Developing a Bio-ID method for elucidating the interactome of Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF)

Bio-ID is a technique that utilizes biotin ligase enzyme to biotinylate proteins that are in close proximity to a target protein, allowing the detection of transient protein-protein interactions. Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) is a neurotrophic factor that has been studied for its growth-promoting properties in the peripheral nervous system (PNS). However, the receptor and the interactome of MANF have not been identified.

In this study, we developed a Bio-ID protocol to study MANF’s interactome. We generated lentiviruses that carry Dox-inducible biotin ligase BirA (L-Dox-BirA) and MANF-BirA (L-Dox-MANFBirA). They were transduced in rat primary Schwann cells. Stably transduced cells were selected with puromycin, and gene expression was induced by Doxycycline supplementation.

Immunostaining and western blot experiments confirmed the expression of BirA and MANF-BirA in these cells. We then supplemented biotin to the cells to biotinylate proteins closely associated with MANF. BirA mediated biotinylation was also confirmed using fluorescence staining and western blot. Finally, the biotinylated proteins were pulled down using streptavidin coated beads for LC-MS/MS analysis and detection of MANF’s interactome. Overall, this study developed a protocol and generated sufficient samples for Bio-ID-based elucidation of MANF’s interactome.