Semisynthesis and Characterization of the First Analogues of Pro‐Neuropeptide Y

Abstract

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).

Strategies and Limitations in Dendrimeric Immunogen Synthesis. The Influenza Virus M2e Epitope as a Case Study

“…Use of the demonstrably superior Rink-amide ChemMatrix resin (42) and double coupling at some positions (Glu residues 16 and 14, plus the entire Glu8-Ser2 stretch, based on MS analysis of the crude from the initial trial, and on prediction of difficult sequences with the Peptide Companion program) ameliorated only partially the situation ( Figure 2B). In a third, successful trial, the above improvements plus replacement of the two pairs of consecutive Ser residues in the M2e sequence with the pseudoproline dipeptide Ser(tBu)-Ser(Ψ Me,Me pro)-OH (43)(44)(45) led to a more acceptable product that could be purified and characterized as the desired M2e peptide in free thiol form ( Figure 2C; see also Figure S1 and Table S1, Supporting Information). The chemoselective ligation of this purified, sparingly soluble peptide to the ClAc 4 -Lys core was carried out in Tris-Cl buffer at pH 7.6 and 50°C, using a 10-12-fold molar excess of M2e peptide in order to drive the reaction into completion.…”

The effect of interleukin‐8 truncations on its interactions with glycosaminoglycans

“…Fusion proteins were cleaved with DTT or MESNa, respectively. The latter thiol yields stable protein thioesters suitable for subsequent Expressed protein ligation (EPL) reactions, while DTT protein thioesters hydrolyzes readily under mild basic conditions. Thus, IL‐8(1‐54) was cleaved off the chitin beads with MESNa while all other variants were cleaved by DTT, hydrolyzed, and refolded to produce the target proteins.…”

Exprimierte enzymatische Ligation zur Semisynthese chemisch modifizierter Proteine

“…Zusammenfassend bestätigen diese Resultate die EEL als eine effiziente Alternative zur NCL und SCFC, die unabhängig von der Anwesenheit Thiol enthaltender wie enzymspezifischer Aminosäurenreste an der Ligationsstelle ist. Die EPL‐Strategie, die wir parallel zur Synthese von proNPY angewendet haben,11 erforderte den Einbau eines artifiziellen Cysteinrests in die Proteinsequenz. Diese wesentliche Einschränkung kann elegant durch die EEL umgangen werden, indem spezifische Thioester‐Substratmimetika für die enzymvermittelte Ligation eingesetzt werden.…”