Regioselective labeling of Nanofitin by using
a phosphorylated peptide tag
Marine Goux
[1,2,3,*]
, Mathieu Cinier
[2]
and Charles Tellier
[1]
[1]Unité de Fonctionnalité et Ingénierie des Protéines, University of Nantes, France ; [2]Affilogic SAS, Nantes, France and [3]Cyclotron Research
Center, University of Liège, Belgium ; * contact: [email protected]
References : [1] Cinier M. et al. (2012), Journal of Biological Inorganic Chemistry, 17, pp. 399–407 ; [2] Martin L. J. et al. (2007), Journal of American Chemistry Society,
129(22), 7106–7113 ; [3] Pardoux R. et al. (2012), PLoS ONE, 7(8).
Recently, new strategies emerged in the field of monoclonal antibodies radiolabeling for PET imaging with the use of positron emitters 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
phosphorylatable peptide tag. A specific phosphorylation step, with the alpha subunit of the human casein kinase II
can generate a nanocluster of phosphate moieties that can interact strongly with metal ions like zirconium[1]. Two
peptides sequences have been used as a starting material, one already described to promote the specific anchoring of protein on zirconium-phosphate surface, the other one selected for its capacity to chelate lantanide ions such as terbium(III).
UFIP UMR CNRS 6286
2 rue de la Houssinière, 44322 Nantes, France AFFILOGIC SAS
2 rue de la Houssinière, 44322 Nantes, France Cyclotron Research Center
Allée du 6 Août, 4000 Liège, Belgium
Small Protein:10kDa
pH stability: 0-12
Temperature Tm≈80°C
Stability:
Production
Low (Generated in vitro and
cost:
produced in bacteria)
Affinity:
nM
C
HELATION AND
L
ANTHANIDE
-B
INDING
T
AG
W
HAT ARE
N
ANOFITINS
?
I
NTRODUCTION
R
ESULTS
We succeeded to generate two types of phosphorylatable tag able to chelate terbium(III). Through competition studies, we have shown evidence for a capacity of chelation of zirconium(IV) and gallium(III). Radiolabeling studies with gallium-68 are on going to evaluate the powerfulness of such a strategy for the chelation of radionuclides.
C
ONCLUSION
1) Adapt the labeling tag to the stereoselective chelation of radionuclides for PET imaging. 2) Genetically fuse the tag to a Nanofitin, a protein scaffold developed as an alternative to
antibodies, to ensure an efficient targeting of the radionuclide.
O
BJECTIVES:
3rd NanoFar Autumn School October 20-24, 2014 University of Louvain, Brussels, Belgium 0 500 1000 1500 2000 2500 3000 3500 0 0,25 0,5 0,75 1 Fl uor es cence 544nm (A U )Equivalent of terbium or terbium/zirconium (1:1) per peptide
Terbium(III) titration of tri-phosphorylated peptide (20µM) and competition with ZrOCl2 in formic acid pH2
(After removal of peptide and terbium fluorescence)
Tri-phosphorylated peptide + TbCl3 Tri-phosphorylated peptide + TbCl3/ZrOCl2 (1:1)
0 10 20 30 40 50 60 70 80 90 100 0 0,5 1 1,5 2 2,5 3 3,5 4 Fl uor es cence 544nm (% ) [Tb3+] (µM)
Terbium(III) titration of protein and competition with Zr(NTA)2 in HEPES buffer pH7
LBT (1µM) (n=4) LBT + Zr4+ (1:1) (1µM) (n=4)
C
HELATION AND PHOSPHORYLATABLE TAG
0 5000 10000 15000 20000 25000 30000 35000 40000 0,00 20,00 40,00 60,00 80,00 100,00 120,00 140,00 160,00 Fl uor es cence 544nm (A U ) [Tb3+] (µM)
Terbium(III) titration of phosphorylated peptides (20µM) in HEPES buffer pH7
(After removal of peptides and terbium fluorescence)
0P 1P 2P 3P 4P
● Affinity for Tb3+ in the micromolar range : 2P>3P≈4P>1P>0P
● Chelation of the Zr4+ by the peptide tag was confirmed by competition.
To optimize the sequence of the phosphorylatable tag, we studied the chelation of different mimetic peptides (from 0 to 4 phospho-serine) with a lanthanide (terbium).
M
ETHOD:
NO FLUORESCENCE UV UV FLUORESCENCE Tb3+M
ETHOD:
+
LBT KD(Tb3+) ≈ nM Nanofitin UV FLUORESCENCE Gene fusion Mutation UV FLUORESCENCE ? In vitro phosphorylation LBT 1S LBT 1SP FLUORESCENCE ?+
UV+
+
[Zr(NTA)2] 2-FLUORESCENCE ?+
UV+
Ga3+● Affinity for terbium(III) in the sub-micromolar range for the lanthanide-binding tag fused to the Nanofitin and in the micromolar range for the mono-phosphorylated.
● Chelation of zirconium and gallium by the peptide tag was observed by a competition study.
To increase the affinity for radionuclide from micromolar to nanomolar, we worked on a sequence derived from calcium-binding proteins to chelate specifically lanthanides[2]. We optimized this sequence by incorporating a phosphate nanocluster to improve the chelation with radionuclides [3].
R
ESULTS
0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 35 40 45 50 Fl uor es cence 544nm (% ) [Tb3+] (µM)Terbium(III) titration of protein and competition with GaCl3 in MES buffer pH5.5
LBT (1µM) (n=2) LBT (1µM) + Ga3+ (1:1) (n=2) LBT (1µM) + Ga3+ (1:1000) (n=2)
Thesis project funded by “Région Pays de la Loire”, into the Erasmus Mundus programme NanoFar Tb3+ Zr4+ Tb3+ Tb3+ 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 Fl uor es cence 544nm (% ) [Tb3+] (µM)
Terbium(III) titration of protein and competition with Zr(NTA)2 in HEPES buffer pH7
LBT 1SP (1µM) (n=4) LBT 1SP + Zr4+ (1:1) (1µM) (n=2) LBT 1SP + Zr4+ (1:4) (1µM) (n=3)
NB : Actually, terbium emits fluorescence intrinsically. In aqueous solution, water molecules quench fluorescence and chelation prevents this quenching by expelling water molecules.
NB : Actually, terbium emits fluorescence intrinsically. In aqueous solution, water molecules quench fluorescence and chelation prevents this quenching by expelling water molecules.
