Identification of small compounds regulating the secretion of extracellular vesicles via a TIM4-affinity ELISA.

Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are required. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system using a high-affinity phosphatidylserine-binder TIM4 to capture EVs and screened a 1567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. Moreover, the three activators, namely cucurbitacin B, gossypol, and obatoclax, had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified four compounds that can regulate the bioactivity of EVs.

Oriented immobilization of rBC2LCN lectin for highly sensitive detection of human pluripotent stem cells using cell culture supernatants.

Human pluripotent stem cells (hPSCs) are considered ideal cell sources for regenerative medicine, but their clinical and industrial applications are hindered by their tumorigenic potential. We previously identified an hPSC-specific lectin, rBC2LCN, that recognizes the podocalyxin glycoprotein secreted by undifferentiated hPSCs into the culture media. Using biotinylated rBC2LCN and a peroxidase-labeled R-10G antibody, we developed a sandwich assay for the detection of tumorigenic hPSCs. In this assay, the lectin is randomly immobilized on streptavidin-coated microplates to capture hPSC-derived podocalyxin. In the present study, rBC2LCN was genetically fused with polystyrene-binding peptides (PS-tags) for direct, site-specific, and oriented immobilization on polystyrene microplates. rBC2LCN lectins fused with PS-tags at the C-terminus were successfully overexpressed as a soluble form in Escherichia coli and then purified by affinity chromatography. We optimized the various parameters (protein and NaCl concentration, buffer pH, and blocking agents) of the sandwich assay by using PS-tagged rBC2LCN and the R-10G antibody. Finally, the lower limit of detection (LLOD) of the sandwich assay for hPSCs was examined. The LLOD was 2.2-fold lower than that achieved with the previous method. Considering that the developed method does not require the precoating of polystyrene microplates with streptavidin, it provides a cost-effective approach for the highly sensitive detection of hPSCs residing in hPSC-derived cell therapeutics.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips.