SpringerLink
Log in

Tag-free protein modification by lipoate ligase A: exploring substrate tolerance

  • Original Paper
  • Published:
Analytical Sciences Aims and scope Submit manuscript

Abstract

This study delves into the functional intricacies of lipoate ligase A (LplA), an enzyme showing great promise in bioconjugation due to its unique capacity for introducing azido groups into proteins without requiring a genetic tag. We aimed to enhance the understanding of LplA's functionality, particularly its substrate tolerance and the reliability of various analytical techniques. A pivotal aspect of our approach was incorporating azido groups into a range of proteins, followed by the addition of the fluorescent molecule Cy3 via click chemistry. Analysis of fluorescent intensity in the altered proteins indicated varying degrees of conjugation. Additionally, phenyl resin-based RP-HPLC facilitated effective separation of modified proteins, unmodified proteins, and remaining fluorescent tags post-separation. SASA analysis provided insights into conjugation trends, guiding the identification of proteins amenable to LplA's tag-free modification. Our findings demonstrate LplA's broad substrate tolerability for protein modification.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Data availability

Not applicable.

References

  1. S. Yamazaki, Y. Matsuda, Tag-free enzymatic modification for antibody−drug conjugate production. ChemistrySelect 7(48), e202203753 (2022). https://doi.org/10.1002/slct.202203753

    Article  CAS  Google Scholar 

  2. A.F. Hussain, A. Grimm, W. Sheng, C. Zhang, M. Al-Rawe, K. Brautigam, M. Abu Mraheil, F. Zeppernick, I. Meinhold-Heerlein, Toward homogenous antibody drug conjugates using enzyme-based conjugation approaches. Pharmaceuticals (Basel) 14(4), 343 (2021). https://doi.org/10.3390/ph14040343

    Article  CAS  PubMed  Google Scholar 

  3. A. Beygmoradi, A. Homaei, R. Hemmati, P. Fernandes, Recombinant protein expression: challenges in production and folding related matters. Int. J. Biol. Macromol.Macromol. 233, 123407 (2023). https://doi.org/10.1016/j.ijbiomac.2023.123407

    Article  CAS  Google Scholar 

  4. Z. Tawfiq, N.C. Caiazza, S. Kambourakis, Y. Matsuda, B. Griffin, J.C. Lippmeier, B.A. Mendelsohn, Synthesis and biological evaluation of antibody drug conjugates based on an antibody expression system: conamax. ACS Omega 5(13), 7193–7200 (2020). https://doi.org/10.1021/acsomega.9b03628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. P.R. Spycher, C.A. Amann, J.E. Wehrmuller, D.R. Hurwitz, O. Kreis, D. Messmer, A. Ritler, A. Kuchler, A. Blanc, M. Behe, P. Walde, R. Schibli, Dual, site-specific modification of antibodies by using solid-phase immobilized microbial transglutaminase. ChemBioChem 18(19), 1923–1927 (2017). https://doi.org/10.1002/cbic.201700188

    Article  CAS  PubMed  Google Scholar 

  6. S. Dickgiesser, M. Rieker, D. Mueller-Pompalla, C. Schroter, J. Tonillo, S. Warszawski, S. Raab-Westphal, S. Kuhn, T. Knehans, D. Konning, J. Dotterweich, U.A.K. Betz, J. Anderl, S. Hecht, N. Rasche, Site-specific conjugation of native antibodies using engineered microbial transglutaminases. Bioconjug. Chem.. Chem. 31(4), 1070–1076 (2020). https://doi.org/10.1021/acs.bioconjchem.0c00061

    Article  CAS  Google Scholar 

  7. A. Hadjabdelhafid-Parisien, S. Bitsch, A. Macarron Palacios, L. Deweid, H. Kolmar, J.N. Pelletier, Tag-free, specific conjugation of glycosylated IgG1 antibodies using microbial transglutaminase. RSC Adv. 12(52), 33510–33515 (2022). https://doi.org/10.1039/d2ra05630e

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. T.W. Morris, K.E. Reed, J.E. Cronan, Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product. J. Biol. Chem. 269(23), 16091–16100 (1994). https://doi.org/10.1016/s0021-9258(17)33977-7

    Article  CAS  PubMed  Google Scholar 

  9. M. Baalmann, M. Best, R. Wombacher, Site-specific protein labeling utilizing lipoic acid ligase (LplA) and bioorthogonal inverse electron demand Diels-Alder reaction. Methods Mol. Biol. 1728, 365–387 (2018). https://doi.org/10.1007/978-1-4939-7574-7_23

    Article  CAS  PubMed  Google Scholar 

  10. J.G. Plaks, J.L. Kaar, Lipoic acid ligase-promoted bioorthogonal protein modification and immobilization. Methods Mol. Biol. 2012, 279–297 (2019). https://doi.org/10.1007/978-1-4939-9546-2_14

    Article  CAS  PubMed  Google Scholar 

  11. S. Puthenveetil, D.S. Liu, K.A. White, S. Thompson, A.Y. Ting, Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. J. Am. Chem. Soc. 131(45), 16430–16438 (2009). https://doi.org/10.1021/ja904596f

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. S. Yamazaki, N. Shikida, K. Takahashi, Y. Matsuda, K. Inoue, K. Shimbo, Y. Mihara, Lipoate-acid ligase a modification of native antibody: synthesis and conjugation site analysis. Bioorg. Med. Chem. Lett.. Med. Chem. Lett. 51, 128360 (2021). https://doi.org/10.1016/j.bmcl.2021.128360

    Article  CAS  Google Scholar 

  13. S. Yamazaki, K. Inoue, Y. Mihara, Y. Matsuda, Tag-free antibody modification mediated by lipoic acid ligase a: application to antibody-drug conjugates production. ChemistrySelect 8(9), e202204706 (2023). https://doi.org/10.1002/slct.202204706

    Article  CAS  Google Scholar 

  14. Y. Matsuda, M. Leung, Z. Tawfiq, T. Fujii, B.A. Mendelsohn, In-situ reverse phased HPLC analysis of intact antibody-drug conjugates. Anal. Sci. 37(8), 1171–1176 (2021). https://doi.org/10.2116/analsci.20P424

    Article  CAS  PubMed  Google Scholar 

  15. E. Durham, B. Dorr, N. Woetzel, R. Staritzbichler, J. Meiler, Solvent accessible surface area approximations for rapid and accurate protein structure prediction. J. Mol. Model. 15(9), 1093–1108 (2009). https://doi.org/10.1007/s00894-009-0454-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. T. Fujii, Y. Matsuda, T. Seki, N. Shikida, Y. Iwai, Y. Ooba, K. Takahashi, M. Isokawa, S. Kawaguchi, N. Hatada, T. Watanabe, R. Takasugi, A. Nakayama, K. Shimbo, B.A. Mendelsohn, T. Okuzumi, K. Yamada, AJICAP second generation: improved chemical site-specific conjugation technology for antibody-drug conjugate production. Bioconjug. Chem.. Chem. 34(4), 728–738 (2023). https://doi.org/10.1021/acs.bioconjchem.3c00040

    Article  CAS  Google Scholar 

  17. Y. Nakahara, B.A. Mendelsohn, Y. Matsuda, Antibody-drug conjugate synthesis using continuous flow microreactor technology. Org. Process Res. Dev. 26(9), 2766–2770 (2022). https://doi.org/10.1021/acs.oprd.2c00217

    Article  CAS  Google Scholar 

  18. P.E. Stein, A.G. Leslie, J.T. Finch, R.W. Carrell, Crystal structure of uncleaved ovalbumin at 1.95 A resolution. J. Mol. Biol. 221(3), 941–959 (1991). https://doi.org/10.1016/0022-2836(91)80185-w

    Article  CAS  PubMed  Google Scholar 

  19. H. Kurokawa, J.C. Dewan, B. Mikami, J.C. Sacchettini, M. Hirose, Crystal structure of hen apo-ovotransferrin. Both lobes adopt an open conformation upon loss of iron. J. Biol. Chem. 274(40), 28445–28452 (1999). https://doi.org/10.1074/jbc.274.40.28445

    Article  CAS  PubMed  Google Scholar 

  20. K.A. Majorek, P.J. Porebski, A. Dayal, M.D. Zimmerman, K. Jablonska, A.J. Stewart, M. Chruszcz, W. Minor, Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Mol. Immunol.Immunol. 52(3–4), 174–182 (2012). https://doi.org/10.1016/j.molimm.2012.05.011

    Article  CAS  Google Scholar 

  21. S.Y. Wu, M.D. Perez, P. Puyol, L. Sawyer, beta-lactoglobulin binds palmitate within its central cavity. J. Biol. Chem. 274(1), 170–174 (1999). https://doi.org/10.1074/jbc.274.1.170

    Article  CAS  PubMed  Google Scholar 

  22. A. Sivertsen, J. Isaksson, H.K. Leiros, J. Svenson, J.S. Svendsen, B.O. Brandsdal, Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin. BMC Struct. Biol.Struct. Biol. 14, 4 (2014). https://doi.org/10.1186/1472-6807-14-4

    Article  CAS  Google Scholar 

  23. M. Muraki, K. Harata, N. Sugita, K. Sato, Origin of carbohydrate recognition specificity of human lysozyme revealed by affinity labeling. Biochemistry 35(42), 13562–13567 (1996). https://doi.org/10.1021/bi9613180

    Article  CAS  PubMed  Google Scholar 

  24. K. Harata, X-ray structure of monoclinic turkey egg lysozyme at 1.3 A resolution. Acta Crystallogr. D Biol. Crystallogr. Crystallogr. D Biol. Crystallogr. 49(Pt 5), 497–504 (1993). https://doi.org/10.1107/S0907444993005542

    Article  CAS  Google Scholar 

  25. N. Noinaj, N.C. Easley, M. Oke, N. Mizuno, J. Gumbart, E. Boura, A.N. Steere, O. Zak, P. Aisen, E. Tajkhorshid, R.W. Evans, A.R. Gorringe, A.B. Mason, A.C. Steven, S.K. Buchanan, Structural basis for iron piracy by pathogenic Neisseria. Nature 483(7387), 53–58 (2012). https://doi.org/10.1038/nature10823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Y. Matsuda, A. Chakrabarti, K. Takahashi, K. Yamada, K. Nakata, T. Okuzumi, B.A. Mendelsohn, Chromatographic analysis of site-specific antibody-drug conjugates produced by AJICAP first-generation technology using a recombinant FcgammaIIIa receptor-ligand affinity column. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1177, 122753 (2021). https://doi.org/10.1016/j.jchromb.2021.122753

    Article  CAS  Google Scholar 

  27. Y. Matsuda, T. Seki, K. Yamada, Y. Ooba, K. Takahashi, T. Fujii, S. Kawaguchi, T. Narita, A. Nakayama, Y. Kitahara, B.A. Mendelsohn, T. Okuzumi, Chemical site-specific conjugation platform to improve the pharmacokinetics and therapeutic index of antibody-drug conjugates. Mol. Pharm. 18(11), 4058–4066 (2021). https://doi.org/10.1021/acs.molpharmaceut.1c00473

    Article  CAS  PubMed  Google Scholar 

  28. T. Fujii, C. Reiling, C. Quinn, M. Kliman, B.A. Mendelsohn, Y. Matsuda, Physical characteristics comparison between maytansinoid-based and auristatin-based antibody-drug conjugates. Explor. Target Antitumor Ther. 2(6), 576–585 (2021). https://doi.org/10.37349/etat.2021.00064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. H. Wu, D. Cao, T. Liu, J. Zhao, X. Hu, N. Li, Purification and characterization of recombinant human lysozyme from eggs of transgenic chickens. PLoS ONE 10(12), e0146032 (2015). https://doi.org/10.1371/journal.pone.0146032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. J. Lescar, H. Souchon, P.M. Alzari, Crystal structures of pheasant and guinea fowl egg-white lysozymes. Protein Sci. 3(5), 788–798 (1994). https://doi.org/10.1002/pro.5560030508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. D. Mercadante, L.D. Melton, G.E. Norris, T.S. Loo, M.A. Williams, R.C. Dobson, G.B. Jameson, Bovine beta-lactoglobulin is dimeric under imitative physiological conditions: dissociation equilibrium and rate constants over the pH range of 2.5–7.5. Biophys. J.. J. 103(2), 303–312 (2012). https://doi.org/10.1016/j.bpj.2012.05.041

    Article  CAS  Google Scholar 

  32. M. Da Silva, S. Beauclercq, G. Harichaux, V. Labas, N. Guyot, J. Gautron, Y. Nys, S. Rehault-Godbert, The family secrets of avian egg-specific ovalbumin and its related proteins Y and X. Biol. Reprod.Reprod. 93(3), 71 (2015). https://doi.org/10.1095/biolreprod.115.130856

    Article  CAS  Google Scholar 

  33. D. Fologea, B. Ledden, D.S. McNabb, J. Li, Electrical characterization of protein molecules by a solid-state nanopore. Appl. Phys. Lett. 91(5), 539011–539013 (2007). https://doi.org/10.1063/1.2767206

    Article  CAS  PubMed  Google Scholar 

  34. D.A. Belinskaia, P.A. Voronina, A.A. Batalova, N.V. Goncharov, Serum albumin. Encyclopedia 1(1), 65–75 (2020). https://doi.org/10.3390/encyclopedia1010009

    Article  Google Scholar 

  35. F. Giansanti, L. Leboffe, F. Angelucci, G. Antonini, The nutraceutical properties of ovotransferrin and its potential utilization as a functional food. Nutrients 7(11), 9105–9115 (2015). https://doi.org/10.3390/nu7115453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. M. Kallsten, R. Hartmann, K. Artemenko, S.B. Lind, F. Lehmann, J. Bergquist, Qualitative analysis of antibody-drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs. Analyst 143(22), 5487–5496 (2018). https://doi.org/10.1039/c8an01178h

    Article  CAS  PubMed  Google Scholar 

  37. Z. Tawfiq, Y. Matsuda, M.J. Alfonso, C. Clancy, V. Robles, M. Leung, B.A. Mendelsohn, Analytical comparison of antibody-drug conjugates based on good manufacturing practice strategies. Anal. Sci. 36(7), 871–875 (2020). https://doi.org/10.2116/analsci.19P465

    Article  CAS  PubMed  Google Scholar 

  38. N. Shikida, S. Yamazaki, K. Takahashi, Y. Matsuda, K. Shimbo, Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide map**. Anal. Bioanal. Chem.Bioanal. Chem. 415, 6461–6469 (2023)

    Article  CAS  Google Scholar 

  39. Y. Matsuda, Z. Tawfiq, M. Leung, B.A. Mendelsohn, Insight into temperature dependency and design of experiments towards process development for cysteine-based antibody-drug conjugates. ChemistrySelect 5(28), 8435–8439 (2020). https://doi.org/10.1002/slct.202001822

    Article  CAS  Google Scholar 

  40. L. Conilh, L. Sadilkova, W. Viricel, C. Dumontet, Payload diversification: a key step in the development of antibody-drug conjugates. J. Hematol. Oncol.Hematol. Oncol. 16(1), 3 (2023). https://doi.org/10.1186/s13045-022-01397-y

    Article  CAS  Google Scholar 

  41. T. Fujii, Y. Matsuda, S Novel formats of antibody conjugates: recent advances in payload diversity, conjugation, and linker chemistry. Expert Opin. Biol. Ther.Opin. Biol. Ther. 23(11), 1053–1065 (2023). https://doi.org/10.1021/jo2010186

    Article  CAS  Google Scholar 

  42. I. Cheng-Sanchez, F. Moya-Utrera, C. Porras-Alcala, J.M. Lopez-Romero, F. Sarabia, Antibody-drug conjugates containing payloads from marine origin. Mar. Drugs 20(8), 494 (2022). https://doi.org/10.3390/md20080494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors would like to thank their colleagues from A**omoto Co., Inc. and A**omoto-Genetika Research Institute., as follows: Dr. Sergey Vasilievich Smirnov, Dr. Ilina Libovna Tokmakova, Mr. Kota Inoue, and Dr. Yasuhiro Mihara for LplA preparation; Dr. Uno Tagami for crystal structure modeling and constructive discussion; Ms. Natsuki Shikida and Dr. Kazutaka Shimbo for protein analysis; Dr. Shigeo Hirasawa, Dr. Mototaka Suzuki and Dr. Tatsuya Okuzumi for helpful comments and suggestions in this study.

Funding

This work was supported by A**omoto Co., Inc.

Author information

Authors and Affiliations

  1. A**omoto Co., Inc., 1-1 Suzuki-cho, Kawasaki, Kanagawa, 210-8681, Japan

    Shunsuke Yamazaki, Kazutoshi Takahashi & Yutaka Matsuda

Authors
  1. Shunsuke Yamazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kazutoshi Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yutaka Matsuda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Shunsuke Yamazaki or Yutaka Matsuda.

Ethics declarations

Conflict of interest

The authors declare no potential conflicts of interest.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (XLSX 148 KB)

Supplementary file2 (DOCX 898 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yamazaki, S., Takahashi, K. & Matsuda, Y. Tag-free protein modification by lipoate ligase A: exploring substrate tolerance. ANAL. SCI. 40, 1111–1119 (2024). https://doi.org/10.1007/s44211-024-00534-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s44211-024-00534-6

Keywords

Search

Navigation