A CXCL8-binding peptide designed from the interaction sites of CXCR1 with CXCL8 serves as a capture agent and inhibits neutrophil migration.
The migration of leukocytes in response to chemokine gradients is an important process in the homeostasis of the human immune system and inflammation. In vivo the migration takes place on the surface of the endothelium to which the chemokine gradient is immobilized via interaction with glycosaminoglycans. To study leukocyte migration in response to surface-bound chemokines, we generated chemokine gradients by a simple stamping method: agarose stamps were soaked with chemokine solution to form continuous chemokine gradients by diffusion. These gradients could be easily transferred to a petri dish surface by stamping. We show that neutrophil granulocytes recognize these gradients and migrate toward increasing chemokine concentrations dependent on the slope of the gradient. Single-cell responses were recorded, and statistical analyses of cell behavior and migration were performed. For analysis of chemotaxis/haptotaxis, we propose a chemotactic precision index that is broadly applicable, valid, and allows for a straightforward and rapid quantification of the precision by which cells follow the direction of a given gradient. The presented technique is very simple, cost-efficient, and can be broadly applied for generating defined and reproducible immobilized gradients of almost any protein on surfaces, and it is a valuable tool to study haptotaxis.
Correction for 'Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL8:CXCR1 complex' by Dorothea Helmer et al., RSC Adv., 2015, 5, 25657-25668. The authors regret that the original article included some results which were subsequently found to be based on a slightly different peptide sequence than the sequence originally reported. This issue is addressed in the following text, which is an update to the original article.Upon further investigation of the IL8 capture peptide IL8-RP-Loops introduced in the original article, we found that during synthesis of the intended peptide sequence AKWRMVLRI-Ahx-ADTLMRTQ we had obtained the peptide AKWRMVLRI-Ahx-ADTLMRTE, in which the C-terminal glutamine was replaced with glutamic acid. This was conrmed by high resolution mass spectrometry. The reported high affinity (0.5 AE 0.3 mM) was reproduced by the E-mutant within experimental error (1.1 AE 0.1 mM) but not for the original sequence ending with glutamine. We conclude that all experiments were performed with the peptide AKWRMVLRI-Ahx-ADTLMRTE.The affected amino acid Q271 was shown to be non-essential for receptor function by Hébert et al.1 So no essential amino acid of the original sequence was omitted in the exchange.In the original publication we claimed that preorganization of the peptide is responsible for the high affinity of the receptorderived peptide, as CD-spectroscopy had revealed a helical structure for the synthesized peptide. To test the effect of the exchange of glutamine with the structurally closely related glutamic acid on the preorganization of the peptide in solution, we ran a new set of molecular dynamics simulations on the peptide containing the original receptor sequence and the actually synthesized peptide IL8-RP-Loops. The helical content of the peptide structure over the course of the simulation was higher for IL8-RP-Loops with C-terminal glutamic acid (23.6%) than for the peptide ending with glutamine (6.0%). Thus, the serendipitous exchange of the polar glutamine at the C-terminus with negatively charged glutamic acid enhanced preorganization and led to the capture peptide with high affinity.In conclusion, a capture peptide could be designed based on a binding region identied from a computational model of the CXCL8:CXCR1 complex. The peptide comprises receptor residues known to form essential contacts with the chemokine ligand and the exchange of the non-essential C-terminal glutamine for glutamic acid enhances the preorganization into a partially helical structure in solution responsible for its high affinity to the chemokine. In the design of receptor-derived capture peptides it is therefore important to examine the propensity to form secondary structure elements in solution to obtain high affinity peptide mimetics. MethodsPeptides were synthesized, puried and tested in uorescence anisotropy binding assays as described in the original publication. High resolution mass spectra were obtained by MALDI-TOF MS on a 4800 Plus MALDI TOF/TOF Analyzer from AB SCIEX.
Molekulare Signale steuern, wohin die Zellen des Immunsystems wandern, wenn sie Pathogene und Entzündungen bekämpfen. Dabei beeinflussen Proteinmuster die patrouillierenden Zellen. Die Frage ist nur: Wie?
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