title: Structural consequences of aglycosylated IgG Fc variants evolved for FcγRI binding.
abstract: In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind FcγRs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, FcγRI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between FcγRI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Förster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for FcγR binding and therapeutic effector functions of IgG antibodies.
authors.affiliation: Department of Bio and Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Man-Seok
authors.initials: MS
authors.lastname: Ju
authors.affiliation: Department of Bio and Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea; Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Na
authors.affiliation: Department of Bio and Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors.forename: Yeon Gyu
authors.initials: YG
authors.lastname: Yu
authors.affiliation: Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
authors.forename: Jae-Yeol
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 136-791, Republic of Korea. Electronic address: che.jeong@kist.re.kr.
authors.forename: Cherlhyun
authors.initials: C
authors.lastname: Jeong
authors.affiliation: Department of Bio and Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea. Electronic address: sjung@kookmin.ac.kr.
authors.forename: Sang Taek
authors.initials: ST
authors.lastname: Jung
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D017452:Receptors, IgG / Q000378:metabolism*; D006023:Glycoproteins
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D050505:Mutant Proteins; D017452:Receptors, IgG; C046179:glycosylated IgG; D006023:Glycoproteins
keywords: Aglycosylated antibody; Alternative laser excitation; Antibody engineering; Effector function; Förster resonance energy transfer; Single-molecule analysis