Abstract
The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the potassium channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (kon = 2.3 ×105 M−1.s−1, koff = 1.3 10−4 s−1), yielding an affinity constant (KD = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications.
Highlights
Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor
This study describes a new method to express full-length complex membrane receptor proteins targeted to exosomes
Successful reconstitution of functional membrane proteins is an empirical process, depending on many factors that must be optimized, including the formation and composition of the lipid environment[2]. We encountered this problem while developing cell-free biosensor assays to measure the affinity and kinetics of BoNT interactions with their membrane receptors
Summary
Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor. The Hc domains of BoNT/A, B and E contain binding sites for a polysialo-ganglioside, in particular GT1b and a protein receptor, which confer neuro-specific binding with KDs in the 0.5 nM range[12,13,14]. This study describes a new method to express full-length complex membrane receptor proteins targeted to exosomes. The exosomes were validated using conformation-dependent ligand binding to the voltage-gated potassium channel Kv1.2 and the G-protein coupled receptor CXCR4 and used to determine the kinetic and equilibrium binding constants of BoNT/B to its receptor
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