Semisynthesis and characterization of the first analogues of pro-neuropeptide y

Détails

ID Serval
serval:BIB_D5CD094A8A00
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Semisynthesis and characterization of the first analogues of pro-neuropeptide y
Périodique
Chembiochem
Auteur(s)
von Eggelkraut-Gottanka  R., Machova  Z., Grouzmann  E., Beck-Sickinger  A. G.
ISSN
1439-4227 (Print)
Statut éditorial
Publié
Date de publication
05/2003
Volume
4
Numéro
5
Pages
425-33
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: May 9
Résumé
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).
Mots-clé
Amino Acid Sequence Biotin/chemistry Cysteine/chemistry/genetics Escherichia coli/metabolism Esters/chemistry Fluoresceins/chemistry Molecular Sequence Data Neuropeptide Y/*biosynthesis/*chemical synthesis/chemistry/genetics Peptide Fragments/chemistry Proline/analogs & derivatives Protein Precursors/*biosynthesis/*chemical synthesis/chemistry/genetics Protein Splicing Recombinant Fusion Proteins/biosynthesis/chemistry/genetics Staining and Labeling/methods Sulfhydryl Compounds/chemistry
Pubmed
Web of science
Création de la notice
25/01/2008 11:55
Dernière modification de la notice
20/08/2019 16:55
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