Presentation Information
[P04-538]Functional enhancement of G protein-coupled receptors on magnetosomes through co-expression of human intramembrane chaperones
○Kanata Yuasa1, Ryoto Tomoe1, Tomoko Yoshino1 (1. Graduate School of Engineering, Tokyo University of Agriculture and Technology (Japan))
Keywords:
Magnetotactic bacterium,Magnetosome,GPCR,Intramembrane chaperone
[Purpose]
Magnetotactic bacterium, Magnetospirillum magneticum, biosynthesizes intracellular magnetic nanoparticles called magnetosomes. Magnetosomes are membrane-enveloped magnetic particles that can be readily recovered from aqueous solutions using an external magnetic field. This property makes them attractive as a platform for high-throughput ligand screening in drug discovery. In our laboratory, we have developed human transmembrane protein–magnetosome complexes by displaying G protein-coupled receptors (GPCRs) on magnetosomes. However, the GPCRs displayed on magnetosomes show reduced ligand-binding affinity. Recent advances in eukaryotic membrane protein biogenesis have revealed important roles of intramembrane chaperones in membrane insertion and folding. EMC facilitates insertion of the first transmembrane domain (TMD1), whereas PAT complexes support insertion of TMD2 and beyond. This study aimed to improve the ligand-binding affinity of magnetosome-displayed GPCRs by introducing human intramembrane chaperones into M. magneticum.
[Method]
Transformants expressing thyroid-stimulating hormone receptor (TSHR) alone or with EMC or PAT were generated in M. magneticum. Magnetosomes isolated from these strains were designated TSHR-mag and TSHR/EMC or PAT-mag, and TSHR expression on magnetosomes was quantified by ELISA. Ligand-binding affinity was evaluated by determining the dissociation constant (Kd) using TSH as the cognate ligand. Ligand binding in impurity-containing samples was further examined in the presence of fetal bovine serum (FBS) as a model matrix. To assess the general applicability of the intramembrane chaperone co-expression strategy, seven GPCRs were selected and the contribution of EMC to TMD1 expression was evaluated by Western blot.
[Results and Consideration]
TSHR expression levels were nearly identical among magnetosomes isolated from each transformant. Ligand-binding assays showed that TSHR/EMC-mag exhibited an approximately 2.8-fold higher ligand-binding affinity than TSHR-mag. This improvement likely reflects EMC-assisted TMD1 insertion, promoting proper topology and folding of GPCRs. Furthermore, ligand-binding assays in the presence of FBS showed that TSHR-mag was more strongly inhibited than TSHR/EMC-mag, suggesting that EMC co-expression improves binding robustness in complex sample environments. In addition, analysis of EMC-dependent insertion of TMD1 from seven GPCRs showed that the contribution of EMC to TMD1 expression increased as TMD1 hydrophobicity decreased.
[Conclusion]
Our results demonstrate that introducing human intramembrane chaperones into M. magneticum improves the functional expression of TSHR on magnetosomes. EMC co-expression enhanced the ligand-binding affinity of TSHR and may support membrane insertion of GPCRs with less hydrophobic TMD1s. This strategy may expand the applicability of magnetosome-displayed GPCRs for drug screening and discovery of novel therapeutic compounds.
Magnetotactic bacterium, Magnetospirillum magneticum, biosynthesizes intracellular magnetic nanoparticles called magnetosomes. Magnetosomes are membrane-enveloped magnetic particles that can be readily recovered from aqueous solutions using an external magnetic field. This property makes them attractive as a platform for high-throughput ligand screening in drug discovery. In our laboratory, we have developed human transmembrane protein–magnetosome complexes by displaying G protein-coupled receptors (GPCRs) on magnetosomes. However, the GPCRs displayed on magnetosomes show reduced ligand-binding affinity. Recent advances in eukaryotic membrane protein biogenesis have revealed important roles of intramembrane chaperones in membrane insertion and folding. EMC facilitates insertion of the first transmembrane domain (TMD1), whereas PAT complexes support insertion of TMD2 and beyond. This study aimed to improve the ligand-binding affinity of magnetosome-displayed GPCRs by introducing human intramembrane chaperones into M. magneticum.
[Method]
Transformants expressing thyroid-stimulating hormone receptor (TSHR) alone or with EMC or PAT were generated in M. magneticum. Magnetosomes isolated from these strains were designated TSHR-mag and TSHR/EMC or PAT-mag, and TSHR expression on magnetosomes was quantified by ELISA. Ligand-binding affinity was evaluated by determining the dissociation constant (Kd) using TSH as the cognate ligand. Ligand binding in impurity-containing samples was further examined in the presence of fetal bovine serum (FBS) as a model matrix. To assess the general applicability of the intramembrane chaperone co-expression strategy, seven GPCRs were selected and the contribution of EMC to TMD1 expression was evaluated by Western blot.
[Results and Consideration]
TSHR expression levels were nearly identical among magnetosomes isolated from each transformant. Ligand-binding assays showed that TSHR/EMC-mag exhibited an approximately 2.8-fold higher ligand-binding affinity than TSHR-mag. This improvement likely reflects EMC-assisted TMD1 insertion, promoting proper topology and folding of GPCRs. Furthermore, ligand-binding assays in the presence of FBS showed that TSHR-mag was more strongly inhibited than TSHR/EMC-mag, suggesting that EMC co-expression improves binding robustness in complex sample environments. In addition, analysis of EMC-dependent insertion of TMD1 from seven GPCRs showed that the contribution of EMC to TMD1 expression increased as TMD1 hydrophobicity decreased.
[Conclusion]
Our results demonstrate that introducing human intramembrane chaperones into M. magneticum improves the functional expression of TSHR on magnetosomes. EMC co-expression enhanced the ligand-binding affinity of TSHR and may support membrane insertion of GPCRs with less hydrophobic TMD1s. This strategy may expand the applicability of magnetosome-displayed GPCRs for drug screening and discovery of novel therapeutic compounds.
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