Isoleucine/leucine<sup>2</sup> is essential for chemoattractant activity of β-defensin Defb14 through chemokine receptor 6

β-Defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1CV derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1CV peptides with substitution of the leucine2 for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1CV L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal α-helix region of this β-defensin is essential for CCR6-mediated chemotaxis.

β-防御素具有抗菌作用，并且能够通过CCR6趋化多种免疫细胞，包括未成熟的树突状细胞和CD4 T细胞。它们是短的阳离子肽，具有高度保守的六个半胱氨酸基序。已经表明，只有第五个半胱氨酸对表达CCR6的细胞的趋化作用至关重要。为了确定与CCR6功能性相互作用所必需的其他残基，我们使用了基于Defb14的肽缺失衍生物库。Defb14 - 1CV衍生物最初两个氨基酸的缺失破坏了其趋化表达CCR6细胞的能力。由于第二个氨基酸是进化上保守的亮氨酸，我们制备了全长的Defb14 - 1CV肽，将亮氨酸2替换为甘氨酸（L2G）、赖氨酸（L2K）或异亮氨酸（L2I）。Defb14 - 1CV L2G和L2K不能趋化表达CCR6的细胞，但半保守性改变L2I具有活性。通过圆二色光谱法，我们没有发现由于这些替换导致二级结构发生显著变化的证据，因此不能将趋化活性的丧失归因于N - 末端螺旋的破坏。我们得出结论，这种β - 防御素N - 末端α - 螺旋区域的异亮氨酸/亮氨酸对CCR6介导的趋化作用是必不可少的。