Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8.
Human interleukin 8 (hIL-8), a neutrophil-activating and chemotactic cytokine, is known to play an important role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. This cytokine belongs to the family of CXC chemokines, mediating the response through binding to the seven-transmembrane helical G protein-coupled receptors CXCR1 and CXCR2. For the first time, we employed the expressed protein ligation (EPL) strategy to chemokine synthesis and subsequent modification. The ligation site was chosen with respect to the position of four cysteine residues within the hIL-8 sequence. Ligation with synthetic peptides that carry cysteine at their N-termini resulted in full-length hIL-8 and the specifically carboxyfluorescein-labelled analogue [K69(CF)]hIL-8(1-77). [K69(CF)]hIL-8(1-77) was fully active as shown by inhibition of cAMP production. Furthermore, this analogue was used to study receptor internalisation in human promyelotic HL60 cells that express CXCR1 and CXCR2 receptors. Binding and quenching studies were performed on HL60 membranes and suggest that the C-terminus of IL-8 is accessible to solvent in the receptor-bound state. Thus, we introduce here a powerful approach that allows the site-specific incorporation of chemical modifications into the sequence of chemokines, which opens new avenues for studying IL-8 function.
David
Ralf
R
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, D-04103 Leipzig, Germany.
Machova
Zuzana
Z
Beck-Sickinger
Annette G
AG
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002686:Chitin / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005452:Fluoresceins / Q000737:chemistry*; D018922:HL-60 Cells; D006801:Humans; D016209:Interleukin-8 / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010720:Phosphines / Q000737:chemistry; D023062:Receptors, Interleukin-8A / Q000378:metabolism; D023063:Receptors, Interleukin-8B / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000737:chemistry
D002686:Chitin; D005576:Colforsin; D000242:Cyclic AMP; D003497:Cyclic N-Oxides; D005452:Fluoresceins; D016209:Interleukin-8; D010446:Peptide Fragments; D010720:Phosphines; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011993:Recombinant Fusion Proteins; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C080938:tris(2-carboxyethyl)phosphine; C024098:6-carboxyfluorescein; C001803:tempol