Introduction:: The blood-brain barrier (BBB) is a highly selective and dynamic interface made up of brain microvascular endothelial cells that regulates the exchange of molecules between the blood and the brain. Protein-protein interactions that occur on the cell surface of the BBB are central to both its function and dysfunction. Understanding these interactions is of interest from both a physiological and therapeutic perspective. Peptide-targeted proximity labeling is a powerful tool for identifying protein-protein interactions but has scarcely been used to study the BBB. Recently, TurboID, a novel proximity labeling system with fast kinetics was introduced. In the presence of ATP and biotin, the TurboID biotin ligase covalently tags proximal lysine residues with biotin. When fused to targeting peptides, the TurboID biotin ligase can be used to tag the peptide binding partners in the native cell environment. Tagged proteins can then be isolated and identified. Here, we designed and tested novel peptide-targeted TurboID fusion constructs to examine cell surface protein-protein interactions at the BBB.
Materials and Methods:: We designed numerous recombinant peptide-targeted TurboID fusion constructs in which the TurboID biotin ligase was fused to peptides purported to interact with key proteins expressed on the BBB surface (Fig. 1A). The peptides introduced into our designs included GRGDSP, AP2, TfR-T12, HAIYPRH and amyloid-beta 1-42. These peptides were incorporated into the fusion constructs with varying valency. The TurboID biotin ligase with no peptide fusion was used as a control. The fusion constructs were recombinantly expressed in E. coli BL21 cells. IMAC purification of the constructs was performed using nickel columns. To assess the intramolecular biotinylation activity of the fused TurboID biotin ligases, the constructs were incubated in solution with sfGFP in the presence or absence of ATP and biotin. Specific peptide-directed binding of these constructs was assessed by performing pull-down assays in which the constructs were immobilized on agarose beads and panned with binding partners. We developed a plate-based assay to assess peptide-directed labeling by these constructs. Binding partners were immobilized and then panned with the TurboID constructs, ATP, and biotin. After rigorous washing, biotinylayion was detected using HRP-streptavidin conjugates. Labeling activity and specificity was tested as a function of pH, assay buffer, and time. For in vitro labeling studies, immortalized brain microvascular endothelial cells (hCMEC/D3 cells) were grown to confluence. The cell monolayers were panned with TurboID constructs, ATP, and biotin for a range of times to allow for surface binding and labeling. After labeling was complete, cells were either fixed for immunofluorescence staining or western blot analysis.
Results, Conclusions, and Discussions:: The TurboID biotin ligase in each of the constructs was functional and could perform intramolecular biotinylation. Of the tested constructs, the GRGDSP-TurboID fusion constructs exhibited the greatest binding specificity to their intended target, αVβ3 integrin. In addition to binding specificity, the GRGDSP constructs also exhibited the highest level of specific biotinylation of their immobilized targets that was concentration-dependent and could be tuned through adjustment of the pH (Fig. 1B). When panned on the surface of hCMEC/D3 cells, biotinylayion of the cell surface could be observed prior to substantial internalization of the constructs. However, less specificity was observed in this format. Overall, this study provides a foundation for using TurboID-mediated proximity labeling for studying protein-protein interactions at the BBB. To the best of our knowledge, this is the first instance in which TurboID fusion constructs were expressed for subsequent cell-surface labeling and the first application of this technology to the BBB. Future studies will focus on improving the specificity of TurboID labeling activity on the cell surface.
