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.

Figure 1.

Model of the human IgG1 C_H3 domain in a dimeric form. The domain structure was truncated from the human antibody IgG1 b12 (Saphire et al. 2001) with a PDB ID of 1HZH. The figures were generated using DS ViewerPro (Accelrys). The viewpoint is parallel (A) and perpendicular (B) to the axis of symmetry with each monomer colored separately, and the Cys and Trp residues are in stick representation. The disulfide linkage of Cys31 and Cys89 in the recombinant C_H3 protein corresponds to that of Cys250 and Cys308 in the human immunoglobulin γ-1 chain C region (Swiss-Prot entry name, IGHG1_HUMAN; primary accession number, P01857). Similarly, Trp45 and Trp81 correspond to the residues at 264 and 300 of the human IgG1 heavy-chain constant region.

Figure 2.

Spectroscopic characterization. (A) Far-UV CD spectra and (B) near-UV CD spectra of reduced (gray) and oxidized (black) forms of human C_H3. The measurements were carried out at 20°C at a protein concentration of 230 μg/mL (8.6 μM dimer). The reduced sample was measured in 2 mM sodium acetate, pH 5.0, and the oxidized sample was in 5 mM sodium acetate, pH 5.0. (C) Fluorescence spectra of reduced (gray) and oxidized (black) forms of human C_H3. The reduced form is shown under native conditions (0.0 M GdmCl; gray solid line) and denaturing conditions (4.8 M GdmCl; gray dotted line). The oxidized form is shown under native conditions (0.1 M GdmCl; black solid line) and denaturing conditions (5.7 M GdmCl; black dotted line). The measurements were performed at 20°C at a protein concentration of 30 μg/mL (1.1 μM dimer). The excitation wavelength was set at 280 nm. The emission maximum occurs in the region of 357 nm in the unfolded state and is shifted to a lower wavelength (∼330 nm) in the folded state. The emission is quenched in the folded state for the oxidized form because of the proximity of the disulfide bond to Trp45 (see Fig. 1).

Figure 3.

(A) Molecular mass determination of reduced (gray) and oxidized (black) human C_H3 homodimer by dynamic light scattering. X-, left Y-, and right Y-axes represent time, molar mass, and UV absorbance at 280 nm (A ₂₈₀), respectively. The inset SE-HPLC chromatograms show the entire chromatograms monitored by A ₂₈₀. (B) SE-HPLC elution times of reduced and oxidized C_H3 (●) were plotted against their molecular masses with the following molecular mass standards: human antibody (IgG, 150 kDa, open squares), bovine serum albumin (BSA, 68 kDa, open triangles), and granulocyte-colony stimulating factor (G-CSF, 14 kDa, open inverted triangles). Both reduced and oxidized C_H3 were eluted at identical elution times.

Figure 4.

(A) Acid-induced denaturation of reduced (△) and oxidized (○) forms of human C_H3 at pH 2.0–8.0. The fluorescence measurements were performed at 20°C at a protein concentration of 70 μg/mL (2.6 μM dimer). The fraction of folded C_H3 was calculated from the red shift of the maximum wavelength of emission spectra at each pH (see details in Materials and Methods). The solid lines were fit to a three-parameter sigmoidal using SigmaPlot software. (B) Thermal unfolding of reduced (△) and oxidized (○) C_H3 domain monitored by CD at 218 nm. The solid lines were fit to a three-parameter sigmoidal plot using SigmaPlot software.

Figure 5.

(A) Guanidinium chloride-induced denaturation of reduced (△) and oxidized (○) forms of human C_H3. Measurements were obtained at 20°C at a protein concentration of 50 μg/mL (1.9 μM dimer) in 10 mM sodium acetate, pH 5.0. Data correspond to emission intensities measured at 357 nm at an excitation wavelength of 280 nm. Signals were normalized relative to unfolded and folded values. The solid lines were fit to the data using Equation 3. (B) Sedimentation equilibrium of reduced (△) and oxidized (○) forms of human C_H3 at varied concentrations of GdmCl. Ultracentrifugation was performed at 20°C at a protein concentration of 50 μg/mL (1.9 μM dimer) in 10 mM sodium acetate, pH 5.0. Data correspond to the molecular mass of C_H3 at equilibrium. Error bars are generated from three data sets.

Figure 6.

Size-exclusion chromatograms of reduced (A) and oxidized (B) human C_H3 with their corresponding molar masses. The Y-axes on the right and left represent UV absorption at 214 nm and molar mass (g/mol), respectively. For reduced C_H3, chromatograms and molar mass are shown at 1.0 M (purple), 1.5 M (blue), and 2.0 M (green) GdmCl. At 1.5 M GdmCl, there is a split peak with two separate molar masses. For oxidized C_H3, chromatograms and molar mass are shown at 1.0 M (purple), 1.5 M (blue), 2.0 M (green), and 2.75 M (red) GdmCl. Approximately 200 μg/mL (7.5 μM dimer) of C_H3 in 10 mM sodium acetate at pH 5.0 was equilibrated at the corresponding GdmCl concentration prior to chromatography.

Figure 7.

Comparison between the C_H3 unfolding monitored by fluorescence (closed symbols; left Y-axis) and the dimer to monomer dissociation monitored by AUC (open symbols; right Y-axis). (A) Reduced form; (B) oxidized form. The fractions of unfolded and dimer were calculated from the data shown in Figure 5, A and B, respectively. The solid lines were fit to a three-parameter sigmoidal plot, (f = a/(1 + exp[−(x − x₀/b)])), using SigmaPlot software.