neuropeptide-y and 6-carboxyfluorescein

Regulatory, receptor-binding peptides are considered as the agents of choice for diagnostic imaging and therapy of cancers, because their receptors are overexpressed in various human cancer cells. It has been recently indicated that there is a putative role of NPY in breast tumors. The expression of the two best-investigated NPY receptor subtypes, Y1 and Y2, in breast tissue shows predominant occurrence of the Y1 receptor subtype in tumors, whereas Y2 receptors are found in nonproliferative tissue. To investigate the usefulness of NPY analogs for tumor diagnosis and therapy, we investigated the metabolic stability of receptor-selective NPY analogs in human blood plasma. NPY analogs were synthesized by Fmoc/t-Bu solid-phase strategy. Prior to the cleavage of peptides from the resin, they were labeled with 5(6)-carboxyfluorescein (CF) either at the N-terminus or at the side chain of Lys4. For the metabolic stability study, the digestion of peptides was monitored by HPLC and the cleavage products were identified by MALDI-ToF mass spectrometry. The data showed that full-length [Phe7, Pro34]NPY analogs, which show high binding affinity to Y1 receptors are enzymatically more stable than centrally truncated analogs, which show high binding affinity to Y2 receptors. Furthermore, the N-terminally CF-labeled Y1 and Y2 receptor-selective peptides were found to be enzymatically more resistant than their counterparts containing the CF label at Lys4 side chain.

Topics: Amino Acid Sequence; Drug Stability; Fluoresceins; Fluorescent Dyes; Half-Life; Humans; In Vitro Techniques; Molecular Sequence Data; Neuropeptide Y; Peptide Fragments; Receptors, Neuropeptide Y

Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).

Topics: Amino Acid Sequence; Biotin; Cysteine; Escherichia coli; Esters; Fluoresceins; Molecular Sequence Data; Neuropeptide Y; Peptide Fragments; Proline; Protein Precursors; Protein Splicing; Recombinant Fusion Proteins; Staining and Labeling; Sulfhydryl Compounds