Selective labelling of stromal cell-derived factor 1α with carboxyfluorescein to study receptor internalisation.
SDF1α plays an important role in the regeneration of injured tissue after ischemia or stroke by inducing the migration of progenitor cells. In order to study the function of this therapeutically relevant chemokine site-specific protein labelling is of great interest. However, modification of SDF1α is complicated because of its complex tertiary structure. Here, we describe the first site-specific fluorescent modification of SDF1α by EPL. We recombinantly expressed SDF1α (1-49) by intein-mediated protein expression. The C-terminal peptide SDF1α (50-68) was synthesised by SPPS and selectively labelled with carboxyfluorescein at Lys(56). In a cell migration assay, M-[K(56)(CF)]SDF1α showed a clear potency to induce chemotaxis of human T-cell leukaemia cells. Microscopic analysis on HEK293 cells transfected with the CXCR4 revealed specific binding of the fluorescent ligand. Furthermore, receptor-induced internalisation of the ligand could be visualised. These results show that site-specific modification of SDF1α yields in a biologically functional molecule that allows the characterisation of CXCR4 production of cells on a molecular level.
Bellmann-Sickert
Kathrin
K
Institute of Biochemistry, Leipzig University, Leipzig, Germany.
Baumann
Lars
L
Beck-Sickinger
Annette G
AG
D000595:Amino Acid Sequence; D002460:Cell Line; D002465:Cell Movement; D054377:Chemokine CXCL12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; C024098:6-carboxyfluorescein