A novel method of preparing the monoform structure of catalytic antibody light chain

Hifumi, Emi; Matsumoto, Shingo; Nakashima, Hiroyuki; Itonaga, Shogo; Arakawa, Mitsue; Katayama, Yoshiki; Kato, Ryuichi; Uda, Takaaki

doi:10.1096/fj.15-276394

Cited by 12 publications

(17 citation statements)

References 34 publications

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“…After ultrasonication of the E coli , a soluble fraction was recovered and applied to Ni‐ion affinity chromatography, as described in Section . After this step, Cu(II) ion was added to make the heterogeneous structures of the light chain (possessing different electrical charges) to the homogeneous (a single charge), which contributes to the efficient production and the high reproducibility (see Reference ). Then, the treatment of EDTA (for taking out Cu from the system) was carried out to keep the stability of the homogeneous structure for a long‐term period.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, one has to be aware of the structural diversity problem of antibodies. We were able to solve the structural diversity issue, and reported how to control for an antibody having a mono‐form structure . With this technology, we can make the catalytic antibody structure take a monomolecular form, which is necessary to preparing the same conformational antibody to attain effective production well as reproducible reactivity.…”

Section: Introductionmentioning

confidence: 99%

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New technologies to introduce a catalytic function into antibodies: A unique human catalytic antibody light chain showing degradation of β-amyloid molecule along with the peptidase activity

et al. 2019

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Since the discovery of a natural catalytic antibody in 1989, many catalytic antibodies targeting peptides, nucleotides, virus and bacterial proteins, and many molecules have been prepared. Although catalytic antibodies should have features superior to non‐catalytic monoclonal antibodies, the reports on catalytic antibodies are far fewer than those on normal (non‐catalytic) antibodies. Nowadays, we can obtain any monoclonal antibody we want, which is not the case for catalytic antibodies. To overcome this drawback of catalytic antibodies, much basic research must be done. As one way to attain this purpose, we have been making a protein bank of human antibody light chains, in which the light chains were expressed, purified, and stored for use in screening against many kinds of antigen, to then get clues to introducing a catalytic function in normal antibodies. As the number of stored light chains in the above protein bank has reached the hundreds, in this study, we screened them against amyloid‐beta (Aβ), which is well‐known as one of the molecules causing Alzheimer's disease. We found two interesting light chains, #7TR and #7GY. The former could degrade both a fluorescence resonance energy transfer‐Aβ substrate and Aβ1‐40 full peptide. In contrast, #7GY, whose sequence is identical to that of #7TR except for the amino acids at the 29th and 30th positions, did not degrade the FRET‐Aβ substrate at all. By using a synthetic substrate, Arg‐pNA, the difference between the chemical features of the two light chains was investigated in detail. In addition, we found that the presence of Zn(II) ion hugely influenced the catalytic activity of the #7TR light chain but not #7GY. Through these facts and the discussion, we propose one of the clues to how to put a catalytic function in a normal (non‐catalytic) antibody.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

New technologies to introduce a catalytic function into antibodies: A unique human catalytic antibody light chain showing degradation of β-amyloid molecule along with the peptidase activity

et al. 2019

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“…Using the FRET-A (26)(27)(28)(29)(30)(31)(32)(33) [MCA-SNKGAIIGK(DNP)rrr-NH 2 ] substrate, we examined catalytic activity by T99wt and T99-Pro95(−) at a temperature of 37°C. The reaction time courses are shown in Fig.…”

Section: Cleavage For Fret-amentioning

confidence: 99%

A new algorithm to convert a normal antibody into the corresponding catalytic antibody

et al. 2020

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Over thousands of monoclonal antibodies (mAbs) have been produced so far, and it would be valuable if these mAbs could be directly converted into catalytic antibodies. We have designed a system to realize the above concept by deleting Pro 95 , a highly conserved residue in CDR-3 of the antibody light chain. The deletion of Pro 95 is a key contributor to catalytic function of the light chain. The S35 and S38 light chains have identical amino acid sequences except for Pro 95 . The former, with Pro 95 did not show any catalytic activity, whereas the latter, without Pro 95 , exhibited peptidase activity. To verify the generality of this finding, we tested another light chain, T99wt, which had Pro 95 and showed little catalytic activity. In contrast, a Pro 95 -deleted mutant enzymatically degraded the peptide substrate and amyloid-beta molecule. These two cases demonstrate the potential for a new method of creating catalytic antibodies from the corresponding mAbs. RESULTS Human antibody light chains S35 and S38 Comparison of amino acid sequences of the V region and a germ lineThis study focuses on two unique light chains, S35 and S38, which belong to subgroup II of the human kappa light chain. The amino acid sequence of 219 amino acid residues of the S35 light chain is a perfect match with that of the germline 2/2D-28*01 of the V region (V: amino acids 1 to 95, see Fig. 1A). This means that no somatic mutations took place in the S35 light chain. On the other hand, the S38 light chain has amino acid sequences identical to S35 except for the Pro 95 residue, which was deleted from S38 during the mutation process. Purification and monoform structureIn the purification process, a Cu(II) ion was added into the solution after Ni-ion affinity chromatography. This step is important and necessary to making a monoform structure from the multiform structures of the light chain (28-30). In the final step, EDTA was added to take out the Cu(II) ion to keep the monoform structure in the long term. Both Cu(II) and EDTA are not concerned to create a catalytic site, but to make and keep the preferable structure. The results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis for S35 and S38 under the reduced and nonreduced conditions are presented in Fig. 1B. Bands at about 46 and 26 kDa corresponding to the dimer and monomer, respectively, were observed under the nonreduced condition. Only a 30-kDa band corresponding to the monomer was detected under the reduced condition. In SDS-PAGE analysis, bands other than the monomer and dimer bands of the light chains were hardly observed, suggesting that the S35 and S38 light chains were highly purified. Peptidase activity (hydrolysis of synthetic substrates)We examined how the light chains with or without Pro 95 affected catalytic properties, by using synthetic substrates. As Matsuura et al. (31) and Durova et al. (32) used the substrate Arg-pNA to evaluate catalytic activity, we also used synthetic substrates, such as Arg-pNA (R-pNA), Glu-pNA (E-pNA), Leu-pNA (L-pNA), Ala-pNA ...

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“…Regarding the amount of Cu 2+ incorporated in the CL, the chemical analysis implies that a ratio of 0.6 eq Cu 2+ to CL was the maximum, suggesting that two CL molecules bind with one Cu 2+ . The binding site of Cu 2+ may be close to the C terminus where His195 and Cys220 are located, as suggested by our previous study on the full length of the human antibody light chains (25). In the constant region of antibody light chain, there is a zinc finger-like motif, which can bind with a divalent metal ion.…”

Section: Discussionmentioning

confidence: 83%

“…We have also reported that molecular heterogeneity is caused by different electrical charges and different molecular sizes in a mouse monoclonal antibody (24). In addition, we recently found similar molecular heterogeneity in recombinant human catalytic antibody light chains (25). An antibody light chain consists of 2 domains: the variable region of light chain and the constant region of the light chain [i.e., the catalytic antibody k light chain (CL)].…”

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Role of the constant region domain in the structural diversity of human antibody light chains

et al. 2017

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Issues regarding the structural diversity (heterogeneity) of an antibody molecule have been the subject of discussion along with the development of antibody drugs. Research on heterogeneity has been extensive in recent years, but no clear solution has been reached. Heterogeneity is also observed in catalytic antibody κ light chains (CLs). In this study, we investigated how the constant region domain of CLs concerns structural diversity because it is a simple and good example for elucidating heterogeneity. By means of cation-exchange chromatography, SDS-PAGE, and 2-dimensional electrophoresis for the CL, multimolecular forms consisting of different electrical charges and molecular sizes coexisted in the solution, resulting in the similar heterogeneity of the full length of CLs. The addition of copper ion could cause the multimolecular forms to change to monomolecular forms. Copper ion contributed greatly to the enrichment of the dimer form of CL and the homogenization of the differently charged CLs. Two molecules of the CL protein bound one copper ion. The binding affinity of the ion was 48.0 μM Several divalent metal ions were examined, but only zinc showed a similar effect.-Hifumi, E., Taguchi, H., Kato, R., Uda, T. Role of the constant region domain in the structural diversity of human antibody light chains.

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A novel method of preparing the monoform structure of catalytic antibody light chain

Cited by 12 publications

References 34 publications

New technologies to introduce a catalytic function into antibodies: A unique human catalytic antibody light chain showing degradation of β-amyloid molecule along with the peptidase activity

New technologies to introduce a catalytic function into antibodies: A unique human catalytic antibody light chain showing degradation of β-amyloid molecule along with the peptidase activity

A new algorithm to convert a normal antibody into the corresponding catalytic antibody

Role of the constant region domain in the structural diversity of human antibody light chains

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