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    • The fact that the BPloopAsn regains

    2024-03-28

    The fact that the α1-BPloopAsn141 regains its α-Btx binding with a Kd similar to that of the wild type α1 ECD [18], suggests that the AChBP Cys-loop does not interfere with the correct conformation of the mutant ECD. Data from analogous mutants of the human neuronal α7 subunit ECD showed improved ligand binding affinities of the BPloop mutant [21]. Recently, studies on the complex between α-Btx and an α7/AChBP chimera revealed the key residues involved in the interaction within loop C of the ECD, namely Tyr184 and its flanking residues, while it appears that the same residues are involved in the α-Btx interaction with the α1 ECD as well [13], [23], [24]. Although the non-α1 subunits do not normally bind any ligands on their own, the improved antibody binding of the BPloop ECDs is again indicative of a conformation close to the native AChR. Such an improvement has been shown in detail for the γ ECD mutants [17]. Taken together, all of the above support that the BPloop mutants can be useful tools for the structural study of the ECDs of the AChR. The AChR forms a ring-shaped heteropentamer, where the inter-subunit interfaces play a crucial role for functions such as subunit assembly and formation of ligand Vancomycin hydrochloride synthesis [24]. Simultaneous expression of the Torpedo californica α, β, γ and δ ECDs in insect cells resulted in the formation of pentameric complexes resembling the entire ECD of the AChR [25]. Unfortunately, the attempts we made to co-express the ECDs in yeast failed to result in any kind of interaction among them. Therefore, we proceeded to covalently link the ECDs to form concatamers. Similar approaches have been used in the past to study other members of the AChR family [26]. Dimers of the whole α4 and β2 neuronal AChR subunits or a complete pentameric β4-β4-α3-β4-α3 concatamer have been used to produce functional α4β2 or α3β4 receptors in oocytes, respectively [27], [28]. More recently, this approach has been successful in creating the ligand binding site between the neuronal α4 and β2 AChR using linked ECDs rather than the whole subunits, expressed in P. pastoris[29]. The concatamers have the added advantage that only pentamers with a specific subunit composition and sequence are produced, allowing for subunit- and receptor type-specific studies [30], [31], [32]. To this end, the two pentameric β1-δ-α1-γ-α1 and β1-δ-α1-ɛ-α1 concatamers were constructed using the BPloop mutants of the ECDs. However, when we expressed these concatamers we encountered a number of difficulties. In more detail, the expression yield was decreased for both proteins, while the chromatograms of the two pentamers revealed the extensive formation of aggregates and the presence of lower molecular weight fragments (Fig. 4b and c). The large size of the pentamer was thought to be responsible for the low yields, while the presence of the flexible linker could make them especially susceptible to proteolytic damage. Furthermore, we did not see an improvement in 125I-α-Btx binding compared to the monomeric α1-BPloopAsn141, suggesting that the concatamer ECDs did not form a proper binding site between two adjacent subunits. Expression of smaller dimer and trimer concatamers that arose as intermediates during the pentamer construction was also examined. Initially, the α1-γ, γ-α1, α1-ɛ, ɛ-α1 and α1-γ-α1 combinations were tested, which are of interest due to the possible formation of the ligand binding site in the inter-subunit interfaces, should they assemble correctly. Unfortunately, none of these concatamers appeared to form a native ligand binding site, as similarly to the pentamers, the binding affinity for 125I-α-Btx was the same as the monomeric α1-BPloopAsn141 (data not shown). On the other hand, expression of the α1-β1 and β1-α1 dimers proved very successful since they both had good yields and very satisfactory size exclusion chromatograms. These dimers were studied because they carry the majority of epitopes involved in MG. Although the individual ECDs are useful in the study of MG pathophysiology, our team is developing a therapeutic strategy for MG, based on the selective depletion of anti-AChR antibodies from patients’ serum [33]. In this respect, the use of a single polypeptide rather than separately expressed ECDs would be highly beneficial. The proteins obtained here could be excellent tools for this application. Therefore, further detailed characterization is underway with respect to their antigenicity and other relevant attributes.