Reference : Regioselective labeling of Nanofitin by using a phosphorylated peptide tag
Scientific congresses and symposiums : Poster
Life sciences : Biotechnology
Regioselective labeling of Nanofitin by using a phosphorylated peptide tag
Goux, Marine mailto [Université de Liège - ULiège > > > Doct. sc. bioméd. & pharma. (Bologne)]
NanoFar Autumn School 2014
du 20 octobre 2014 au 24 octobre 2014
[en] Nanofitine ; Chelation ; LBT
[en] Recently, new strategies emerged in the field of monoclonal antibodies radiolabeling for PET imaging with the use of positron emitter such as zirconium-89 or gallium-68. Despite their important role in the therapeutic world, antibodies have many disadvantages related to their structure. Moreover, conjugation of chelating agent often occurs on lysines, which is non-regioselective and leads to a heterogeneous mixture of products. In addition, the slow clearance of antibodies can be a problem to obtain a good contrast when they are used in imaging. To address these different limitations, we developed a chemistry-free chelating system consisting of a highly phosphorylatable peptide tag. A specific phosphorylation step, with the alpha subunit of the casein kinase II, generates a nanocluster of 4 phosphates that can interact strongly with metal ion like zirconium. This strategy has already demonstrated its powerfulness for the stable and specific anchoring of protein on zirconium phosphonate-based microarray [1]. We are now adapting the use of this labeling tag to the stereoselective chelation of radionuclides for PET imaging.
As described previously, functionalizing the tag with a phosphate nanocluster is a key step to provide the capacity to specifically chelate metal ions such as zirconium or gallium. In order to optimize the sequence of the phosphorylatable tag, we have used mimetic peptides with different phosphorylation states (from 0 to 4 phospho-serine). We have shown the complexation between the phosphorylatable tag and a lanthanide: terbium(III). With a competition study between terbium(III) and zirconium(IV), we showed that the peptide tag preferably chelate zirconium with an affinity in the micromolar range. Considering this tag has been created to specifically anchoring protein on zirconium phosphonate-based microarray, it is possible that the sequence is not optimized for our application. In order to increase the affinity from micromolar to nanomolar, we are currently working on a sequence derived from calcium-binding proteins to chelate specifically lanthanides[2]. As described by Pardoux et al.[3], our strategy is to functionalize this sequence with a phosphate nanocluster able to chelate zirconium or gallium. Our first results shown that a mono-phosphorylatable tag phosphorylated in vitro is able to chelate terbium(III) with a lower affinity than the wild type. Considering that terbium(III) is bigger than gallium(III) or zirconium(IV), we can suppose that the cage obtained is too small but suitable for other ions. In order to validate our hypothesis, we have planned to radiolabel those tags and determine their affinity for gallium(III).
[1] M. Cinier, M. Petit, F. Pecorari, D. R. Talham, B. Bujoli, and C. Tellier, “Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays.,” J. Biol. Inorg. Chem., vol. 17, no. 3, pp. 399–407, Mar. 2012.
[2] L. J. Martin, “Development of Lanthanide-Binding Tags (LBTs) as powerful and versatile peptides for use in studies of proteins and protein interactions,” 2008.
[3] R. Pardoux, S. Sauge-merle, D. Lemaire, P. Delangle, L. Guilloreau, J. Adriano, and C. Berthomieu, “Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides : Effect of Phosphorylation,” PLoS One, vol. 7, no. 8, 2012.
Unité de Fonctionnalité et Ingenierie des Protéines (UMR CNRS 6286)
Région Pays de la Loire

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