Intramolecular isopeptide bonds are amide bonds formed spontaneously between amino acid side chains that confer significant structural, thermal, and proteolytic stability to the parent protein, previously used in Synthetic Biology as a means to ligate protein tags1. The objective of the Domain project was to demonstrate the ability of isopeptide bonds as a protein engineering tool for increasing stability, with particular focus on antibody proteins due to their wider relevance as therapeutics and diagnostics.
To do this we employed machine-learning assisted protein structure prediction and a rational design approach based on the only previous work engineering such a bond from Kwon et al. (2017)2, which lead us to a wider bioinformatics search of the Protein Data Bank (PDB) in an effort to broaden the applicability of the method outlined by Kwon and colleagues.
Whilst intermolecular isopeptide bonds ligating proteins occur widely throughout life3,4,5,6,7,8, intramolecular isopeptide bonds are only known from the surface proteins of gram-positive bacteria, where they have been found in most species examined to date2. Here, these bonds confer unusually high thermal, mechanical, and in some cases proteolytic stability to the parent proteins9,10, allowing these typically long and thin proteins to survive the stresses of the extracellular environment.
In all known cases, the bond-forming residues are restricted to a Lysine (Lys) and either an Aspartate (Asp) or more commonly an Asparagine (Asn), with a conserved acidic (Aspartate or Glutamate) residue always being found adjacent to the bond (figure 1). The bonds form spontaneously, and analysis of bond formation has revealed that this is due to the conserved adjacent acidic residue acting as a catalytic ‘proton shuttle’ (figure 1). As this requires the acidic residue to be protonated, it is worth noting that this reaction can only occur in the hydrophobic core of the protein.
Figure 1: The reaction mechanism forming an isopeptide bond between an Asparagine and a Lysine. The Glutamate residue acts as a catalyst shuttling protons from the Lysine to Asparagine, with Ammonia as the leaving group. Note the Glutamate here could be substituted for an Aspartate, as all that is required is an acidic group. Similarly the Asparagine could be substituted for an acidic group, with water as a leaving group, or for a Glutamine.
Our idea is to introduce such a bond into an antibody, with the intent to impart these highly stable features to it. As this has never been achieved in a mammalian protein, we think our project, if successful, would be a major proof-of-concept to the field of antibody engineering.
Inspiration to work on immunotherapies came from members of the team with autoimmune diseases, who have first-hand experience with the cost associated with immunotherapy. We were thus inspired to work on a project which would at least lower the cost, and ideally broaden the accessibility of immunotherapeutics.
Seeking to improve the economics of immunotherapies, we postulated that engineering an antibody with these enhanced stabilisation features shown in isopeptide proteins would reduce the frequency of dosage and give a better quality of treatment to those taking repeated medications. A recent review highlighted that despite inherent stability issues, humanised monoclonal antibodies (mAbs) are the fastest growing group in clinical trials, suggesting that this is an area of key interest in healthcare11. Equally, the World Health Organisation have acknowledged the growing importance of mAbs in a therapeutic context for cancer, autoimmune and chronic diseases12.
Following consultations with healthcare professionals and assay company Antibody Analytics, we decided to look at the broader uses of mAbs beyond medical applications, namely in prolonging the shelf-life of diagnostic assays which use mAbs. This decision was reinforced by feedback we received from our Discussion Forum, wherein attendees were invited to ask questions and discuss our project and their current understanding of mAbs. A key takeaway from this forum was that the public felt strongly about the waste reduction potential for more stable assays, as well as the ability to improve diagnostic abilities in resource limited settings. Consequently, we have chosen to focus our project on a proof of concept, as we felt that the stabilisation of mAb has the potential to have far-reaching impact beyond simply therapeutics.
Sources marked * are of wider context to the project in Synthetic Biology, sources marked ** are of direct relevance to, or had influence on, the project.
- Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012) – ‘Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin’ PNAS 109(12), pp. E690-E697 (https://doi.org/10.1073/pnas.1115485109) *
- Kwon, H., Young, P.G., Squire, C.J., and Baker, E.N (2017) – ‘Engineering a Lys-Asn isopeptide bond into an immunoglobulin-like protein domain enhances its stability’ Scientific Reports 7, e42753 https://doi.org/10.1038/srep42753 **
- Turner, B.M. (2002) – ‘Cellular Memory and the Histone Code’ Cell 111(3), pp. 285-291 https://doi.org/10.1016/S0092-8674(02)01080-2
- Gill, G. (2004) – ‘SUMO and ubiquitin in the nucleus: different functions, similar mechanisms’ Genes & Development 18, pp. 2046-2059 https://doi.org/10.1101%2Fgad.1214604
- Ariens, R.A.S., Lai, T.S., Weisel, J.W., Greenberg, C.S., and Grant, P.J. (2002) – ‘Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms’ Blood 100, pp. 743-754 https://doi.org/10.1182/blood.V100.3.743
- Griffin, M., Casadio, R., and Bergamini, C.M. (2002) – ‘Transglutaminases: Nature’s biological glues’ Biochemical Journal 368(2), pp. 377-396 https://doi.org/10.1042%2FBJ20021234
- Marraffini, L., DeDent, A.C., and Schneewind, O. (2006) – ‘Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria’ Microbiology and Molecular Biology Reviews 70(1), pp. 192-221 https://doi.org/10.1128%2FMMBR.70.1.192-221.2006
- Kudryashov, D.S., Durer, Z.A.O., Ytterberg, A.J., Sawaya, M.R., Pashkov, I., Prochazkova, K., Yeates, T.O., Loo, R.O.O., Loo, J.A., Satchell, K.J.F., and Reisler, E. (2008) – ‘Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin’ PNAS 105(47), pp. 18537-18542 https://doi.org/10.1073/pnas.0808082105
- Kang, H.J., Coulibaly, F., Clow, F., Proft, T., and Baker, E.N. (2007) – ‘Stabilizing Isopeptide Bonds Revealed in Gram-Positive Bacterial Pilus Structure’ Science 318(5856), pp. 1625-1628 https://doi.org/10.1126/science.1145806
- Wang, B., Xiao, S., Edwards, S.A., and Grater, F. (2013) – ‘Isopeptide Bonds Mechanically Stabilize Spy0128 in Bacterial Pili’ Biophysical Journal 104(9), pp. 2051-2057 https://doi.org/10.1016/j.bpj.2013.04.002
- Awwad, S. & Angkawinitwong , U., (2018) – ‘Overview of Antibody Drug Delivery’ Pharmaceutics 10(3), p. 83 https://doi.org/10.3390/pharmaceutics10030083 **
- Sparrow, E., Friede , M., Sheikh, M. & Torvaldsen, S. (2016) – ‘Therapeutic antibodies for infectious diseases’ Geneva: WHO https://doi.org/10.2471/BLT.16.178061 **