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Invest Ophthalmol Vis Sci. 2020 Apr 9;61(4):16. doi: 10.1167/iovs.61.4.16.
Corneal abrasion is a common eye injury, and its resolution can be seriously complicated by bacterial infection. We showed that topical application of the cationic antimicrobial protein of 37 kDa (CAP37) promotes corneal re-epithelialization in mice, and peptides derived from CAP37 can recapitulate the antibacterial and wound-healing effects of the full-length protein. The current study was designed to identify the molecular mechanisms mediating the wound-healing effect of CAP37 and derived bioactive peptides.
We used a TriCEPS-based, ligand-receptor glycocapture method to identify the binding partners of CAP37 on live human corneal epithelial cells using the hTCEpi cell line. We used an ELISA method to confirm binding with identified partners and test the binding with CAP37-derived peptides. We used a reporter cell line to measure activation of the identified membrane receptor by CAP37 and derived peptides.
We pulled down S100 calcium-binding protein A9 (S100A9) as a binding partner of CAP37 and found that CAP37 and four derived peptides encompassing two regions of CAP37 bind S100A9 with high affinities. We found that CAP37 and the S100A9-binding peptides could also directly interact with the Toll-like receptor 4 (TLR4), a known receptor for S100A9. CAP37 and one peptide partially activated TLR4. The other three peptides did not activate TLR4. Finally, we found that CAP37 and all four peptides could inhibit the activation of TLR4 by S100A9.
This study identifies a mechanism of action for CAP37 and derived antimicrobial peptides that may restrain inflammatory responses to corneal injury and favor corneal re-epithelialization.
角膜擦伤是一种常见的眼部损伤，其愈合过程可能会因细菌感染而严重复杂化。我们已经证明，阳离子抗菌蛋白 37kDa（CAP37）的局部应用可促进小鼠角膜再上皮化，并且源自 CAP37 的肽可重现全长蛋白的抗菌和伤口愈合作用。本研究旨在确定介导 CAP37 及其衍生生物活性肽的伤口愈合作用的分子机制。
我们使用基于 TriCEPS 的配体-受体糖捕获方法，使用 hTCEpi 细胞系在活的人角膜上皮细胞上鉴定 CAP37 的结合伴侣。我们使用 ELISA 方法确认与鉴定的伙伴的结合，并测试与 CAP37 衍生肽的结合。我们使用报告细胞系来测量 CAP37 和衍生肽对鉴定的膜受体的激活作用。
我们拉下 S100 钙结合蛋白 A9（S100A9）作为 CAP37 的结合伴侣，并发现 CAP37 和包含 CAP37 两个区域的四个衍生肽与 S100A9 具有高亲和力结合。我们发现 CAP37 和 S100A9 结合肽还可以直接与 Toll 样受体 4（TLR4）相互作用，TLR4 是 S100A9 的已知受体。CAP37 和一个肽部分激活 TLR4。其他三个肽则不能激活 TLR4。最后，我们发现 CAP37 和所有四个肽都可以抑制 S100A9 对 TLR4 的激活。
这项研究确定了 CAP37 和衍生抗菌肽的作用机制，该机制可能限制对角膜损伤的炎症反应，并有利于角膜再上皮化。
