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agttCCATGGATRTTGTGATGACYCAG-39) with an NcoI site and first-reverse were used. In the PCR amplification for the C51 light chain, the forward primer ...
The FASEB Journal • Research Communication

A novel method of preparing the monoform structure of catalytic antibody light chain Emi Hifumi,*,†,1 Shingo Matsumoto,‡,§ Hiroki Nakashima,‡ Shogo Itonaga,‡ Mitsue Arakawa,{ Yoshiki Katayama,§ Ryuichi Kato,k and Taizo Uda†,‡ *Research Promotion Institute and ‡Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita, Japan; †Nanotechnology Laboratory, Institute of Systems, Information Technologies, and Nanotechnologies (ISIT), Fukuoka, Japan; §Graduate School of System Life Science, Kyushu University, Fukuoka, Japan; {Tottori College of Nursing, Tottori, Japan; and kHigh Energy Accelerator Research Organization, Tsukuba, Japan Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet–visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.—Hifumi, E., Matsumoto, S., Nakashima, H., Itonaga, S., Arakawa, M., Katayama, Y., Kato, R., Uda T. A novel method of preparing the monoform structure of catalytic antibody light chain. FASEB J. 30, 895–908 (2016). www.fasebj.org ABSTRACT

Key Words: charge heterogeneity • 2D electrophoresis • pI copper ion



Along with clarification of the mechanisms and functions of catalytic antibodies, their potential as new drugs

Abbreviations: 2D, 2 dimensional; AFM, atomic force microscope; AMC, 7-amino-4-methylcoumarin; BPB, bromophenol blue; CBB, Coomassie brilliant blue; CHAPS, 3-[(3cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; CL, constant region of light chain; CR, constant region domain; Cu:Lc, ratio of Cu to full-length light chain; eq, (continued on next page)

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for antirabies virus (1), anti-influenza virus (2), antiHelicobacter pylori (3), anti-HIV (4–6), and anti-Alzheimer’s disease (7, 8) has become more likely. In addition to this research, the idea of passive vaccination against glycoprotein (gp)120 of HIV is another unique approach (9). Apart from these developments, the structural diversity of antibodies has raised increasing interest. From the structural point of view, some of the catalytic antibodies play the same role as the whole structure of IgG (10–13), IgA (14), or IgM (15–17). On the other hand, in some cases, the subunits (light chain or heavy chain) exhibit unique functions (1–3, 7, 8, 18–20). Once the antibody subunits are separated, the structure of the light or heavy chain becomes flexible and has a tendency to possess structural diversity, whose phenomena were found, even in a whole antibody about 20 years ago (21, 22). For example, an antibody has different structures (not a monoform structure) related to the various electrical charges. Capillary isoelectric focusing (22) and 2-dimensional (2D) gel electrophoresis (23, 24) recently showed that the isoelectric point (pI) spreading for a whole antibody is attributable to the presence of charge-related isoforms of the heavy and light chains. This phenomenon has an adverse effect on preparation efficacy, high reproducibility, and practical application of antibodies. In addition, structural diversity leads us to ask what structure plays the most important role in catalytic antibody functions. It also raises the subject of how we can best make a dependable structure with high reproducibility and productivity. Solving the subject of structural diversity is crucial in the future development of catalytic antibodies. Because of its high degree of complexity, this has been considered a difficult subject for a long time, and only a few studies have focused on it. Herein, we report on a novel method of preparing a catalytic antibody light chain that possesses a single defined structure, by using a metal ion that substantially 1

Correspondence: Research Promotion Institute, Oita University, 700 Dan-noharu, Oita-shi, Oita 870-1192, Japan. E-mail: [email protected] doi: 10.1096/fj.15-276394 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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contributes to solving the structural heterogeneity problem. MATERIALS AND METHODS Cloning, sequencing, and expression Genes of the human light chains used in this study were obtained by using as the template cDNA that we prepared in our earlier study (1) and PCR amplification. For the cloning of #4 and #7 light chains, detailed procedures are stated in our earlier study. In brief, DNA fragments encoding human light chains were amplified from the cDNA by semi-nested PCR. For the first PCR, the primers first-forward (59-agttCCATGGAGCTCCTGGGGCTGCTAATG39) with an NcoI site (italics) and first-reverse (59-ccgtCTCGAGACACTCTCCCCTGTTGAAG-39) with an XhoI site (italics) were used. For the second PCR, the primers second-forward (59agttCCATGGATRTTGTGATGACYCAG-39) with an NcoI site and first-reverse were used. In the PCR amplification for the C51 light chain, the forward primer (59-cgtctaCCATGGAAATTGTGTTGACACAGTC-39, 59-cgtctaCCATGGAAATAGTGATGACGCAGTC-39) with an NcoI site (italics) and reverse primer (59-atgtgaCTCGAGACACTCTCCCCTGTTGAAG-39) with an XhoI site (italics) were used in the following incubation conditions: 60 s at 98°C, 28 cycles of 10 s at 98°C, 30 s at 72°C for annealing, and 30 s at 72°C for extension using Phusion DNA Polymerase (Finnzymes, Vantaa, Finland). PCR products of 674 bp were purified with a QIAprep PCR Purification kit (Qiagen, Hilden, Germany). A fragment encoding human light chains was digested by restriction enzymes NcoI and XhoI (New England BioLabs, Beverly, MA, USA). The resulting DNA fragments were inserted between the same restriction sites of expression vector pET20b (+) (Novagen, Madison, WI, USA). To express the light chains, the expression vectors were transformed into BL21 (DE3) pLysS (Novagen). The constant region of the light chain was obtained by amplifying the gene corresponding to the constant region of human k light chain and expressing in a BL21 (DE3) pLysS system. Site-directed mutagenesis (C51 C220A) was performed by PCR with a KOD Plus Mutagenesis Kit (Toyobo, Osaka, Japan) with a mutagenic primer and an antisense primer (forward: 59GCGCTCGAGCACCACCAC-39 or 59-GCTCTCGAGCACCACCAC-39, reverse: 59-CTCTCCCCTGTTGAAGCT-39), and pET20b (+) vector containing a DNA fragment encoding the wild-type light chain as a template. After the inverse PCR reaction, the template DNA was specifically digested with DpnI. The PCR fragments were phosphorylated, self-ligated, cloned into Escherichia coli strain DH5a, and sequenced, to ensure that no unexpected mutation would occur. Culture, recovery, and purification The transformant was grown at 37°C in 1 L Luria-Bertani medium containing 100 mg/ml ampicillin to an absorbance at (continued from previous page) equivalent; MALDI-TOF, matrix-assisted laser desorption/ ionization–time of flight; MCA, peptidyl-4-methyl-coumaryl7-amide substrates (combined with amino acids EAR, GluAla-Arg; GLPGP, Asn-Leu-Pro-Asn-Pro; QAR, Gln-Ala-Arg; VPR, Val-Pro-Arg); MS, mass spectroscopy; Ni-NTA, nickel– nitroloacetic acid; pI, isoelectric point; SA/TA (solution), sinapinic acid with a mixture of 0.1%TFA and acetonitrile; UV/VIS, ultraviolet-visible spectroscopy; VL, variable region of light chain

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600 nm of 0.6 and then incubated with 0.01 mM isopropyl-b-Dthiogalactopyranoside (IPTG; Wako Pure Chemicals, Osaka, Japan) for 20 h at 18°C. The cells were harvested by centrifugation (3500 g, 4°C, 10 min) and then resuspended in a 100 ml solution of 250 mM NaCl and 25 mM Tris-HCl; pH 8.0). (In some cases, an aliquot of a solution of 50 mM CuCl2 (final concentration: 50 mM; CuCl2·2H2O; Wako Pure Chemical Industries) was added to the cell suspension. The cells were lysed by ultrasonication 3 times for 2 min each in an ice bath, followed by centrifugation (14,000 g, 4°C, 20 min). The expressed human light chain was recovered as the supernatant. The supernatant was first subjected to Ni-NTA column chromatography (Takara, Otsu, Japan) equilibrated with 25 mM Tris-HCl (pH 8.0), containing 250 mM NaCl. Elution was performed by increasing the concentration of imidazole from 0 and/or 30 to 300 mM. After the Ni-NTA column chromatography was completed, in the case of addition of copper (Cu) ion, an aliquot of a solution of 50 mM CuCl2 was added into the eluent, based on the calculation that the absorbance at 600 nm of 1.0 was regarded as ;1 mg/ml (40 mM light chain). The solution including the light chain and copper ion was dialyzed against a 50 mM Na acetate buffer (pH 5.5) for 22–24 h and subjected to cationexchange chromatography on an SP-5PW column (Tosoh, Tokyo, Japan), with a gradient of NaCl solution (from 0.05 to 0.6 M) on the purification apparatus (AKTA System; GE Healthcare, Tokyo, Japan). The eluent was dialyzed against Tris-HCl buffer (pH 8.5) for 15–20 h, followed by solution concentration with an Amicon Ultra 1000 (EMS-Millipore, Billerica, MA, USA). The concentrated solution was dialyzed against PBS (pH 7.4) twice, first for 6 h and then for 16 h. After filtration with the 0.2 mm filter (Millex-LG, 0.2 mm, 13 mm; EMS-Millipore), the solution was stored at 4°C. Size-exclusion chromatography was performed with an SEC3000 preparatory size-exclusion HPLC column on the AKTA system, with PBS (pH 7.4) used as the solvent. Protein concentrations were determined by the Bradford and Lowry methods, using a detergent compatible protein assay kit (Bio-Rad, Hercules, CA, USA).

Hydrolysis of synthetic peptidyl substrates: peptidase activity To avoid contamination in cleavage assays, most glassware, plasticware, and buffer solutions used in this experiment were sterilized by heating (180°C, 2 h), autoclaving (121°C, 20 min), or filtration through a 0.20-mm sterilized filter, as much as possible. Most of the experiments were performed in a biologic safety cabinet to avoid airborne contamination. Cleavage of the amide bond linking 7-amino-4-methylcoumarin (AMC) to C-terminal amino acid in peptide-MCA substrates (Peptides International, Louisville, KY, USA) was measured at 37°C in PBS containing 0.05% sodium azide in 96-well plates. Purified light chain (100 ml, 10 mM) was mixed with a synthetic substrate (100 ml, 100 mM). AMC released from the substrate catalyzed by the light chain was detected by measurement of fluorescence emission at 470 nm when excited at 360 nm with a microplate reader (SpectraFluor Plus; Tecan, Maennedorf, Switzerland). The concentration of the released AMC was estimated by using the fluorescence emission of 50 mM AMC as a standard.

AFM observation The MFP-3D-BIO apparatus (Asylum Research, Santa Barbara, CA, USA) was used. A mica surface was treated with (3-aminopropyl) trimethoxysilane (Sigma-Aldrich). Cleaved mica was put in a 1.5-L

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Figure 1. Amino acid sequences of light chains. Black: variable region of antibody light chain. Violet: constant region of antibody light chain. Methionine was adducted at the N terminus. At the C terminus, 6 histidine residues are present as a His-tag for purification. Leu-Glu residues were inserted because of the XhoI site. Three mutated positions: C1; C51CA, C51 C220A; C51AAA, C51 H195A/H204A/C220A (3 mutated positions).

desiccator with15 ml 98% (3-aminopropyl)trimethoxysilane and then left in a vacuum created with a DAP-60 vacuum pump (Ulvac, Kanagawa, Japan) for 60 min. A light chain sample (5 mg/ml) was then put onto the treated mica surface. After incubation for 180 min, the sample was gently rinsed with 500 ml PBS (pH 7.5) buffer and moved to the AFM chamber to prevent evaporation of the buffer. All AFM images were obtained in PBS (pH7.5) buffer by using the MFP-3D-BIO AFM in AC mode at room temperature. The cantilevers used for all AFM imaging were BL-AC40TS (spring constant: 0.1 N·m; Olympus, Tokyo, Japan).

Mass spectroscopy Mass spectra were recorded by using a matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) system (Autoflex; Bruker Daltonics, Billerica, MA, USA). All measurements were performed in linear mode and in positive polarity. Each C51 light chain sample was prepared with 0.1% TFA/H2O. The final concentration of the sample was 2.0 mg/ml. One microliter of the sample was mixed with 2.0 ml of 0.2% TFA/H2O and 7.0 ml of SA/TA solution (SA, sinapinic acid; TA, a mixture of 0.1%TFA and acetonitrile). Then, 1.0 ml of the mixture was loaded on a gold target plate and allowed to air dry at room temperature. After the target was inserted in the MALDI-TOF mass spectrometer, mass calibration was performed by using Standard II (Bruker Daltonics) mixed with SA/TA solution. The mass values given in the text are monoisotopic. Ultraviolet–visible spectroscopy Ultraviolet-visible spectroscopy (UV/VIS) spectra were obtained with an MPS-2400 spectrophotometer (Shimadzu, Kyoto, Japan), in the range of 900–220 nm, with a bandwidth of 1 nm. Chemical analysis of Cu ion Before the analysis of the Cu ion, we conducted 2 experiments: inductively coupled plasma–mass spectrometry (ICP-MS) analysis and a commercially available chemical analysis kit for Cu ion, to ascertain the accuracy of the chemical analysis. Both analyses were in good agreement, with or without the Cu ion (data not shown).

STRUCTURAL DIVERSITY OF CATALYTIC ANTIBODY LIGHT CHAIN

In brief, in a chemical analysis of Cu(II), an assay kit (Copper Low Concentration Assay Kit, AKJ, CU21M; Metallogenics Co., Ltd., Chiba, Japan) was used. Before the experiment, all reagents and samples were allowed to incubate at 37°C. Most experiments were performed in accordance with the kit protocol. First, a standard curve was made by diluting the standard solution of 80 mg/dl. A preparation of chromogenic substrate was made by mixing 140 ml of R-A buffer solution with 5 ml of R-R chelate reagent for each sample, and 140 ml of the chromogenic substrate solution was added to 100 mL of a sample, mixed by pipetting, and allowed to stand for 10 min at room temperature. Then, 180 mL of the mixture was put into a well of the plate (Nunc Immunoplate/MaxiSorp; Thermo Fisher Scientific, Roskilde, Denmark) and the absorbance was measured by using an Immuno-Mini NJ-2300 plate reader (Nalge Nunc International K. K., Tokyo, Japan) at l = 590 nm. The concentration of Cu(II) was derived from Eq. 1: CuðmMÞ ¼ 0:1573 3 absorbance=slope  of  standard  curve (1) Molecular modeling The samples of the light chains were sequenced with the ABIautomated DNA sequencer Model 3100 (Thermo Fisher ScientificApplied Biosystems, Foster City, CA, USA), with the universal primer of a T7 promoter. Genetix 8 software (Genetix, Tokyo, Japan) was used for sequence analysis and deduction of amino acid sequences. Computational analyses of the antibody structures were performed from the deduced antibody light chain amino acid sequences by using Discovery Studio (Accelrys Software, San Diego, CA, USA). For homology modeling, the template structures were made by a BLAST search. The resulting Protein Data Bank (PDB; www.rcsb.org) data were used to modify the complementaritydetermining region (CDR) structures defined by the Kabat numbering system and to minimize the total energy of a molecule by using the CHARMm algorithm.

2D gel electrophoresis 2D electrophoresis was performed in an Ettan IPGphor 3 system (GE Healthcare). In the first-dimension run, the running

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Figure 2. Purification, chromatogram, and SDS-PAGE for the C51 light chain. A) Conventional flow of the purification process. B) Ni-NTA column chromatogram. Fractions 29–42 in the C51 light chain were collected and subjected the cation-exchange chromatography. Abs, absorbance. C ) SDS-PAGE. Fraction 35 was the monomeric form of C51 light chain under both the reduced and nonreduced conditions. D) Cation-exchange chromatogram of C51 by the conventional method [without Cu(II)]. E ) SDS-PAGE of each peak observed in the chromatogram in (D). Peak 1 was the monomer. Peak 2 included both the monomer and dimer. Peak 3, with a shoulder, was the dimer. F ) Cation-exchange chromatogram of C51 with the addition of 20 mM of Cu (II) to the cell suspension. Peaks 1, 2, and 3 were detected, but they were small. Peak 4 corresponds to peak 3 in (D). G) SDSPAGE for the chromatogram in (F ). Peak 4 was the dimer of the C51 light chain. H ) Cation-exchange chromatogram of C51 with the addition of 20 mM of Cu(II) to the Ni-NTA eluent. Peaks 1 and 2 substantially decreased, and peak 3 became a simple peak. I ) SDS-PAGE for the chromatogram in (H ). Peak 3 was the dimer of the C51 light chain. In the cation-exchange chromatograms, m, monomer; di, dimer. buffer contained 8 M urea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS), 0.002% bromphenol blue (BPB), 2.8% DTT, and 0.5% immobilized pH

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gradient (IPG) buffer. Three aliquots of the buffer were mixed with 1 aliquot of a sample (8 M urea, 4% CHAPS, and 40 mM Tris). The following 4 steps were performed after the sample

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(C51 light chain) and Immobiline DryStrip (GE Healthcare) were loaded into the IPGphor 3: 1) hold at 300 V for 120 min; 2) gradient 1000 V for 30 min; 3) gradient 5000 V for 90 min; and 4) hold at 5000 V for 30 min. For the second-dimension run, the gel strip was cut and incubated for 15 min in equilibration buffer A [50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 30% glycerol, 0.25% DTT, and 0.002% BPB] at room temperature, followed by 15 min in equilibration buffer B [50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 30% glycerol, 4.5% iodoacetamide, and 0.002% BPB]. The running buffer contained 25 mM Tris base, 0.2 M glycine, and 0.1% SDS. The gel strip was drained briefly and placed on a 10 cm 3 10 cm 3 1 mm 12% polyacrylamide gel. The seconddimension separation was performed under 250 V in the running buffer containing 25 mM Tris base, 0.2 M glycine, and 0.1% SDS. The gels were stained with Coomassie brilliant blue (CBB; Wako Pure Chemical Industries).

RESULTS Amino acid sequences of light chains The amino acid sequences of human antibody k light chains of C51—its mutants (C51 C220A (abbreviated as C51CA) and C51 H195A/H204A/C220A (abbreviated as STRUCTURAL DIVERSITY OF CATALYTIC ANTIBODY LIGHT CHAIN

C51AAA)—and #4, #7, and constant region (CR) domain are summarized in Fig. 1. The proteins were expressed in an E. coli system, in accordance with the protocol described in Materials and Methods. A methionine residue at the N terminus was confirmed by amino acid sequence analysis. The residue was not included as an amino acid of the light chain. Leu and Glu residues were inserted at the XhoI site before a histidine tag (His 6X), which was adducted for purification. Purification of the C51 light chain and the effect of Cu(II) After the E. coli cells were recovered by centrifugation, they were sonicated. Then, the soluble fraction was subjected to purification, as shown in Fig. 2A, where 2 purification steps using nickel–nitroloacetic acid (Ni-NTA) affinity chromatography and cation-exchange chromatography were used. Fig. 2B shows the result of Ni-NTA affinity column chromatography for the C51 light chain, which was eluted in fractions 29–42. The collected fractions were analyzed by SDS-PAGE with CBB staining (Fig. 2C), where a monomeric form of C51 light chain was observed in both reduced and nonreduced conditions. 899

(In the nonreduced condition, either the monomer or the dimer form was observed in many cases, depending on the characteristics of the light chain.) Fractions 29–42 were collected and subjected to cation-exchange chromatography. Figure 2D shows the chromatogram, where several peaks were observed. The SDS-PAGEs of the peaks are shown in Fig. 2E. Peaks 1, 2, and 3 were a monomer, a mixture of monomer and dimer, and a dimer, respectively. The large peak 3, the dimer, had a shoulder. Twenty micromolar CuCl2 [Cu(II)] was added to either the cell suspension after recovery of the cells (cell suspension) or the eluent of Ni-NTA chromatography (Ni-NTA eluent), Cu, was added at the points indicated with dotted arrows in Fig. 2A. By the addition of Cu(II) to the cell suspension, the chromatogram changed to that shown in Fig. 2F, where a main peak (peak 4) was observed at the retention time of 25 min, but peaks 1–3 were small. SDS-PAGE (Fig. 2G) showed a dimer at peak 4. In Fig. 2H, 20 mM of Cu(II) (0.5 equivalent to the light chain) was added to the Ni-NTA eluent. In this case, only a main peak (peak 3) was observed at the retention time of 25 min, but peaks 1 and 2 were scarcely detected. SDS-PAGE showed a dimer for peak 3 (Fig. 2I). The addition of Cu(II) made the dimer dominant. MS analysis was performed for the main peaks. The result for the sample of C51 light chain, which was prepared by the conventional method without the addition of Cu(II), is shown in Fig. 3A. A monomeric light chain was detected at 25,000 m/z and a dimer at 49,000 m/z. A small trimer and tetramer were also detected at 74,000 and 98,000 m/z, respectively. By the addition of Cu(II) to the cell suspension or the Ni-NTA eluent, the MS spectra changed, and the signal for the monomer was substantially reduced in both Fig. 3B, C (with Cu) in comparison with that of Fig. 3A (without Cu). The addition of Cu(II) was effective for the formation of the dimer. To examine the pI of the light chain, we performed 2D gel electrophoresis with C51 light chain, with and without the addition of Cu(II) (Fig. 3D, E). Without Cu(II), many spots at different pIs were observed with the same molecular mass of 31 kDa (Fig. 3D). The pI spots were widely located from 6.12 to 10.00. A strong spot was observed at pI 6.45–6.73. In contrast, with the addition of Cu(II), the spots gathered on the strongest spot at pI 6.57, and 2 faint spots were detected at ;6.32 and 6.90 (Fig. 3E). It is evident that the electrical charges of the molecule became monoform with the addition of Cu(II). Other light chains The other light chains (4, 7, C51 mutants, and CR) were also investigated to confirm whether the observations were general phenomena. As stated earlier, the chromatogram in Ni-NTA purification is similar for many light chains. Thus, the following explanations are focused on the results of cation-exchange chromatography, which are very different in each light chain used. The chromatograms for #4 light chain, without and with Cu(II), are shown in Fig. 4A, B, respectively. In the former case, there were several peaks. The results of SDSPAGE (nonreduced condition) corresponding to the other 3 peaks are also shown. The observed peaks were a mixture of monomer and dimer. Several structurally 900

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different light chains caused by different electrical charges coexisted in the solution. However, in the latter case, when 20 mM of Cu(II) (0.5 equivalent to the light chain) was added to the Ni-NTA eluent, a single peak (mainly the dimer) was obtained (Fig. 4B). Cation-exchange chromatography of the #7 light chain gave 3 large peaks without Cu(II) (Fig. 4C). The peak of the retention time at 9 min was mainly monomer. The peak at 13 min was a mixture of monomer and dimer. The peak at 22 min was also a mixture. Light chains possessing different molecular sizes and electrical charges coexisted in the solution. When 15 mM of Cu(II) (0.38 eq) was added to the Ni-NTA eluent, 2 peaks (peak A at 12 min and peak B at 23 min; Fig. 4D) were observed in the chromatogram. The results of SDS-PAGE analysis indicated that both peaks A and B were dimers. Specifically, in this case, 2 kinds of dimers with different electrical charges were found. When 40 mM Cu(II) (1.0 eq) was added to the same eluent (Fig. 4E), only peak C, which corresponds to peak A in Fig. 4D, was observed at 12 min, and peak B was not detected. We concluded that peak B moved to peak A in Fig. 4D. As a consequence, the dimeric light chain of a single electrical charge was obtained. A chemical analysis of Cu(II) gave interesting results. The Cu:light chain (Lc) ratio was 0.48, 0.03, and 0.64 for peaks A, B, and C, respectively. When the Cu:Lc ratio was 0.48 in Fig. 4D and 0.64 in Fig. 4E, the dimer was eluted at 12 min. In contrast, at a ratio of 0.03, the retention time was 23 min. Note that whenever enough Cu(II) was present in the solution, an electrically homogeneous light chain was observed at a retention time of 12 min. Peaks of A, B, and C were collected and subjected to AFM analysis, as shown in Fig. 4F (peak A), 4G (peak B), and 4H (peak B). A clear image of the dimeric light chain is encircled in red. The size of the dimer was roughly estimated at a length of ;20 nm, width of ;10 nm, and height of ;4 nm. The lateral and height length are comparable with the AFM image of IgG by Ouerghi et al. (25) We could not identify the position of the Cu ion residing in the light chain from this AFM analysis. To clarify the Cu-binding site, we introduced several mutations in the C51 light chain. Both histidine and cysteine residues are considered to be the most plausible candidates for the binding site. Therefore, the residues His195, His204, and Cys220, which are present in the CR domain of the C51 light chain were mutated to Ala, and 2 mutants were made: Cys220Ala (C220A) and is His195Ala, His204Ala, and Cys220Ala (H195A/H204A/C220A) (Fig. 5A). No histidine residue is present in the variable domain of the C51 light chain. The locations of the mutated residues are shown in Fig. 5B. These mutants were similarly expressed and purified as stated earlier. Fifty micromolar Cu(II) (1.25 eq) was added to both the cell suspension and the Ni-NTA eluent, where all light chains gave a single peak in cation-exchange chromatography. The Cu uptake by the wild-type and each mutant was chemically analyzed, and the results are presented in Table 1. The wild-type possessed 0.75 atoms of Cu(II) per C51 light chain. The C220A and H195A/H204A/C220A mutants possessed 0.54 and 0.25, respectively. The Cu uptake of the C220A mutant was lower than that of the wild-type by 0.21 atoms,

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Figure 3. MS and 2D gel electrophoresis. Arrow in insets: fraction selected for MS analysis. A) MS spectrum corresponding to peak 3 in Fig. 2D, without Cu(II). The monomer (25,000 m/z) and dimer (50,000 m/z) gave strong, comparable signals. The signals of the trimer (74,000 m/z) and tetramer (98,000 m/z) were detectable but small. B) MS spectrum corresponding to peak 4 in Fig. 2F, with Cu(II) in the cell suspension. The signal corresponding to the monomer was substantially reduced in comparison with that in (A). C ) MS spectrum corresponding to peak 3 in Fig. 2H, with Cu(II) in Ni-NTA eluent. Very similar results were obtained as in (B). D) 2D gel electrophoresis of C51 light chain without Cu(II). Many different spots of pI are seen at 31 kDa, from pI 6.12 to 10.0. The most intense spot was observed at pI 6.45–6.73. E ) 2D gel electrophoresis of C51 light chain with Cu(II). The spots gathered at pI 6.57 (theoretical pI 6.44).

suggesting that the C-terminal cysteine has an uptake of 0.21 atoms. The uptake of H195A/H204A/C220A mutant was lower than that of the C220 mutant by 0.29 atoms, which was the contribution by the 2 histidine residues. The Cu uptake by these 3 residues is considered to be 0.5 (0.75–0.25) atoms, which reaches 67% of the total uptake by the C51 wild-type. From molecular modeling (Fig. 5B), the location of His195 is close to Cys220, whereas that of His204 is more distant. His195 and Cys220 may work cooperatively for the uptake of Cu ion. STRUCTURAL DIVERSITY OF CATALYTIC ANTIBODY LIGHT CHAIN

To examine the contribution of the constant region of the light chain to structural diversity, the CR domain (Fig. 1) for the human k light chain was similarly expressed and purified and exhibited the results in Ni-NTA chromatography shown in Supplemental Fig. S1A, B. The results of cation-exchange chromatography without and with the addition of Cu(II) are shown in Supplemental Figs. S2, S3. In Supplemental Fig. S2 [without Cu(II)], several peaks were observed: peaks 1 and 2 were monomer rich, and peak 3 was dimer rich. 901

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Figure 4. Light chains #4 and #7. A) The #4 light chain without Cu(II). The peaks observed at retention times 5–23 min were mainly mixtures of monomer and dimer. B) The #4 light chain with Cu(II) in the Ni-NTA eluent. The single peak observed at the retention time of 18 min was a mixture of the monomer and dimer, but was dimer rich. Addition of Cu(II) effectively influenced the charge heterogeneity of the light chain. C ) The #7 light chain without Cu(II). The peaks observed at retention times of 5–25 min were monomers and/or mixtures of monomer and dimer. D) The #7 light chain with 15 mM of Cu(II) (0.38 eq into Ni-NTA eluent). Two peaks were observed at retention times of 12 (peak A) and 23 (peak B) min. They were mainly dimers. The ratio of Cu:Lc for peaks A and B were 0.48 and 0.03, respectively. E ) The #7 light chain with 40 mM of Cu(II) (1.0 eq in Ni-NTA eluent). One peak (peak C) was detected at the retention time of 12 min, and it was mainly dimer. Cu:Lc ratio, 0.64. AFM analysis of the #7 light chain: F ) Image for peak A (D), which included Cu(II). Cu:Lc ratio, 0.48. G) Image for peak B (D). This sample of peak B did not include Cu(II) (Cu:Lc ratio, 0.03). H ) Image for peak C (E ), including Cu(II) (Cu:Lc ratio, 0.64). Red circles: AFM images of the dimer of the #7 light chain. The size of the dimer was roughly estimated at a length of ;20 nm, width of ;10 nm, and height of ;4 nm. m, monomer; di, dimer in the cation-exchange chromatograms.

The Cu:CR ratio was 0:0 in all peaks. With the addition of Cu(II) [50 mM (1.25 eq) addition to the Ni-NTA eluent], the peak became nearly single and was mainly a dimer (Supplemental Fig. S3). The Cu:CR ratio was 0.62. The CR exhibited behaviors similar as those of full-length light chains (C51, #4, and #7). 902

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The UV/VIS spectrum for peak 1 in Supplemental Fig. S3 is presented in Fig. 5C, where an absorbance at ;550 nm was clearly observed. The uptake of Cu ion was also confirmed by spectroscopy, as the ion has a tendency to coordinate with the oxygen, nitrogen, and sulfur atoms of the amino acid residues (26, 27). With

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the addition of EDTA to the fraction of peak 1, no absorbance was detected, suggesting that Cu(II) was removed by EDTA. Size-exclusion chromatography To investigate the molecular size of the C51 light chain possessing a homogeneous electrical charge, we performed STRUCTURAL DIVERSITY OF CATALYTIC ANTIBODY LIGHT CHAIN

size-exclusion chromatography. The Cu(II)-bound C51 light chain, which was prepared as shown in Supplemental Fig. S4, gave a single, sharp, dimer-rich peak. The peak was subjected to size-exclusion chromatography (Supplemental Fig. S5). At a retention time of 70–90 min, a large peak with a small shoulder was observed. The main peak at 74 min corresponded to the dimer and the shoulder at 84 min to the monomer. 903

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Figure 5. Mutants and the CR domain. A) Mutated positions in the C51 light chain. B) The relation of the mutated positions by molecular modeling. Blue: b-sheet, red: a-helix. His195 is located close to Cys220. C ) UV/VIS spectra for the CR, measuring the spectrum of peak 1 in (Supplemental Fig. S3). Absorbance at ;550 nm was clearly observed. With the abolishment of Cu(II) by EDTA, absorbance was not detected.

The result of SDS-PAGE showed that a small amount of the monomer was included, indicating that, in some cases, the molecular sizes were not yet homogeneous, even though the electrical charge had become monoform. The same expression and purification were performed for the C51 C220A mutant. Size-exclusion chromatography of the mutant showed a clear single peak (no dimer was detected) appeared at 85 min, indicating the simple monomeric structure with a single charge (i.e., a defined structure) was obtained (Supplemental Fig. S6).

Peptidase activity Several synthetic peptidyl-MCA substrates were used to investigate the peptidase activity of the C51 dimeric light chain with and without Cu(II). The results are presented in Fig. 6A–D for Gln-Ala-Arg (QAR)-MCA, Val-Pro-Arg (VPR)-MCA, Glu-Ala-Arg (EAR)-MCA, and Asn-Pro-Leu-Asn-Pro (GPLGP)-MCA, respectively. Regarding QAR-MCA, VPR-MCA, and EAR-MCA, the cleavage of the peptide bond was observed, regardless of the presence of the Cu ion. For the GPLGP-MCA substrate, no cleavage was observed, with or without the

TABLE 1. Cu(II) uptake by each light chain C51 light chain

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Cu:Lca

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Form

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+ CL Figure 6. Peptidase activity and the intramolecular interaction of the C51 light chain. A–D) Peptidase activity. The highly purified C51 dimeric light chain (100 mL, 10 mM) with and without Cu(II) was mixed with an equal volume of 100 mM synthetic substrates QAR-MCA, VPR-MCA, EAR-MCA, and GPLGP -MCA and incubated at 37°C. The AMC released from the substrate catalyzed by the light chains was detected by measurement of fluorescence emission at 470 nm/excitation at 360 nm. Regarding QAR-MCA, (continued on next page)

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Cu ion. No cleavage occurred in the Lys-MCA, Arg-MCA, and Ala-Pro-Ala-MCA substrates (data not shown). Although the catalytic activity of the C51 light chain was low by one-tenth-fold compared with that of Leu 12 found by Durova et al. (5) and 22F6 found by Hifumi et al. (2), there was no difference with or without Cu(II). DISCUSSION As stated in the introduction, the structural diversity of monoclonal antibodies has been studied for .20 yr. Recently, the discussion has concentrated on clarifying the causes of the heterogeneity resulting in C-terminal lysine variants (28) and elongation or truncation of the N terminus of the protein (29). In the case of our light chains, variations such as lysine variants and elongation were excluded, because lysine variants appear in the C terminus of the heavy chain (in this study, only the light chain was used), and no elongation was observed at the N terminus by the amino acid sequence analysis. Regarding the charge heterogeneity, Harris et al. (23) and Nebija et al. (24, 30) have extensively studied this aspect with the recombinant monoclonal antibody. The former paper pointed out a deamidation of Asn residues in the protein. In our study, the possibility of deamidation was excluded because the addition of the Cu ion caused a reverse reaction of the deamidation and heterogeneity was lost. The latter papers showed multiple spots of pI spreading in the heavy and light chains, caused by generation of charge-related isoforms. The pI-spreading pattern in 2D gel electrophoresis (30) is similar to observations. The effects of sugar chains are also taken into account for molecular heterogeneity. As it is well known that antibody light chains have no sugar chains, their effect is excluded from our discussion. Thus, we conclude that the structural diversity is mostly due to the heterogeneity of the different electrical charges. In the Ni-NTA chromatography, the chromatogram patterns were similar to those of many light chains (Fig. 2B). In cation-exchange chromatography, several peaks were observed, even for single light chains such as C51, #4, and #7. Considering that the peaks were generated from a single light chain and not from other irrelevant proteins, several different conformational light chains must coexist. When Cu (II) was added to the Ni-NTA eluent, the peak in the cation-exchange chromatography became nearly 1 peak altogether for the C51, #4, and #7 light chains. The single peak was mainly a dimer. The Cu(II) accelerates the formation of the dimeric light chain and

then contribute to making an electrically monoform structure from the multiform. The dimerization proceeds perfectly within 10 h after the addition of the Cu ion. Next, we must consider the role of Cu(II). What amount of Cu(II) is taken up in 1 molecule of the light chain? What site of the light chain interacts with Cu(II)? The concentration dependency of the Cu ion was examined. In the cation-exchange chromatography for the #7 light chain, 2 dimer peaks (Fig. 4D, peak Am and Fig. 4E, peak C) were considered to have the same electrical charge. The Cu:Lc ratio of peaks A and C was 0.48 and 0.64, respectively. In contrast, the single peak of the CR (Supplemental Fig. S3) gave a Cu:CR ratio of 0.62. Considering these facts, the Cu:Lc ratio was at ;0.5, indicating that 2 monomeric molecules (one dimer) of the light chain take up 1 Cu(II). To investigate the site of interaction of Cu(II) with the light chain, we made 2 mutants of the C51 light chain, and the Cu uptake was chemically analyzed. The results in Table 1 show that the cysteine at the C terminus and the histidines located at residue 195 (and/or 204) may play an important role in the binding with Cu(II) via the cooperative function. This possibility means that the Cu ion is not incorporated in the S—S bond such as S—Cu—S, but it may be taken up in the light chain by interacting with the one or both of the nitrogen of His and sulfur of Cys residues. The possibilities of His-tag and other residues such as Asp and Arg are not excluded. Ponomarenko et al. (31) succeeded in performing X-ray diffraction analysis of their A17 catalytic antibody (reactibody) and proposed a binding site of the antigen and the antibody. In our method, X-ray diffraction analysis must be performed for the assignment, although the crystallization of the light chain is difficult. Furthermore, we must consider the possibility of a noncovalent interaction force that causes multimers such as dimers, trimers, tetramers, and hexamers to form. Thus, we examined noncovalent interaction by using molecular modeling for the C51 light chain, wherein electrostatic potential and molecular conformation were calculated. The results are presented in Fig. 6E, where red indicates the hydrophobic region, yellow the positive charge (+), and green the negative charge (2). The hydrophobic region of the variable light chain (VL) is indented; conversely, the constant light chain (CL) region protrudes. Thus, the VL and CL regions fit well, like a lock and key. The area of the positive charge (+) in VL and that of the negative charge (2) in the CL are characteristic. These opposite charges may accelerate the noncovalent binding between VL and CL. As a consequence, the C51 light

VPR-MCA, and EAR-MCA, the cleavage of the peptide bond was observed regardless of the presence of the Cu ion. For the GPLGP-MCA substrate, no cleavage was observed, with or without the Cu ion. Catalytic activity was not influenced with the presence of Cu(II). E ) Intramolecular interaction of the C51 light chain. Electrostatic potential and molecular conformation were calculated for VL and CL by using a molecular modeling system. Red: hydrophobic region; yellow: positive charge (+) region; green: negative charge (2) region. The hydrophobic region of VL is indented and that of CL protrudes, suggesting that the regions of VL and CL fit well. The areas of positive charge (+) in VL and that of negative charge (–) in CL are characteristic. This feature may accelerate the noncovalent binding between VL and CL, resulting in the formation of a monomer by the intramolecular interaction between VL and CL.

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chain tends to become a monomer by the intramolecular noncovalent interaction between VL and CL. This characteristic depends on the amino acid sequence of the light chain. Sharma et al. (32) found an interesting phenomenon, in which the dimer and tetramer of their human catalytic antibody light chain showed low and high reactivities to cleave a TNF-a molecule, respectively. It is likely that the noncovalent interaction caused by the heterogeneity formed a tetramer from a dimer and then derived the different reactivities from them. Regarding the metal ions, we have already pointed out the importance of the function of a catalytic antibody (33). In that study, the Cu ion contributed substantially to convert the light chain’s structure from monoform to multiform, indicating the high advantages of reproducibility and the efficacy of the production. Recently, Smirnov et al. (13), Bezuglova et al. (34), and Nishiyama et al. (7) also found the significance of metal ions, in supporting the catalytic function of their catalytic antibodies. In our study, Zn(II) showed the interesting feature of being effective in inducing charge heterogeneity (data not shown). We conclude that the metal ion may contribute to the stability of the preferable conformation of the catalytic antibodies. The novel method reported herein will accelerate the development of catalytic antibody research. The authors thank Dr. H. Sugasawa (Oxford Instruments KK, Tokyo, Japan), Ms. E. Saito, Ms. S. Takemoto, and Ms. Y. Akiyoshi (Oita University) for assistance. This study was supported by the Japan Science and Technology Agency (CREST/Establishment of Innovative Manufacturing Technology Based on Nanoscience; Super Highway: accelerated research to bridge university intellectual properties and practical use), and Grant-in-Aid 24350085 for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to E.H. and T.U.). The authors declare no conflicts of interest.

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Received for publication May 26, 2015. Accepted for publication October 19, 2015.

HIFUMI ET AL.

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