...
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Exosomes secreted by HeLa cells shuttle on their surface the plasma membrane-associated sialidase NEU3 Lucia Paolini, Flavia Orizio, Sara Busatto, Annalisa Radeghieri, Roberto Bresciani, Paolo BERGESE, and Eugenio Monti Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00665 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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Biochemistry

Exosomes secreted by HeLa cells shuttle on their surface the plasma membraneassociated sialidase NEU3

Lucia Paolini§, Flavia Orizio§, Sara Busatto, Annalisa Radeghieri, Roberto Bresciani, Paolo Bergese, Eugenio Monti*

Department of Molecular and Translational Medicine (DMTM), University of Brescia, 25123 Brescia, Italy.

§

These authors contributed equally to this work

*Corresponding author: Eugenio Monti Department of Molecular and Translational Medicine (DMTM), Section of Biotechnology, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Phone: +39 030 3717544 Fax: +39 030 3701157 e-mail: [email protected]

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Abstract Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids and glycoproteins. The plasma membrane-associated sialidase NEU3 is involved in the fine-tuning of sialic acid containing glycans directly on the cell surface and plays relevant roles in important biological phenomena such as cell differentiation, molecular recognition and cancer transformation. Extracellular vesicles are membranous structures with a diameter of 0.03-1 µm released by cells and detectable in blood, urine, as well as in culture media. Among extracellular vesicles, exosomes play roles in intercellular communication, maintenance of several physiological and pathological conditions, including cancer, and could represent a useful diagnostic tool for personalized nanomedicine approaches. Using inducible expression of the murine form of NEU3 in HeLa cells, a study of the enzyme association with exosomes released in the culture media has been carried out. Briefly, NEU3 is associated with highly purified exosomes and localizes on the external leaflet of these nanovesicles, as demonstrated by enzyme activity measurements, Western-blot and dot blot analysis using specific protein markers. Based on these results it is plausible that NEU3 activity on exosome glycans enhances the dynamic biological behaviour of these small extracellular vesicles by modifying the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on exosomal surface could represent a useful marker for the detection of these nanovesicles and a tool to better understand the biology of these important extracellular carriers in physio/pathological conditions.

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Introduction Sialidases (EC 3.2.1.18) are a family of glycohydrolases that specifically remove terminal sialic acid residues from various glycoconjugates such as glycoproteins, glycolipids and oligosaccharides 1. In higher vertebrates four genes encode for sialidases with different subcellular localization and substrate specificities, namely the lysosomal sialidase NEU1, the cytosolic sialidase NEU2, the plasma membrane-associated NEU3 and, finally, the mitochondrial/intracellular membrane-associated NEU4 1. An in-silico study carried out on 21 different species of Metazoa has shown the sialidase family origins from the ancestral sialidase NEU1, providing insights into the mechanisms involved in the substrate specificity and identifying loop portions that might be involved in the evolution of some specific properties of sialidases 2. The plasma membrane-associated NEU3 has been first cloned from bovine brain 3, subsequently from mouse brain 4 and finally, using an expressed sequence tag (EST) 5, from human skeletal muscle 6. NEU3 recognizes gangliosides as preferential substrates and can act on sialylated glycans that reside on the same membranes of the enzyme (cis activity) as well as on the lipid bilayers of neighbouring cells (trans activity) 7. NEU3 is involved in a broad range of biological events, from cell differentiation/maintenance 8-11 to cancer initiation and promotion 12. NEU3 shows high degree of homology with the human cytosolic sialidase NEU2, the only mammalian sialidase whose 3D structure has been solved, so far 13. Using the NEU2 as template, homology modelling approaches allowed the prediction of the NEU3 3D model that, as expected, resulted similar to the cytosolic enzyme, lacking any typical membrane anchorage feature(s) 2, 14. In addition, NEU3 behaves as a peripheral membrane protein and recycles on the cell surface following the endosomal route 15. Recently, a study on NEU3 of

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human origin has demonstrated that the enzyme is S-acylated 16. In this perspective, the association of NEU3 with the membrane is mediated by a post-translational modification, although further experiments are needed to elucidate how a modification that is restricted to the cytosolic side of the membrane(s) can be responsible for the anchorage of an enzyme that is present on the cell surface 7. From the beginning of the new century an increasing number of researchers have focused their interest on extracellular vesicles, membranous vesicles with a diameter ranging from 30 nm to 1 µm that cells release from their surface and are detectable in eukaryotic fluids including blood and urine, as well as in cell culture media 17, 18. By considering biogenesis, size, density and protein content, extracellular vesicles can be broadly divided into small extracellular vesicles, or exosomes, and large extracellular vesicles, or microvesicles 19. Exosomes are intraluminal vesicles contained in multivesicular bodies, which are released into the extracellular environment upon fusion of multivesicular bodies with the plasma membrane. Their diameter ranges from 30 to 150 nm and they have a buoyant density within 1.11 and 1.19 g/ml. Instead, microvesicles are directly assembled and released from the plasma membrane through budding and reach up to 1 µm in diameter (note that extracellular vesicles classification is an open subject and the one adopted here complains with the latest indications 20, 21). Despite their different biogenesis, exosomes and microvesicles share biological functions and significance, including: i) their role in intercellular communication as carriers of macromolecules (proteins, lipids, mRNA, ncRNA and DNA) and metabolites 22; ii) their role in spread and maintenance of several pathological conditions, including cancer and metabolic diseases; iii) their translational potential for personalized nanomedicine − which aims at health care that is tailored on the basis of an individual’s genes, lifestyle and environment − for example by enabling liquid biopsy 23-25.

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In 2016, a proteomic study has been published on extracellular vesicles released by 60 different human cancer cells derived from 9 distinct tissue types collected by the National Cancer Institute (NCI-60) 26. This study allowed the identification of more than six thousand proteins associated to extracellular vesicles among which sialidase NEU3 has been detected (http://microvesicles.org/gene_summary?gene_id=10825). Using the inducible TET/Off HeLa tTA2 cells model 27 we report here the first direct evidence for the presence of NEU3 on the surface of exosomes released by cells in the culture media.

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Materials and methods Chemicals and reagents All chemicals are molecular biology-grade (SIGMA-Aldrich) unless specified. Protein concentration was determined using the Coomassie Protein Assay Reagent (Sigma).

Cell Culture HeLa cells, overexpressing the murine form of NEU3 tagged with the HA epitope at the Cterminus under the control of the Tetracycline promoter (Tet Off) 27 were cultured in high glucose Dulbecco’s modified Eagle’s medium (EuroClone) containing 4 mM L-glutamine, 100 Units/ml Penicillin, 100 µg/ml Streptomycin, 10% (v/v) Fetal Bovine Serum (FBS), 0.25 mg/ml G418 and 1 ng/ml Doxycycline and were maintained at 37°C and 5% CO2 in a humidified incubator. Expression of NEU3-HA was achieved by removing doxycycline from culture medium. To obtain the media from which extract the exosomes, 500,000 cells were seeded in 20 dishes (2r = 10 cm) in presence (HeLa Off) or absence (HeLa On) of doxycycline and grown until they reached 70-80% of confluence. Cells were then washed twice with PBS (GIBCO) and incubated for further 24 h with 5 ml Serum-Free OptiMEM (GIBCO). The media were collected and further processed to isolate the different extracellular vesicles populations.

Extracellular vesicles separation from culture media One hundred ml of culture media from HeLa On or HeLa Off cells were processed with serial centrifugation steps as previously described 28. This protocol allows the separation of different elements in biological fluids based on their size and density exploiting the gravity force 29. Briefly, media were centrifuged at 800 g for 30 min, 16,000 g for 45 min and finally

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100,000 g for 2h. The 800 g centrifuge step allows sedimentation of cell debris and large vesicles like apoptotic bodies, the 16,000 g step allows to pellet microvesicles, while the final 100,000 g ultracentrifugation enriches smaller extracellular vesicles, including exosomes. To eliminate smaller contaminant residues, the microvesicle pellet was further processed by 500 µl PBS washings, followed by 16,000 g centrifugation for 45 min. The supernatant resulting from the microvesicle pellet was concentrated using a Centricon Plus70 Centrifugal Filter Units 100,000 NMWL (Merck Millipore) following manufacturer instructions. The resulting concentrated medium sample was finally centrifuged at 100,000 g for 2 h (rotor TLA-55, Beckman Optima MAX, Backman). The supernatant after this step is referred as SN3, whereas the final pellet is hereafter referred as exosomes (Figure 1A).

Nanoplasmonic colorimetric assay and Atomic Force Microscopy (AFM) imaging Exosomes were resuspended in 100 µl of H2O (Milli-Q, Merck Millipore) and checked for purity and quantified using the nanoplasmonic colorimetric assay as previously described 30. AFM imaging and image analysis were performed adapting the protocols we developed in previous works 28, 31. Briefly, exosomes were resuspended in 100 µl of H2O (Milli-Q, Merck Millipore) and further diluted 1:300 with H2O. Five μL of samples were then spotted onto freshly cleaved mica sheets (Grade V-1, thickness 0.15 mm, size 15 × 15 mm), allowed to dry at room temperature (RT) and imaged by a Nanosurf NaioAFM (Nanosurf AG), equipped with Tap190AI-G tips (Budget Sensors). Images were snapped in tapping mode. The scan size ranged from 0.5 to 15 µm and the scan speed ranged from 0.6 to 1.5 s x line 32. Vesicles size distributions were obtained by image analysis of at least 5 representative AFM images of scan size 8.7 µm X 8.7 µm (> 2200 vesicles overall). Image analysis was carried out using WSxM 5.0 software (http://www.wsxmsolutions.com).

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Sialidase activity Cells were washed twice with PBS, collected by scraping in PBS, centrifuged at 800 g for 10 min at 4°C and the pellet was resuspended in PBS containing Proteases Inhibitors Cocktail. Crude cell extracts, obtained by gentle sonication, were centrifuged at 800 g for 10 min to eliminate unbroken cells and nuclear components Cell media were centrifuged as previously described to separate microvesicle and exosome populations. The resulting pellets, resuspended in PBS containing Proteases Inhibitors Cocktail, were analysed together with crude cell extracts to evaluate the enzyme activity. Sialidase activity was measured using 4methylumbelliferyl α-N-acetylneuraminic acid (4MU-NeuAc) as substrate 6. All reaction mixtures were set-up in triplicate with 10 µl of sample in a final volume of 100 µl in the presence of 12.5 mM sodium citrate/phosphate buffer, pH 3.8, and 0.12 mM 4MU-NeuAc. Samples were incubated at 37°C for 30 min and enzyme reaction was stopped with 1.5 ml 0.2 M Glycine/NaOH, pH 10.8. The amount of sialic acid released was evaluated by spectrofluorimetric measurement of 4-methylumbelliferone.

SDS-PAGE and Western-blot SDS-PAGE and Western-blot were carried out by standard procedures on total cell homogenate and fractions derived from extracellular vesicle purification procedure. Briefly, 30 µg of total cell homogenates was loaded on SDS/10% PAGE. In case of exosomes from On and Off HeLa tTA2 cells, fractions were resuspended in 100 µl of H2O (Milli-Q, Merck Millipore) and quantified using the nanoplasmonic colorimetric assay 30. Finally, equal volume of extracellular vesicles (microvesicles and exosomes) and SN3 were loaded on SDS/10% PAGE. After electrophoresis, the proteins were transferred to a HybondTM-P PVDF membrane (GE Healthcare). Membranes were then blocked with 5% (w/v) non-fat dried

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skimmed milk in PBS, washed three times with PBS containing 0.1% Tween 20 (PBST) and incubated with primary antibodies diluted in PBST containing 1% (w/v) non-fat dried skimmed milk for 1 h at RT. After three washes with PBST, membranes were incubated with HRP-conjugated secondary antibody diluted in PBST for 45 min at RT. The following primary antibodies and dilutions were used: rabbit anti-HA 1:1000 (Sigma), mouse anti-tumor susceptibility gene 101 protein (TSG101) 1:500 (Abcam), mouse anti-Annexin V 1: 500 (Santa-Cruz), Rabbit anti Annexin XI 1: 500 (Genetex), rabbit anti-ADAM10 1:500 (Origene), rat anti-Argonaute2 (AGO2) 1: 1000 (Sigma) and mouse anti-Golgi matrix protein GM130 1:250 (BD Transduction Laboratories). The following HRP-conjugated secondary antibodies and dilutions were used: donkey anti-rabbit 1:5000 (Santa Cruz), goat anti-mouse and goat anti-rabbit 1:3000 (Bethyl) and goat anti-rat 1:3000 (Santa Cruz). Samples were normalized for exosomes concentration when possible 30 or, alternatively, equal volumes of each sample were loaded on SDS-PAGE. Detection of the immunocomplexes was performed using an enhanced chemiluminescence-based system (Western Blot Luminol Reagent; Santa Cruz), followed by densitometric analysis using GelPro 3.1 software (Media Cybernetics). Alternatively, images were acquired using a G:Box Chemi XT Imaging system and quantified using Gene Tools software (Syngene) 33.

Dot Blot assay Exosomes were re-suspended in 100 µl of 100 mM Tris, 150 mM NaCl, 1 mM EDTA (buffer A) for incubation with anti-TSG101 and anti-HA antibodies. Five µl of exosomes diluted 1:1 and 1:5 (v/v) with buffer A were spotted on a nitrocellulose membrane and let dry at RT for 1h without shaking. To detect Ago2, pellets were resuspended in 10 µl of buffer A and 3 µl of exosomes and exosomes diluted 1:5 (v/v) were spotted and treated as described above.

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The resulting membranes were then incubated with TBS + 5% milk (w/v) containing or not Tween-20 0.1% (v/v) for 1h at RT and subsequently incubated with rabbit anti-HA, mouse anti-TSG101 and rat anti Ago2 in TBS + 1% milk (w/v) containing or not Tween-20 overnight at 4°C. After incubation with the primary antibodies, membranes were washed 3 times with TBS containing or not Tween-20 and incubated with HRP conjugated secondary antibodies, namely, goat anti-mouse (Bethyl), goat anti-rabbit (Bethyl) and goat anti rat diluted 1:2000 in TBS + 1% milk with or without Tween-20 for 1h at RT. Finally, membranes were washed 3 times with TBS in presence or without Tween-20 and blot were detected as previously described 34. The antibody dilutions were the same used for Western-blot analysis.

Statistical analysis. Statistically significant differences between On/Off datasets were determined with Student’s t-test (GraphPad Software, Inc.). P values of less than 0.05 were considered statistically significant with **p < 0.01 and ***p < 0.001. Values were shown as mean ± Standard Error of the mean (SEM) of at least 2 experiments.

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Results NEU3 distribution in the extracellular vesicle fractions To assess the possibility that sialidase NEU3 is associated to extracellular vesicles, HeLa tTA2 cells expressing, in an inducible manner, the murine enzyme tagged with the HA epitope at the C-terminus have been used. This cell system has been previously well characterized and allows the expression of NEU3-HA under the control of the tetracycline promoter (TET-Off) 27

. In other words, growth in absence of the tetracycline analogue doxycycline (dox) allows

the expression of NEU3-HA (HeLa-On) while presence of dox in the medium blocks the expression (HeLa-Off). In addition, the murine form of NEU3 has been extensively studied 1 and, in comparison to the human orthologue 35, is more active toward 4MU-NeuAc the artificial fluorescent substrate used in this study to detect sialidase activity. As shown in figure 1 B, after the isolation of two extracellular vesicle populations enriched in microvesicles or exosomes from conditioned media obtained by Hela tTA2-NEU3-HA cells grown in presence (Off) or absence (On) of dox, sialidase activity was measured in each sample. A significant increase of sialidase activity was detected in both microvesicles and exosomes released from HeLa On cells compared to the corresponding fractions obtained from the conditioned media of HeLa Off cells. The ratios between the activity values observed in microvesicles On vs Off and exosomes On vs Off resulted roughly 8- and 6-fold, respectively (Figure 1 B). These observations were confirmed by Western-blot analysis of microvesicle and exosome fractions where the band signal intensities correlate with the sialidase activities measured (Figure 1 C). NEU3-HA was detected only in microvesicles and exosomes On, whereas no immunoreactive material was detected in the corresponding fractions obtained from media of HeLa Off cells. These data indicate that sialidase NEU3 is

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secreted into the growth medium associated to extracellular vesicles maintaining its enzymatic activity. Interestingly, the low but well detectable sialidase activity observed in microvesicles and exosomes Off suggests the presence of endogenous NEU3 in these extracellular vesicles.

Figure 1. A) Schematic representation of the protocol used for isolation of extracellular vesicle populations from conditioned media of HeLa On and HeLa Off cells. B) NEU3 activity, expressed as nanomoles (nmol)/h mg, obtained measuring the 4-methylumbelliferone released after incubation at 37°C for to 30 min, in microvesicles (MV) On/Off and exosome (Exo) On/Off samples. Values represent the average of two independent experiments. Significant differences among On and Off culture conditions were determined with Student’s t –test. P value: ** p< 0.01; *** p< 0.001. C) Western-blot analysis and

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densitometry of microvesicle (MV) and exosome (Exo) pellets from HeLa On and HeLa Off cells for NEU3-HA signal. Results are the mean ± SEM of three independent experiments.

Exosome biophysical and biochemical characterization Since the higher NEU3-HA activity and signal was detected in the exosome fraction, we decided to characterize this vesicle population by using a combination of biophysical and biochemical assays. Exosome size and morphology was determined by AFM. In exosomes isolated from cell culture media in absence (On) or presence (Off) of dox we found the presence of spherical objects with diameters ranging from tens to hundreds nanometers 22 (Figure 2 A and C) matching the typical diameters of exosomes 30, 36-40. In addition, the average thickness of about 30 nm was compatible with the thickness of solid surface supported partially dehydrated membrane particles. The sub-nanometer flatness of the background suggests the absence of smaller nanostructures and protein aggregates. Interestingly, the size distributions obtained from the AFM images (Figure 2 B and D) show that the On and Off exosome populations have different size distributions, characterized by average sizes of 98 ± 22.9 nm and 78 ± 18.7 nm, respectively. These differences, analysed using Student’s t-test, are statistical highly significative with p