Biotinylated synthetic chemokines: their use for the development of nonradioactive whole-cell binding assays.
A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen.
Thierry
Anne-Christine
AC
Dictagene, Lausanne, Switzerland.
Perrenoud
Geneviève
G
Pinaud
Stéphane
S
Bigler
Nicolas
N
Denis
Bérangère
B
Roggero
Mario
M
Moulon
Corinne
C
Demotz
Stéphane
S
D001710:Biotin / Q000737:chemistry / Q000494:pharmacology; D019921:Biotinylation; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement; D002469:Cell Separation; D054422:Chemokine CCL22; D019742:Chemokines, CC / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D008024:Ligands; D008264:Macrophages / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D054398:Receptors, CCR4; D019707:Receptors, Chemokine; D012685:Sepharose / Q000737:chemistry; D019809:Streptavidin / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
C515482:CCL22 protein, human; C515354:CCR4 protein, human; D054422:Chemokine CCL22; D019742:Chemokines, CC; D008024:Ligands; D010455:Peptides; D054398:Receptors, CCR4; D019707:Receptors, Chemokine; D014157:Transcription Factors; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin