Identification of a potential modification site in human stromal cell-derived factor-1.
Selective modification of proteins is an important tool to study their function. However, it is still challenging to identify the best position to avoid a loss of activity. By using a 6-nitroveratryl (Nvoc)-modification approach, we facilitate the identification of a potential modification site as Nvoc can be removed in situ by UV irradiation and accordingly allows directly the comparison of the biological activity of the modified and the unmodified protein derived from the same precursor. As a test system, we used stromal cell-derived factor-1 (SDF-1), which is involved in a wide range of physiological functions, mainly hematopoiesis and embryonic organ development. This chemokine is a potential candidate in regenerative medicine because of its capability to attract stem cells to distinct localizations. First, we synthesized the wildtype and the Nvoc-modified C-terminal segments SDF-1(50-68) and studied their secondary structure formation by circular dichroism spectroscopy. By using the intein-mediated purification with a affinity chitin binding tag system, we then expressed the peptide thioester M-[A(49)]-SDF-1(1-49)-MESNA recombinantly, in which the valine at position 49 was replaced by a more suitable alanine residue to allow improved cleavage and ligation. After ligation and refolding, the biological activity was proven in a cell-based inositol phosphate accumulation assay prior and after Nvoc removal, which showed that neither the alanine 49 nor the attached Nvoc group impair the activity of the analog. The study shows that lysine 56 is a potential site to introduce labels site-specifically in SDF-1.
Baumann
Lars
L
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig University, Brüderstrasse 34, D 04103 Leipzig, Germany.
Beck-Sickinger
Annette G
AG
D000818:Animals; D001547:Benzaldehydes / Q000737:chemistry; D019556:COS Cells; D002465:Cell Movement / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000737:chemistry* / Q000494:pharmacology; D002522:Chlorocebus aethiops; D006801:Humans; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D044968:Regenerative Medicine / Q000379:methods; D013234:Stem Cells / Q000378:metabolism; D014466:Ultraviolet Rays
D001547:Benzaldehydes; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; C402308:NVOC-chloride; D010455:Peptides