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. 2011 May 16;74(6):796-804.
doi: 10.1016/j.jprot.2011.02.024. Epub 2011 Mar 8.

Protein profile of exosomes from trabecular meshwork cells

W D Stamer  1 , E A Hoffman, J M Luther, D L Hachey, K L Schey
Affiliations

Protein profile of exosomes from trabecular meshwork cells

W D Stamer et al. J Proteomics. .

Abstract

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.

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Figures

Figure 1
Figure 1. Venn Diagrams showing protein profile overlap between TM and urine exosomes and ExoCarta database
Panel A compares TM (blue) and urine (yellow) proteins identified by Sequest/ProteoIQ analysis using UniProt accession numbers. Panel B compares the TM (blue) and urine (yellow) proteins to those in the entire ExoCarta database (green) using Gene Symbols. Panel C compares the 143 most commonly observed exosomal proteins in ExoCarta database with expression profile observed for TM and urine exosome proteome. Total TM and urine proteins that met the search criteria were compared to the ExoCarta database using the Venn diagram generator found at: http://bioinfogp.cnb.csic.es/tools/venny/index.html
Figure 2
Figure 2. Analysis of exosomal marker proteins in extracellular membranes prepared from conditioned media taken from human trabecular meshwork cell monolayers
Membranes isolated by differential centrifugation were floated into linear sucrose gradients, fractions were collected and proteins were analyzed by SDS-PAGE/Western blotting (panel A). Shown are representative blots from one TM cell strain of three that were tested. Protein content of fractions was determined by probing with antibodies specific to Annexin-2, Annexin-5, GAPDH, Emilin-1, Myocilin and Neuropilin-1. Relative protein distributions across fractions were quantified by densitometry and graphed as percent of total abundance for each protein across gradient (panel B). Linearity of gradients was verified by refractometry (inset, panel B).
Figure 3
Figure 3. Effect of dexamethasone or dexamethasone plus ionomycin on release of exosomes from trabecular meshwork cell monolayers
Exosome release was monitored by abundance of exosomal marker proteins in the lipid organizing class (annexin-1, -2, -5 and flotilin-1) in exosomal fraction by SDS-PAGE/Western blotting (panel A). Marker protein content was compared to samples of total cell lysate (CL) prepared from human trabecular meshwork cell monolayers. Effects of drug treatments on protein abundance in exosome fraction were determined using densitometry (panel B). Thus, dexamethasone (dex; 100 nM for 5 days) and dex plus ionomycin (iono; 100 μM for 3 hours) treatments were compared to untreated controls (con). Shown are combined data from a total of 5 independent experiments testing 5 different TM cell strains.
Figure 4
Figure 4. Effect of treatments on abundance of marker proteins in exosomes prepared from trabecular meshwork cell monolayers
Exosome release was monitored by abundance of exosomal marker proteins in the tetraspanin category (CD-9, CD-81 and syntenin-1) plus GAPDH in exosomal fraction by SDS-PAGE/Western blotting (panel A). Marker protein content was also monitored in total cell lysates (CL) prepared from human trabecular meshwork cell monolayers. Effects of drug treatments on protein abundance in exosome fraction were determined using densitometry (panel B). Thus, dexamethasone (iono; 100 nM for 5 days) and dex plus ionomycin (iono; 100 μM for 3 hours) treatments were compared to untreated controls (con). Shown are combined data from a total of 3–5 independent experiments testing 3–5 different TM cell strains.
Figure 5
Figure 5. Effect of treatments on appearance of TM-specific markers in exosomes prepared from trabecular meshwork cell monolayers
In response to treatment with dexamethasone (dex) or dexamethasone plus ionomycin (iono), abundance of TM-specific proteins (cyclophilin-B, emilin-1, myocilin and neuropilin-1) in exosomes was monitored by SDS-PAGE/Western blotting (panel A). Marker protein content was also monitored in total cell lysates (CL) prepared from human trabecular meshwork cell monolayers. Effects of drug treatments on protein abundance in exosome fraction were determined using densitometry (panel B). Thus, dexamethasone (100 nM for 5 days) and dex plus ionomycin (100 μM for 3 hours) treatments were compared to untreated controls (con). Shown are combined data from a total of 5 independent experiments testing 5 different TM cell strains.
Figure 6
Figure 6. Comparison of exosomal proteins present in human TM exosomes to those prepared from human urine and aqueous humor
Exosomes from TM conditioned media, aqueous humor (AH) and urine (UR) were prepared by differential centrifugation and analyzed by SDS-PAGE/Western blotting. Panel A shows comparison of lipid organizing protein content (annexin-1, -2 and -5 and flotillin-1); panel B shows comparison of tetraspanin proteins (CD-9, CD-81 and syntenin-1) and GAPDH content; while panel C shows TM-specific (cyclophilin-B, emilin-1, myocilin, neuropilin-1) versus urine-specific (podocalyxin) protein profile. Data shown are representative examples of exosomal samples that were prepared from 5 different TM cell strains. Urine samples and aqueous humor samples were pooled from several different donors before exosomal preparation and analysis. During preparation of samples, purified exosomes from each cell strain/bodily fluid were concentrated to 100X original volume before loading onto gels.
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