N. Bracke

Analytical characterization of NOTA-modified somatropins

N. Bracke, E. Wynendaele, M. D'Hondt, R. Haselberg, G.W. Somsen, E. Pauwels, C. Van de Wiele, B. De Spiegeleer
Journal of Pharmaceutical and Biomedical Analysis
96, 1–9


Chemical modification of biomolecules like the introduction of metal-chelators into proteins can lead to heterogeneous product formation. The nature and extend of the modification is important in interpreting the biological properties of the bioconjugate, given their possible influence on the pharmacokinetics as well as on the binding affinity to the target. The present study describes the synthesis and analytical characterization of somatropin modified on its lysine's ɛ-amino groups with the acylating chelator S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). Direct separation and identification techniques (i.e. RP-MS and CE-MS) and peptide mapping after trypsin and chymotrypsin digestion demonstrated that the use of higher amounts of p-SCN-Bn-NOTA during synthesis leads to a complex product composition with higher order substitution degrees (i.e. multiple NOTA-moieties per somatropin molecule), as well as the presence of different position isomers. From the nine lysine (Lys) residues in somatropin, Lys-70 was experimentally found to be the modification hotspot under our synthesis conditions (pH = 9.0). This was supported by the in silico calculated lowest pKa value of 8.3 for Lys-70. Based on the crystal structure of somatropin in complex with the extracellular parts of the growth hormone receptor, the Lys-70 residue is positioned outside the binding pockets and will therefore not directly interfere with receptor binding. Gallium chelation by NOTA-somatropin resulted in a 100% complexation. The synthesis of NOTA-somatropin using p-SCN-Bn-NOTA and somatropin under our operational conditions is therefore a suitable synthesis procedure for the production of a target-specific radiopharmaceutical for further investigation towards treatment and visualization of growth hormone-specific cancers.

Human IL-34 and CSF-1 Establish Structurally Similar Extracellular Assemblies with Their Common Hematopoietic Receptor

J. Felix, J. Elegheert, I. Gutsche, A. Shkumatov, Y. Wen, N. Bracke, E. Pannecoucke, I. Vandenberghe, B. Devreese, D.I. Svergun, E. Pauwels, B. Vergauwen, S.N. Savvides
21 (4), 528-539


The discovery that hematopoietic human colony stimulating factor-1 receptor (CSF-1R) can be activated by two distinct cognate cytokines, colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34), created puzzling scenarios for the two possible signaling complexes. We here employ a hybrid structural approach based on small-angle X-ray scattering (SAXS) and negative-stain EM to reveal that bivalent binding of human IL-34 to CSF-1R leads to an extracellular assembly hallmarked by striking similarities to the CSF-1:CSF-1R complex, including homotypic receptor-receptor interactions. Thus, IL-34 and CSF-1 have evolved to exploit the geometric requirements of CSF-1R activation. Our models include N-linked oligomannose glycans derived from a systematic approach resulting in the accurate fitting of glycosylated models to the SAXS data. We further show that the C-terminal region of IL-34 is heavily glycosylated and that it can be proteolytically cleaved from the IL-34:hCSF-1R complex, providing insights into its role in the functional nonredundancy of IL-34 and CSF-1.

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