Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles

Proceedings of the National Academy of Sciences of the United States of America
David TuckerJoseph B Rucker

Abstract

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states o...Continue Reading

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Citations

Jun 9, 2019·The Journal of Biological Chemistry·Amira KlipDavid E James
Feb 9, 2020·Nature Communications·Sabarinath V RadhakrishnanDjordje Atanackovic
May 4, 2021·Journal of Parasitic Diseases : Official Organ of the Indian Society for Parasitology·Aya Al-NaseriNoha Mahana
Jul 17, 2021·Trends in Pharmacological Sciences·Ellen GulezianSunyia Hussain
Sep 18, 2021·Proceedings of the National Academy of Sciences of the United States of America·Shengya CaoNadia Martinez-Martin

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