| Autor: |
Miyajima R; Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan., Tanegashima K; Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan., Naruse N; Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan., Denda M; Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan., Hara T; Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.; Graduate School of Science, Department of Biological Science, Tokyo Metropolitan University, Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan., Otaka A; Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan. |
| Abstrakt: |
CXCL14 is a primordial CXC-type chemokine that transports CpG oligodeoxynucleotides (ODN) into endosomes and lysosomes in dendritic cells, thereby leading to the activation of the Toll-like receptor 9 (TLR9)-mediated innate immune system. However, the underlying molecular mechanism by which the CXCL14-CpG ODN complex enters cells remains elusive. Herein, we describe the chemical synthesis of CXCL14-derived photoaffinity probes and their application to the identification of target receptors for CXCL14 using quantitative proteomics. By utilizing native chemical ligation and maleimide-thiol coupling chemistry, we synthesized site-specifically modified CXCL14-based photoaffinity probes that contain photoreactive 2-aryl-5-carboxytetrazole (ACT) and a hydrazine-labile cleavable linker. CXCL14-based probes were found to be capable of binding CpG ODN to immune cells, whose bioactivities were comparable to native CXCL14. Application of CXCL14-derived probes to quantitative proteomic experiments enabled the identification of dozens of target receptor candidates for CXCL14 in mouse macrophage-derived RAW264.7 cells, and we discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for CXCL14 by competitive proteome profiling. We further showed that disruption of LRP1 affected the incorporation of the CXCL14-CpG ODN complex in the cells. Overall, this report highlights the power of synthetic CXCL14-derived photoaffinity probes combined with chemical proteomics to discover previously unidentified receptors for CXCL14, which could promote an understanding of the molecular functions of CXCL14 and the elaborate machinery of innate immune systems. |
