title: Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain.
abstract: Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody.
authors.affiliation: Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, California 91320, USA.
authors.forename: Arnold
authors.initials: A
authors.lastname: McAuley
authors.forename: Jaby
authors.initials: J
authors.lastname: Jacob
authors.forename: Carl G
authors.initials: CG
authors.lastname: Kolvenbach
authors.forename: Kimberly
authors.initials: K
authors.lastname: Westland
authors.forename: Hyo Jin
authors.initials: HJ
authors.lastname: Lee
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Brych
authors.forename: Douglas
authors.initials: D
authors.lastname: Rehder
authors.forename: Gerd R
authors.initials: GR
authors.lastname: Kleemann
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
authors.forename: Masazumi
authors.initials: M
authors.lastname: Matsumura
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D019281:Dimerization; D004220:Disulfides / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G