Structural Diversity Problems and the Solving Method for Antibody Light Chains

Hifumi, Emi; Taguchi, Hiroaki; Kato, Ryuichi; Arakawa, Mitsue; YoshikiKatayama,; Uda, Takaaki

doi:10.5772/intechopen.72516

Cited by 5 publications

(11 citation statements)

References 35 publications

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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%

“…In 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)(29)(30). In the final step, EDTA was added to take out the Cu(II) ion to keep the monoform structure in the long term.…”

Section: Purification and Monoform Structurementioning

confidence: 99%

“…The concentration change of the Arg-pNA substrate below 5% was regarded as the initial rate of the reaction. For FRET-A substrate T99-Pro95(−) light chain for FRET-A: The concentration of T99-Pro95(−) light chain was fixed at 5 M, and that of the FRET-A (26)(27)(28)(29)(30)(31)(32)(33) substrate varied from 5 to 60 M at 37°C in the TGT buffer (pH 7.7). The concentration change of the FRET-A (26)(27)(28)(29)(30)(31)(32)(33) substrate within 10% conversion was regarded as the initial rate of the reaction.…”

Section: Kineticsmentioning

confidence: 99%

“…For FRET-A substrate T99-Pro95(−) light chain for FRET-A: The concentration of T99-Pro95(−) light chain was fixed at 5 M, and that of the FRET-A (26)(27)(28)(29)(30)(31)(32)(33) substrate varied from 5 to 60 M at 37°C in the TGT buffer (pH 7.7). The concentration change of the FRET-A (26)(27)(28)(29)(30)(31)(32)(33) substrate within 10% conversion was regarded as the initial rate of the reaction. The measurements were performed in triplicate.…”

Section: Kineticsmentioning

confidence: 99%

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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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Section: Cleavage For Fret-amentioning

confidence: 99%

Section: Purification and Monoform Structurementioning

confidence: 99%

Section: Kineticsmentioning

confidence: 99%

Section: Kineticsmentioning

confidence: 99%

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

et al. 2020

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“…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%

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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A new catalytic site functioning in antigen cleavage by H34 catalytic antibody light chain

Hifumi

Nonaka

Taguchi

et al. 2022

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The cleavage reactions of catalytic antibodies are mediated by a serine protease mechanism involving a catalytic triad composed of His, Ser, and Asp residues, which reside in the variable region. Recently, we discovered a catalytic antibody, H34 wild type (H34wt), that is capable of enzymatically cleaving an immune-check point PD-1 peptide and recombinant PD-1; however, H34wt does not contain His residues in the variable region. To clarify the reason behind the catalytic features of H34wt and the amino acid residues involved in the catalytic reaction, we performed site-directed mutagenesis focusing on the amino acid residues involved in the cleavage reaction, followed by catalytic activity tests, immunological reactivity evaluation, and molecular modeling. The results revealed that the cleavage reaction by H34wt proceeds through the action of a new catalytic site composed of Arg, Thr, and Gln. This new scheme differs from that of the serine protease mechanism of catalytic antibodies.

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Structural Diversity Problems and the Solving Method for Antibody Light Chains

Cited by 5 publications

References 35 publications

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

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

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 catalytic site functioning in antigen cleavage by H34 catalytic antibody light chain

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