Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain

BBAPAP-39544; No. of pages: 10; 4C: 4, 5, 7, 8 Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap

Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain Kazumasa Sakurai a,b,⁎, Ryosuke Nakahata b, Young-Ho Lee b, József Kardos c,d, Takahisa Ikegami b,1, Yuji Goto b a

High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Biochemistry, Eötvös Loránd University, Pázmány P. sétány 1/C, Budapest, H-1117 Hungary d MTA-ELTE NAP B Neuroimmunology Research Group, Eötvös Loránd University, Pázmány P. sétány 1/C, Budapest, H-1117 Hungary b c

a r t i c l e

i n f o

Article history: Received 8 December 2014 Received in revised form 13 February 2015 Accepted 26 February 2015 Available online xxxx Keywords: NMR Aggregation Immunoglobulin fold Disulfide Calorimetry

a b s t r a c t Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The CH3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the CH3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the CH3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetry (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a lessspecifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. Antibody-dependent cellular cytotoxicity, which results from the binding of an antibody to a targeted marker protein, has been applied to the treatment of various kinds of cancer [1]. However, protein-based drugs are generally more prone to aggregation than chemical compoundbased drugs, and previous studies have investigated the aggregation propensities of antibodies under various conditions [2–7]. Of the different antibody classes, IgG1 is mainly used for antibody preparation. An IgG1 molecule consists of two light chains and two heavy chains. The former and latter consist of 2 and 4 immunoglobulin domains, respectively. Each domain has a single disulfide bond. It is historically known that an antibody molecule can be digested by papain into three fragments, two Fab and one Fc fragments. The Fc fragment is composed of two identical polypeptide chains, each of which consists of

⁎ Corresponding author at: High Pressure Protein Research Center, Institute for Advanced Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan. Tel.: +81 736 77 0345x5004. E-mail address: [email protected] (K. Sakurai). 1 Present address: Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.

the CH2 and CH3 domains. Sumi and Hamaguchi [8] reported that refolding of the Fc fragment was biphasic; local folding at the CH3 region was followed by that at the CH2 region. Latypov et al. [4] also showed that the CH2 domain was less stable than CH3 and the aggregation propensity of Fc was dependent on the stability of the CH2 domain. However, other studies suggested the relevance of the CH3 domain in the formation of antibody aggregates. The disulfide bonds in some CH3 domains are known to be reduced in recombinant human monoclonal antibodies [9,10]. The lack of an intramolecular disulfide bond may decrease the stability and increase the aggregation propensity of the antibody molecule. One CH3 domain consists of two β-sheets connected by the intramolecular disulfide bond between Cys 27 and Cys 85, which is located in the hydrophobic core. CH3 domains form homodimers in their native state. The dimerization of the CH3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. Five residues, Thr 26, Leu 28, Phe 65, Tyr 67, and Lys 69, appear to be relevant to the stability of the CH3 dimer. Furthermore, the isomerization of Pro 34 has been suggested to control dimerization [11]. McAuley et al. [12] reported that the reduced form of CH3 had similar secondary and tertiary structures to the oxidized form and retained a dimer structure. However, guanidine hydrochloride (GdnHCl) denaturation experiments indicated that the dimerization affinity as well as stability of the monomer structure of reduced CH3 was lower than those of

http://dx.doi.org/10.1016/j.bbapap.2015.02.020 1570-9639/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

2

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

the oxidized form. Their findings suggested that disulfide bonds bring not only increases in stability but also favorable alterations in the protein conformation for certain functions (e.g., the tight dimerization for CH3). In the present study, we investigated the effects of the reduction of the internal disulfide bond in the CH3 domain on conformational stability and aggregation propensity. To address the stability of the oxidized and reduced CH3 domains, we performed 2D NMR measurements, hydrogen/deuterium (H/D) exchange experiments, ANS fluorescence measurements, and thermal unfolding experiments monitored by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). Thermal unfolding measurements showed that the stability of the reduced form was lower than that of the oxidized form as expected. The reversibility of the reduced form was also significantly lower than that of the oxidized form. It should be noted that, although a dominant fraction of the reduced form retained a fairly stable dimeric structure, there were also less-specifically associated oligomers characterized by more labile interactions between monomers and a large dimension. Formation of such oligomeric adducts might be caused by the monomers with an incompletely folded conformation due to the removal of the disulfide bond. 2. Materials and methods 2.1. Expression and purification of the CH3 domain An expression vector for Pichia pastoris was constructed cDNA encoding the human IgG1 CH3 domain was amplified by PCR using the primers 5′-gtatctctcgagaaaagagaggctgaagcttacgtaggctcctcg and 5′cttgctgcgaattctcatttacccggag. The amplified DNA fragment was digested with Xho I and Not I, and cloned into the P. pastoris expression vector pPIC9 (Invitrogen), resulting in pPIC/CH3. pPIC/CH3 was digested with Aatl and integrated into the genomic DNA of the P. pastoris GS115 strain. The transformant that showed the largest expression in a small-scale test tube culture was selected. Fermentation of the selected strain in the synthetic medium was carried out at 30 °C in a 2-L jar fermentor (Mitsuwa Biosystem) as described previously [13]. After the wet cell weight exceeded 150 mg mL−1, methanol was added to the medium for the induction of protein expression. The expressed protein was secreted in the bulk medium. The supernatant of the harvested culture was desalted using a G-25 column. The protein fraction was applied to CM-sepharose equilibrated with 20 mM sodium acetate (pH 4.5) and eluted with a 0–1.0 M NaCl gradient. After the CH3 fraction was dialyzed against 20 mM Tris (pH 8.0), the solution was applied to DEAE-sepharose and eluted with a 0–0.5 M NaCl gradient. The purified protein was then dialyzed against distilled water and lyophilized. The expressed CH3 domain was confirmed to be in the oxidized state by reverse-phase HPLC. The sample solution was applied to a C4 column and eluted with a 10–90% acetonitrile gradient with 0.5% trifluoroacetic acid. The oxidized and reduced forms were previously shown to be eluted at 24.0 min and 25.0 min, respectively. MALDI-TOF mass spectrometry (Bruker Daltonics) revealed that the expressed species had 5 (Tyr-Val-Gly-Ser-Ser-) additional amino acid residues to the Nterminal (Gly) of the authentic CH3 domain. Additional residues were derived from the signal sequence in the vector.

desired buffers for subsequent experiments. The reduction of the disulfide bond was confirmed by reverse-phase HPLC. 2.3. Fluorescence and static light scattering measurements Fluorescence was measured on a F7000 spectrofluorometer (Hitachi, Tokyo, Japan) with an excitation wavelength of 295 nm at 4.0 μM protein in 50 mM phosphate buffer (pH 6.0) at 20 °C. Static light scatterings were measured with F7000 spectrofluorometer at 25 °C. The excitation and detection wavelengths were 500 nm. Protein solutions of 4.0 μM were added to 50 mM phosphate buffer (pH 6.0). 2.4. CD measurements CD spectra were measured with a J720 spectropolarimeter (JASCO, Tokyo, Japan). Regarding far-UV CD spectra, the protein concentration was 0.20 mg mL − 1 and a 1-mm path-length quartz cuvette was used. Regarding near-UV CD spectra, the protein concentration was 1.0 mg mL− 1 and a 1-cm path-length quartz cuvette was used. Measurement conditions were 50 mM phosphate buffer (pH 6.0) and 20 °C. The spectrum of the buffer solution was subtracted from the sample spectra. The sample conditions for the measurement of thermal unfolding were 0.20 mg mL − 1 C H 3 and 50 mM phosphate buffer (pH 6.0). A 1-mm path-length quartz cuvette was used. Far-UV CD spectra were measured at 20 °C and samples were then heated from 20 °C to 90 °C with a 0.5 °C min− 1 scanning rate by monitoring the CD value at 218 nm. Far-UV CD spectra were then measured at 90 °C. 2.5. ANS fluorescence experiments The GdnHCl concentration dependence of 1-anilino-8naphthalenesulfonate (ANS) fluorescence was measured with excitation at 350 nm in the presence of CH3 molecules. The measurement conditions were 8.2 μM (oxidized form) or 6.5 μM (reduced form) protein, 10 μM ANS, 50 mM phosphate buffer (pH 6.0), and 25 °C. 2.6. Analytical ultracentrifuge analysis Sedimentation equilibrium measurements were performed on a Beckman XL-A instrument (Beckman) at 20 °C, and UV absorbance was monitored at 278 nm. The protein concentrations and solvent conditions were 0.3, 0.6, 0.9 mg mL-1, and 50 mM phosphate buffer (pH 6.0), respectively. Samples were centrifuged at a rotor speed of 25,000 rpm for 24 h (oxidized form) or 21 h (reduced form). The data were globally fitted to the theoretical equation [14] for the apparent molecular weight. 2.7. DSC experiments DSC measurements were performed on a VP-DSC instrument (MicroCal Inc.). A CH3 concentration of 0.30 mg mL− 1 was used in 50 mM phosphate buffer (pH 6.0). If not specified, the scan rate was 1 °C min−1. The data obtained were analyzed using the software of Igor Pro. In the pH titration experiments, the buffer reagents used were 50 mM phosphate buffer (pH 6.0 and 6.5) or 50 mM sodium acetate (pH 4.0, 4.5, 5.0 and 5.5).

2.2. Preparation of the reduced CH3 domain 2.8. NMR experiment The purified protein was dissolved and incubated overnight in 6 M urea and 100 mM Tris–HCl (pH 8.0) with 50 mM TCEP (tris(2carboxyethyl)phosphine) at room temperature. Urea and TCEP were removed using a PD-10 column and the protein was refolded in 50 mM phosphate buffer (pH 6.0) containing 0.45 M L-arginine for 1 h at room temperature. The solvent of the sample was exchanged to the

Sequential assignments of the main-chain atoms of oxidized CH3 were performed on 15N, 13C double-labeled CH3 at pH 6.5 using an Avance-I 800 MHz spectrometer equipped with a TXI cryogenic probe. The NMR pulse sequences used for the assignments were CBCA(CO)NH, HNCACB, HNCO, and HNCACO. Other NMR measurements for the

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

oxidized and reduced forms were performed at 37 °C on a Bruker AV400M spectrometer or DRX-500 spectrometer equipped with a room temperature probe. Spectra were processed using nmrPipe [15] and analyzed with Sparky (Goddard and Kneller, SPARKY 3, University of California, San Francisco). Regarding HSQC measurements, the sample conditions were 1 mg mL-1 in 20 mM phosphate buffer (pH 6.0) in 10%(v/v) D2O. 2.9. H/D exchange experiments with NMR H/D exchange experiments were performed at 37 °C. In the case of oxidized CH3, the exchange was initiated by the manual dissolution of lyophilized protein in 20 mM phosphate buffer (pH* 5.6) in D2O at a protein concentration of 6.0 mg mL−1. In the case of reduced CH3, the exchange was initiated by a buffer exchange to 20 mM phosphate buffer (pH* 5.6) in D2O using a PD-10 column. The protein concentration was 0.8 mg mL−1. The dead times for the oxidized and reduced forms were 10 min and 30 min, respectively. Approximately 60 residues had disappeared for both forms by the first measurement (Fig. 7A, B, down triangles). The exchange periods were one and a half months. We calculated the lowest protection factors (PF), considering the total length of the experimental time, for the residues whose peak intensity did not decay less than 80% of the initial intensity (Fig. 7A, B, upper triangles). Regarding residues that showed an analyzable decay of signal intensity, decays were fit to a single exponential to give the apparent rate constants of exchange (kex). The intrinsic rate constants of exchange (kint) were calculated using the SPHERE program (http://www.fccc.edu/research/labs/ roder/sphere/). PF was calculated as kint/kex. 3. Results 3.1. The structure of CH3 expressed by Pichia pastoris We attempted to express CH3 in P. pastoris to obtain recombinant CH3 samples because the secreted expression system of P. pastoris is known to assist in disulfide bond formation in the expressed protein [16], which appeared to be suitable for the expression of CH3. We obtained a relatively large amount of CH3 (~100 mg/1 L broth on average). The CH3 molecules were obtained in the oxidized form (Fig. 1A). To

3

prepare reduced CH3, the sample was treated with TCEP. The reduction of the disulfide bond was confirmed by a longer retention time in reverse-phase HPLC (Fig. 1A) and larger fluorescence intensity (Fig. 1B), which are common characteristics in immunoglobulin proteins [17–20]. This process was in contrast to those from the E. coli expression system described by McAuley et al. [12], in which CH3 was expressed as the reduced form and its oxidized form was prepared from the obtained CH3. The spectra of Trp fluorescence, far-UV CD, and near-UV CD of our samples (Fig. 1B-D) were almost identical to that of the CH3 samples obtained from the E. coli system (Fig. 2A–C in McAuley et al. [12]). In addition, the sedimentation equilibrium analysis estimated the molecular weights of the oxidized and reduced forms to be 26.8 and 25.3 kDa, respectively, suggesting that both forms may have existed in the dimeric state (Fig. 1E, F), which is also consistent with previous reports [12]. Thus, we concluded that the CH3 sample obtained from P. pastoris is identical to that from E. coli. However, the reduced form of CH3 occasionally formed visible aggregates in solution (Fig. 2). These aggregates appeared after the reduced form had been prepared, indicating difficulties in controlling the formation of aggregates. These visible aggregates were removed by filtration prior to the following measurements. 3.2. Thermal unfolding experiments To investigate the stability of both forms of the CH3 domain, thermal unfolding was monitored by CD (Fig. 3) and DSC (Fig. 4) at pH 6.0. CD measurements showed that both forms became completely unfolded above 90 °C (Fig. 3A, B). Although both forms showed cooperative thermal unfolding transitions (Fig. 3C), the temperature range of the transition of the reduced form was wider than that of the oxidized form, indicating that the thermal transition of the reduced form was less cooperative. (This is later confirmed quantitatively by a lower ΔH value for the transition. See Table 2.) The apparent melting temperatures (Tm) of the oxidized and reduced forms were 82.3 °C and 73.9 °C, respectively. DSC measurements showed that the thermograms of both forms had one major peak (Fig. 4A, B, black lines). The Tm values of the oxidized and reduced forms were 80.9 °C and 74.6 °C, respectively, which was

Fig. 1. Comparison of structural properties between oxidized and reduced CH3. (A) The assay for the redox state of the disulfide bond using reverse-phase HPLC. The sample expressed in the P. pastoris system and the reduced sample after the reduction treatment were eluted at 24 min and 25 min, respectively. (B) Fluorescence spectra of both forms. (C, D) The far- and near-UV CD spectra of the oxidized and reduced forms. In panels A–D, data from the oxidized and reduced forms are drawn in continuous and broken lines, respectively. (E, F) Data of ultracentrifuge measurements for the oxidized (E) and reduced (F) forms. The continuous lines are theoretical curves fitted to the data for the apparent molecular weight.

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

4

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Fig. 2. Formation of visible aggregates upon the preparation of the reduced form of CH3. (A) Pictures of the solutions of the oxidized and reduced forms of CH3. The solution of the reduced form was turbid. (B) Static light scatterings of the oxidized and reduced forms. The error bars in (B) are 5σ of the deviation during a 10-second measurement.

consistent with the values obtained by CD, and confirmed that the oxidized form was thermally more stable than the reduced form. Thermal unfolding of the oxidized form was highly reversible (2nd scan: 87%, 3rd scan: 74% in ΔH values, Fig. 4A), whereas that of the reduced form was irreversible because of precipitation, as indicated by the severe drops on the DSC thermogram (Fig. 4B). It is noted that the thermogram of the reduced form (Fig. 4B, black line) contained a minor peak at a lower temperature than the main

peak (around 63 °C, indicated by an arrow). When the scan rate was decreased from 60 °C/h to 30 °C/h, the minor peak of the reduced form became larger (Fig. 4D, blue line). Such a phenomenon was not observed for the oxidized form (Fig. 4C). At a higher concentration of the reduced CH3, the minor peak became larger, suggesting intermolecular interactions (Fig. 4E). From these observations, the minor peak may indicate the existence of an oligomeric state distinct from the main components. We discuss the minor peak in more detail below. We estimated ΔCp values from differences in the baselines of thermograms of the folded and unfolded states extrapolated to the temperature of Tm. The calculated value of the oxidized form was 1.40 ± 0.06 kcal mol− 1 K− 1 (or 5.86 ± 0.25 kJ mol− 1 K−1: error is the fitting error) (Fig. 4F, double headed arrow). On the other hand, we could not estimate ΔCp for the reduced form because of a distortion in the thermogram, which was attributed to irreversible precipitation. To obtain the ΔCp value in another way, we changed the pH to a range of pH 4.0 to 6.5 and obtained the Tm and ΔH values of the oxidized form using DSC. ΔH values were plotted against Tm and fit to the relationship, ΔCp = (∂ΔH/∂Tm)p. The ΔCp value was 1.99 ± 0.26 kcal mol −1 K −1 (or 8.33 ± 1.09 kJ mol− 1 K−1) (Fig. 4H, black line), similar to the value estimated above. We also performed the same measurements for the reduced form. However, we failed to obtain reliable ΔCp value because we could not obtain a thermogram below pH 5.0 because of irreversible precipitation and, even above pH 5.5, the thermograms were highly distorted and reliable ΔH values were not obtained (Fig. 4H, red markers). 3.3. Model fitting of thermal denaturation data We performed global fittings of experimental data for the thermodynamic parameters of thermal unfolding. We considered three unfolding models, and analytically derived theoretical equations for thermal denaturation curves based on respective models in the following way. Model A postulated a transition between the monomeric native state and monomeric unfolded state (i.e., N ⇄ U), expressing the equilibrium constant, KA, as [U]/[N]. Model B was a two-state transition between the dimeric native state and monomeric unfolded state (i.e., N2 ⇄ 2U). The equilibrium constant for model B was calculated as follows: KB ¼

½U  ½N 2 1=2

ð1Þ

This equilibrium constant was expressed in terms of a change in the state of one CH3 molecule (i.e., 1/2N2 ⇄ U). We treated the equilibrium for the dimer dissociation model in this form to obtain ΔH values per one monomer. Model C was a three-state transition including the dimeric native, monomeric native, and monomeric unfolded states (N2 ⇄ 2N ⇄ 2U). The equilibrium constants for the first and second steps were the same as KB and KA, respectively. The temperature dependence of the equilibrium constants was calculated as follows;
 ΔH 0 ð1−T=T 0 Þ−ΔC p ððT 0 −T Þ þ T ln ðT=T 0 ÞÞ K ðT Þ ¼ exp − RT

Fig. 3. (A, B) Far-UV CD spectra of the oxidized (A) and reduced forms (B). The spectra obtained at 20 °C before temperature scans are shown in black. The spectra obtained at 95 °C (oxidized form) or 90 °C (reduced form) are shown in red. (C) Thermal unfolding reactions of the oxidized (black) and reduced forms (red) were monitored at 218 nm. Blue lines represent the theoretical curves obtained from the global fitting analysis based on Eq. (3) in the main text.

ð2Þ

where R is the gas constant, T is absolute temperature, T0 is the temperature where K = 1, and ΔH0 is the enthalpy change in the reaction at T0. Regarding the monomer–dimer transition (i.e., model B and the first step of model C), Tm ≠ T0, and Tm was dependent on the total protein concentration. We calculated the molar fraction, f, of each state with K obtained from Eq. (2). See Supplementary Methods for how to introduce f. The CD curves were expressed as follows; SðT Þ ¼

X

f X ðSX þ mX T Þ

ð3Þ

X

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

5

Fig. 4. DSC profiles of the CH3 domain under various conditions (A–E) and results of fitting analyses (F–H). (A, B) Reversibility check of thermal unfolding for the oxidized (A) and reduced (B) forms, respectively. The black, red, and blue lines indicate the 1st, 2nd, and 3rd scans of the same sample. In panel (B), the arrow indicates the position of the minor peak. (C, D) Scanrate dependences of thermograms for the oxidized (C) and reduced (D) forms. The black, red, and blue lines indicate thermograms obtained at scan rates of 60, 30, and 90 °C/h, respectively. (E) Concentration dependences of thermograms for the reduced form. The black and red lines indicate thermograms obtained at protein concentrations of 0.3 and 0.45 g/L, respectively. (F,G) Comparison of fitting models for the oxidized (F) and reduced (G) forms, respectively. Data obtained at 60 °C/h and 0.3 g/L were used. The theoretical curves of the best-fit model for each set of data are indicated by red lines. The broken and dotted green lines in (F) are pre- and post-transition baselines for the evaluation of ΔCp values. The amplitude of ΔCp was shown by the double-headed arrow. (H) Plots of ΔH values against Tm at various pH. Data from the oxidized and reduced forms are colored in black and red, respectively. The black line indicates the fitting result for the ΔCp value of the oxidized form.

where X indicates the conformational state, and SX and mX are the spectroscopic signals and its temperature dependences of the corresponding state, respectively. The DSC curves for model A and B were expressed as follows; SðT Þ ¼ −

df initial ΔH ðT Þ þ baseline dT

ð4Þ

where ΔH(T) is enthalpy change at T. The actual forms of the theoretical equations for model B and C and the precise derivations of them were summarized in Supplementary Methods. Although the CH3 samples in the present study showed some irreversibilities, we ignored them and fit the data as if unfolding had been reversible. In model fitting for both forms, the ΔCp value was fixed to 1.40 kcal mol −1 K −1, which was the value obtained for the oxidized form above, because ΔCp for the reduced form was not determined experimentally. This may be valid because no significant differences were observed in structures or dimerization properties (see below). Another immunoglobulin protein

was previously reported to show similar ΔCp values even upon removal of the disulfide bond [21]. In the case of the oxidized form, the model fittings with model A and B gave similar χ2 values (Table 1), where previous studies indicated model B [12]. Furthermore, the theoretical curve of model B assumed an asymmetric shape whereas that of model A was almost symmetric (Fig. 4F). The experimental data appeared as an asymmetric shape.

Table 1 χ2 values of global fittings on each model. Model

A

B

C

B′

9.64

10.6

–

–

10.6

2.00

2.00

0.71

Oxidized form

Reduced form All values were factorized by 10−7.

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

6

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Thus, we concluded that thermal unfolding of the oxidized form followed model B. On the other hand, the DSC data of the reduced form showed a minor peak prior to the main endothermic peak (Fig. 4B), suggesting that an intermediate state may have occurred in the unfolding process (model C). However, we could not obtain lower χ2 with model C. We obtained a better fit by assuming that there were two parallel unfolding processes along with model B (Table 1, Fig. 4G). Thus, we concluded that a sample of the reduced form involved two species with different ΔH values and these species unfolded along with model B (this model is denoted as model B′). The values of ΔH and T0 obtained are summarized in Table 2. 3.4. NMR experiments To determine the effects of reducing the disulfide bond on conformation, we investigated changes in 1H–15N HSQC spectra. Prior to the examination of spectral changes, we assigned 97% of signals (95 out of 98) of the oxidized form with 13C- and 15N-labeled sample (Fig. 5A). We also measured the HSQC spectrum of the reduced form (Fig. 5B, red). The spectrum of the reduced form showed a wide dispersion of cross peaks, each of which had a sharp line, indicating that the reduced form also retained a well-defined structure. To investigate which residues undergo conformational changes upon the disulfide reduction, we calculated chemical shift differences (Δδ) (Fig. 6A) according to the following equation [22]; Δδ ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Δδ2H þ ðΔδN =7Þ2

ð5Þ

where ΔδH and ΔδN are the chemical shift differences between the oxidized and reduced forms in ppm in terms of the 1H and 15N axes, respectively. The obtained Δδ values were mapped onto the crystal structure (Fig. 6C). The residues with high Δδ values gathered near the disulfide bond, indicating that the effects of cleaving of the disulfide bond to the structure were limited to the proximity of the disulfide bond. Fig. 6B shows the relative intensity of reduced CH3 to that of oxidized CH3. Although the pattern was almost flat, the values were less than 1, possibly because of signal broadenings. Signal broadenings can be caused by a restricted global molecular motion, i.e., increase in the apparent molecular weight. Thus, this result also implies the existence of some oligomeric states of reduced CH3. 3.5. H/D exchange experiments We also performed H/D exchange experiments at pH* 5.6 and 37 °C for both forms to obtain protection factors (PF) for each residue (Fig. 7A, B). Considering the present experimental periods, the analyzable PF range was from ~ 102 to ~ 108. In Fig. 7, the residues whose PF value was beyond or below the analyzable range were indicated by upper and down triangles, respectively. Fig. 7C shows the mapping of PF values on crystal structures. The highly protected residues of the oxidized and reduced forms were located at the dimer interface and

near the disulfide bond, indicating that the overall structure of the reduced state remained unchanged from that of the oxidized form. Most of the residues with PF values over the upper limits were involved in the internal core or dimer interface, indicating the similarity of the structures in the native states. 3.6. Changes in hydrophobic properties of the molecular surface We investigated ANS fluorescence in the presence of oxidized or reduced CH3. Fig. 8 shows changes in ANS fluorescence during the GdnHCl titration. The spectrum of the oxidized form remained unchanged, even as the concentration of GdnHCl increased, indicating that oxidized CH3 assumed either a rigidly packed form or completely unfolded form, both of which had no hydrophobic patch or cleft at which the ANS molecule could bind to emit a specific fluorescence. On the other hand, reduced CH3 showed enhanced fluorescence and a shift in λmax at 0 M GdnHCl. These results indicate that less-specifically associated oligomers occur under these conditions and cause ANS bindings because the hydrophobic interactions between the monomers of these oligomers were not tight. The enhanced fluorescence was decreased as the GdnHCl concentration increased and ceased at 1.5 M GdnHCl. McAuley et al. [12] reported that, in the GdnHCl titration experiment of reduced CH3, the dissociation of native dimer into native monomers occurred at 0–1 M GdnHCl prior to the unfolding of the monomeric structure. Similar dissociations could occur for the oligomers at this concentration region, leading to the loss of ANS binding. The packing of the interface in the oxidized form was so rigid that there was no room to accommodate the ANS molecules, whereas the reduction of the disulfide bridge induced alterations in the monomeric structure, leading to loosening of the tight packing between them. 4. Discussion 4.1. The hierarchy of the dimeric structure of the CH3 domain At first, we review the characters of the dimeric structure of the CH3 domain. As mentioned above, CH3 assumes a homodimer, where two folded monomers are interacting with each other through the outsides of βB and βE as illustrated in Fig. 6C. The dimerization is maintained mainly by hydrophobic interactions. The thin dotted line in Fig. 9 shows the intrinsic solubility of the CH3 domain calculated from its amino acid sequence based on Sormanni et al. [23], indicating that there are several less-soluble regions (i.e., βA, βB, βD, βE and βF regions). This method can also calculate the change in the local solubility upon folding considering how each hydrophobic residue is buried in the folded structure. Inputting the monomeric and dimeric structures from the X-ray data of CH3 (PDB ID: 1E4K), we calculated the structurally corrected solubility score of monomer and dimer. It is found that, although the solubility scores for the monomeric state were largely raised, some regions, βA, βB, βD and βE, still showed negative solubility scores (Fig. 9, the thin dotted line), whereas, in the structurally corrected solubility scores for dimer, these regions were increased to

Table 2 Obtained thermodynamic parameters. ΔH (T0)† /kJ mol−1

ΔCp† /J mol−1K−1

T0† /°C

Tm /°C

ΔH (Tm) /kJ mol−1

Fraction†

Oxidized form 508.9

5875⁎

92.68

80.56

437.7

Reduced form Minor peak 301.1 Major peak 456.7

1953

82.61

63.81

264.4

0.11

5875⁎

87.10

73.83

378.8

0.89

ΔHcal /kJ mol−1

ΔH (310K) /kJ mol−1

ΔS (310K) /J mol−1K−1

ΔG (310K) /kJ mol−1

502.7

180.9

418.3

51.26

211.7

577.4

32.71

161.5

385.3

42.09

366.2

†

These parameters were directly determined in the analyses. Other values were deduced from the original parameters. ⁎ These parameters were fixed in the analyses.

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

7

Fig. 5. Comparison between HSQC spectra of the oxidized and reduced forms. (A) The results of the signal assignment of the CH3 domain in the oxidized form at pH 6.5 and 37 °C. (B) HSQC spectra of the oxidized and reduced forms are shown as black and red peaks, respectively.

be almost zero (Fig. 9, the continuous line). These results indicate that, although some hydrophobic residues are still oriented outside on the folded monomer, these residues are completely buried upon dimerization. These observations infer that the steric complementarity of the dimer interface is sophisticated not to leave any spaces between the interfaces. Such a complementarity should be achieved by a specific backbone conformation as well as appropriate arrangement of side chains. 4.2. Structure and thermodynamic stability of the reduced CH3 domain The NMR and analytical ultracentrifugation results indicated that the reduced CH3 domain is able to assume a well-defined dimeric structure similar to the oxidized form. The spectrum of the reduced form showed a wide dispersion of cross peaks, each of which had a sharp line. The patterns of the protection factors of the two forms were globally similar, indicating that the dimer interface of the reduced CH3 domain was also highly protected from the H/D exchange. Indeed, the positions of highly protected residues (Fig. 7A, B) agreed well with those of residues whose solubility scores were significantly enhanced upon dimerization (Fig. 9, from thick dotted line to continuous line), indicating that these hydrophobic residues on these regions are significantly buried in the dimer structure. The lack of a disulfide bond generally causes a significant decrease in conformational stability and makes proteins unfoldable (e.g., ribonuclease T1 [24], BPTI [25] and lysozyme [26]). On the

other hand, most immunoglobulin proteins are foldable even in the reduced form, although their stabilities are significantly decreased. Their high stabilities have been attributed to stable intramolecular hydrophobic interactions around the conserved disulfide bonds. However, among the immunoglobulins, the stability of the reduced CH 3 was distinctive. Although DSC data demonstrated that T m of the reduced form (73.8 °C) was lower than that of the oxidized state (80.6 °C), it was still higher than those of the other immunoglobulin-fold proteins, such as the CL domain (57 °C) and β2m (63 °C) [27]. Furthermore, the decrease in Tm of CH3 upon reduction was significantly smaller than those of the CL domain and β2m (ΔT m,CH3 = − 6.8 °C, ΔT m,CL = − 25 °C, ΔT m,β2m = − 26 °C) [27]. Reduced C H3 was previously shown to fold spontaneously into its native form with the two cysteine residues in the same position as that of the disulfide bond in the oxidized form [28]. These findings indicated that the structure of CH3 was mainly stabilized by intramolecular and intermolecular hydrophobic interactions and the contribution of the disulfide bond was relatively small. However, a structural perturbation appeared to occur around the core region upon the reduction of the disulfide bond. The core residues, which showed high PF values, gathered around the intramolecular disulfide bond (Fig. 7A, B). Residues with significant chemical shift changes also gathered around the disulfide bond (Fig. 6A, C). Furthermore, slight differences were observed in the set of the core residues for the oxidized and reduced forms. For example, Val 23, Ser 63, Phe

Fig. 6. Comparison of HSQC signals between the oxidized and reduced forms. (A, B) Chemical shift perturbations (Δδ) (A) and intensity ratio (B) between oxidized and reduced CH3 are plotted. The secondary structural elements determined on the basis of the X-ray structure are shown schematically at the top of the panels. The circles on the transverse axes indicate the position of the cysteine residues (Cys 27 and Cys 85). (C) Mapping of Δδ on the crystal structure according to the color gauge. The two cysteine residues are drawn in balls and sticks in cyan.

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

8

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Fig. 7. Results of the H/D exchange experiments. (A, B) The PF values for the oxidized (A) and reduced (B) forms are plotted against the residue number. The PF values of the residues whose kinetics could not be traced because of a very fast exchange within the dead time are shown by down triangles. Upper triangles signify lower limits of the residues whose intensity did not change during the incubation time. (C) Mapping of the results of the H/D exchange experiment. Residues showing high PF values in both the oxidized and reduced forms are indicated in green. The residues whose PF values in the oxidized form were significantly higher and lower than those in the reduced form are blue and red, respectively.

64, Phe 65, and Val 87 showed stronger protection in the oxidized form (Fig. 7C, blue residues), whereas Ser 43, Tyr 51, Asp 73, Gln 78, Val 82 and L103 showed stronger protection in the reduced form (Fig. 7C, red residues). The mapping result in Fig. 7C indicated that the span of the core region in the oxidized form was slightly wider than that of the reduced form. The difference in the ΔH values between the reduced form (161.5 kJ mol− 1 for the major peak) and oxidized form (180.9 kJ mol−1) at 37 °C appeared to have been caused by the structural change upon the reduction of the disulfide bond. 4.3. The process of thermal unfolding of the reduced CH3 domain In the present study, we analytically derived the theoretical equations of thermal denaturation curves for the supposed models (models

A–C) and used them for the data fitting. As these theoretical equations provide the detailed shapes of the thermogram, by comparing them to the experimental data, we can distinguish the appropriate model. Based on the results of the model fittings, we concluded that the sample of the reduced form involved two species with different ΔH values and these species unfolded along model B (designated as model B′, see Section 3.3.). The major process of the parallel scheme is the unfolding of the native dimer directly into the unfolded monomers. This conclusion was in contrast with the results of GdnHCl unfolding, in which dissociation of the dimer and unfolding of the monomeric structure were not coupled [12]. The differences observed in the processes may be attributed to the hydrophobic nature of the dimer interface. Hydrophobic interaction are generally strengthened at higher temperatures, but are

Fig. 8. GdnHCl dependencies of ANS fluorescence. (A, B) The spectra of ANS fluorescence at various GdnHCl concentrations for the oxidized (A) and reduced (B) forms of CH3. The thick and thin broken lines indicate the spectra obtained at 0 and 3.6 M GdnHCl, respectively. (C, D) Plots of the GdnHCl concentration dependencies of signal intensities at 493 nm (C) and maximal wavelengths (D) for oxidized (solid markers) and reduced (open markers) CH3, respectively.

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

9

Fig. 9. Solubility score plot of the CH3 domain based on Sormanni et al. [23]. The calculation was performed on the CamSol site (URL: http://www-mvsoftware.ch.cam.ac.uk/index.php/ camsol), inputting the amino acid sequence and PDB structure (PDB ID: 1E4K) of CH3. The thin dotted line indicates the result of the intrinsic solubility score calculated from the sequence. The continuous and thick dotted lines indicate the structurally corrected solubility scores considering the dimeric and monomeric structure based on the PDB data, respectively. The circles on the transverse axis indicate the position of the cysteine residues (Cys 27 and Cys 85).

weakened in the presence of a denaturant. Therefore, the affinity for dimeric associations may be strengthened at higher temperatures until the monomer structure becomes thermally unfolded. In addition, the reversibility of the thermal denaturation of the reduced form was found to be significantly lowered with respect to the oxidized form. One plausible reason was an increase in hydrophobicity in the unfolded state. The intrinsic solubility score of the CH3 domains had two low regions (24–30, 80–87) (Fig. 9, thin dotted line). In the case of the oxidized form, these regions were linked by the intramolecular disulfide bond, Cys 27–Cys 85, and buried to some extent even in the unfolded state. On the other hand, these regions were extensively exposed to the solvent in the reduced form, causing reduced CH3 molecules to become prone to interacting with each other. Brych et al. [29] suggested that free cysteines may make an aggregation core by interacting covalently. However, at high temperature, intermolecular interactions between the exposed hydrophobic regions may have contributed to the increased propensity for irreversible aggregation even without of intermolecular disulfide bond formation. 4.4. Characteristics of the oligomeric adduct of the reduced CH3 domain The model fitting of the thermal unfolding data also suggested that there may be another state in the refolded sample of the reduced form. Our results suggested this state as less-specifically associated oligomers or oligomeric adducts. The enhanced light scattering and turbid solution of the reduced CH3 domain indicated that the maximum size of the oligomeric adducts reaches to a size similar to the wavelength of the probe light (several hundreds of nanometers). As such oligomers are invisible in the HSQC spectra and caused the decreases in the relative intensity (Fig. 6B), the remaining signals came only from the rigid dimer species. The oligomeric adducts may have been generated during the preparation of the reduced sample, especially when the denaturant or antiaggregating reagents (i.e., Arg) were removed. Some of the reduced CH3 molecules misfolded into an incomplete monomeric structure. Such incomplete monomers interacted with each other in a less specific manner because of the low integrity of the dimer interfaces in these molecules. Since interactions between monomers in the oligomeric adducts are not tight and specific, these residues were exposed to the solvent, allowing ANS binding. This ANS fluorescence was ceased at the higher GdnHCl concentration of 1 M because the oligomeric adducts dissociated into monomers. The minor endothermic peak at a temperature lower than that of the major peak in the thermogram of the reduced form also indicated the existence of the oligomeric adducts. This state seems distinct to amorphous aggregations and involves specific, rigid structures, because this species gave comparable thermodynamic parameters to those of the major species (Table 2). This species melted at a lower temperature than the major species and may also form irreversible precipitates more easily.

This state was assumed to be in equilibrium with the native dimer state. The amplitude of the minor peak in the DSC curve was dependent on the scan rate and concentration. If the amount of the oligomeric state were unchanged in the unfolding experiment, such scan rate- and concentration-dependences would not have been observed. It is assumed that, since the timescale of the exchange between the oligomeric adduct and rigid dimer was comparable to the DSC measurement time, with a slower scan rate, the unfolding of the oligomer occurred at ~65 °C with a population shift from the native dimer to the oligomeric state, leading to an enhancement in the minor peak (Fig. 4D). The accumulation of the unfolded monomers increased the frequency of the formation of irreversible precipitates, which caused significant decreases in the thermogram at higher temperatures. For example, the DSC curve at the scan rate of 30 °C min−1 showed a large decrease in the thermogram over 80 °C (Fig. 4D). Interestingly, Thies et al. [30] reported that even oxidized CH3 assumes an oligomeric state with alternatively folded conformation at acidic pH in the presence of moderate concentrations of salt. Although the authors suggested that the monomeric conformation was distinct from that of the native state, the oligomeric state in their report showed similar properties to the oligomeric adduct in this paper, such as cooperative thermal unfolding and ability to bind ANS [30]. These results indicate that, whereas alternatively folded conformations were allowed only at acidic pH for oxidized CH3, they were allowed even in neutral pHs for the reduced form. The present results indicated that, upon refolding of the reduced form of CH3, a small amount of the molecules formed the oligomeric adducts, in which the CH3 molecule assumes an almost correct monomeric structure but does not accompany the perfect dimer assembly. In the preparation processes of antibody medicines, protein molecules undergo necessary transient unfolding stresses, such as acid treatments [31,32] and exposure to air–water or liquid–solid interfaces [3,33]. Thus, aggregation may be induced if the reduced form is present. This aggregation process should be taken into consideration for quality control of antibody preparation. 4.5. Conclusions We performed several measurements in order to determine the effects of a disulfide bond reduction in the CH3 domain on conformational stability and aggregation propensity. The analyses of the experimental results indicated that, although a dominant fraction of the reduced form retained a fairly stable dimeric structure, some molecules were present in the oligomeric adducts, which consists of CH3 molecules with almost correct monomeric structure. The suppression of aggregation by reduced CH3 will improve the storage of antibody medicines. However, the information regarding the structural and thermodynamic properties of the aggregated state is limited. Thus, the effects of aggregate formation by the reduced CH3

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020

10

K. Sakurai et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

domain on the qualities and aggregation propensities of monoclonal IgG1 molecules have yet to be elucidated in detail. The results of the present study have contributed to the quality control of monoclonal antibodies. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgments We thank Dr. Masazumi Matsumura for his valuable comments and Ms. Miyo Sakai (Institute for Protein Research) for her instructions regarding analytical ultracentrifuge measurements. This work was supported by JSPS KAKENHI Grant Numbers 15076101 and 23107719 (K.S.) and by Hungarian National Research, Development and Innovation Office Grant Number KTIA_NAP_13-2-2014-0017 grant (J.K.). Appendix A. Supplementary data Supplementary Methods to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2015.02.020. References [1] G. Cartron, L. Dacheux, G. Salles, P. Solal-Celigny, P. Bardos, P. Colombat, H. Watier, Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene, Blood 99 (2002) 754–758. [2] S. Hermeling, D.J. Crommelin, H. Schellekens, W. Jiskoot, Structure-immunogenicity relationships of therapeutic proteins, Pharm. Res. 21 (2004) 897–903. [3] H.C. Mahler, W. Friess, U. Grauschopf, S. Kiese, Protein aggregation: pathways, induction factors and analysis, J. Pharm. Sci. 98 (2009) 2909–2934. [4] R.F. Latypov, S. Hogan, H. Lau, H. Gadgil, D. Liu, Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc, J. Biol. Chem. 287 (2012) 1381–1396. [5] P.A. Barthelemy, H. Raab, B.A. Appleton, C.J. Bond, P. Wu, C. Wiesmann, S.S. Sidhu, Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains, J. Biol. Chem. 283 (2008) 3639–3654. [6] K. Dudgeon, R. Rouet, I. Kokmeijer, P. Schofield, J. Stolp, D. Langley, D. Stock, D. Christ, General strategy for the generation of human antibody variable domains with increased aggregation resistance, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 10879–10884. [7] X. Wang, T.K. Das, S.K. Singh, S. Kumar, Potential aggregation prone regions in biotherapeutics: a survey of commercial monoclonal antibodies, MAbs 1 (2009) 254–267. [8] A. Sumi, K. Hamaguchi, Denaturation by guanidine hydrochloride of the Fc(t) and pFc′ fragments of human immunoglobulin G, J. Biochem. 92 (1982) 823–833. [9] G.D. Pipes, A.A. Kosky, J. Abel, Y. Zhang, M.J. Treuheit, G.R. Kleemann, Optimization and applications of CDAP labeling for the assignment of cysteines, Pharm. Res. 22 (2005) 1059–1068. [10] W. Zhang, M.J. Czupryn, Free sulfhydryl in recombinant monoclonal antibodies, Biotechnol. Prog. 18 (2002) 509–513. [11] M.J. Thies, J. Mayer, J.G. Augustine, C.A. Frederick, H. Lilie, J. Buchner, Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization, J. Mol. Biol. 293 (1999) 67–79. [12] A. McAuley, J. Jacob, C.G. Kolvenbach, K. Westland, H.J. Lee, S.R. Brych, D. Rehder, G.R. Kleemann, D.N. Brems, M. Matsumura, Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain, Protein Sci. 17 (2008) 95–106. [13] K. Sakurai, Y. Goto, Manipulating monomer-dimer equilibrium of bovine βlactoglobulin by amino acid substitution, J. Biol. Chem. 277 (2002) 25735–25740.

[14] P. Schuck, Analytical ultracentrifugation as a tool for studying protein interactions, Biophys. Rev. 5 (2013) 159–171. [15] F. Delaglio, S. Grzesiek, G.W. Vuister, G. Zhu, J. Pfeifer, A. Bax, NMRPipe: a multidimensional spectral processing system based on UNIX pipes, J. Biomol. NMR 6 (1995) 277–293. [16] T.R. Kim, Y. Goto, N. Hirota, K. Kuwata, H. Denton, S.Y. Wu, L. Sawyer, C.A. Batt, Highlevel expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties, Protein Eng. 10 (1997) 1339–1345. [17] Y. Goto, T. Azuma, K. Hamaguchi, Refolding of the immunoglobulin light chain, J. Biochem. 85 (1979) 1427–1438. [18] Y. Goto, K. Hamaguchi, The role of the intrachain disulfide bond in the conformation and stability of the constant fragment of the immunoglobulin light chain, J. Biochem. 86 (1979) 1433–1441. [19] J.W. Longworth, C.L. McLaughlin, A. Solomon, Luminescence studies on Bence–Jones proteins and light chains of immunoglobulins and their subunits, Biochemistry 15 (1976) 2953–2958. [20] Y. Ohhashi, Y. Hagihara, G. Kozhukh, M. Hoshino, K. Hasegawa, I. Yamaguchi, H. Naiki, Y. Goto, The intrachain disulfide bond of beta(2)-microglobulin is not essential for the immunoglobulin fold at neutral pH, but is essential for amyloid fibril formation at acidic pH, J. Biochem. 131 (2002) 45–52. [21] Y. Hagihara, S. Mine, K. Uegaki, Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region, J. Biol. Chem. 282 (2007) 36489–36495. [22] F.A. Mulder, D. Schipper, R. Bott, R. Boelens, Altered flexibility in the substratebinding site of related native and engineered high-alkaline Bacillus subtilisins, J. Mol. Biol. 292 (1999) 111–123. [23] P. Sormanni, F.A. Aprile, M. Vendruscolo, The CamSol method of rational design of protein mutants with enhanced solubility, J. Mol. Biol. 427 (2015) 478–490. [24] C.N. Pace, G.R. Grimsley, J.A. Thomson, B.J. Barnett, Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds, J. Biol. Chem. 263 (1988) 11820–11825. [25] D.J. States, T.E. Creighton, C.M. Dobson, M. Karplus, Conformations of intermediates in the folding of the pancreatic trypsin inhibitor, J. Mol. Biol. 195 (1987) 731–739. [26] K. Matsuo, H. Watanabe, S. Tate, H. Tachibana, K. Gekko, Comprehensive secondarystructure analysis of disulfide variants of lysozyme by synchrotron-radiation vacuum-ultraviolet circular dichroism, Proteins 77 (2009) 191–201. [27] Y. Hagihara, T. Matsuda, N. Yumoto, Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains, J. Biol. Chem. 280 (2005) 24752–24758. [28] Y. Goto, K. Hamaguchi, Formation of the intrachain disulfide bond in the constant fragment of the immunoglobulin light chain, J. Mol. Biol. 146 (1981) 321–340. [29] S.R. Brych, Y.R. Gokarn, H. Hultgen, R.J. Stevenson, R. Rajan, M. Matsumura, Characterization of antibody aggregation: role of buried, unpaired cysteines in particle formation, J. Pharm. Sci. 99 (2010) 764–781. [30] M.J. Thies, R. Kammermeier, K. Richter, J. Buchner, The alternatively folded state of the antibody C(H)3 domain, J. Mol. Biol. 309 (2001) 1077–1085. [31] D. Ejima, K. Tsumoto, H. Fukada, R. Yumioka, K. Nagase, T. Arakawa, J.S. Philo, Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies, Proteins 66 (2007) 954–962. [32] R.L. Fahrner, H.L. Knudsen, C.D. Basey, W. Galan, D. Feuerhelm, M. Vanderlaan, G.S. Blank, Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography processes, Biotechnol. Genet. Eng. Rev. 18 (2001) 301–327. [33] K. Hasegawa, D. Ozawa, T. Ookoshi, H. Naiki, Surface-bound basement membrane components accelerate amyloid-beta peptide nucleation in air-free wells: an in vitro model of cerebral amyloid angiopathy, Biochim. Biophys. Acta 1834 (2013) 1624–1631.

Abbreviations pH*: pH meter reading GdnHCl: guanidine hydrochloride DSC: differential scanning calorimetry NMR: nuclear magnetic resonance HSQC: heteronuclear single quantum coherence H/D exchange: hydrogen/deuterium exchange PF: protection factor ANS: 1-Anilino-8-naphthalenesulfonate

Please cite this article as: K. Sakurai, et al., Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbapap.2015.02.020