Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK.
Exp Eye Res. 2022 Jun;219:109081. doi: 10.1016/j.exer.2022.109081. Epub 2022 Apr 21.
The human cornea is responsible for approximately 70% of the eye's optical power and, together with the lens, constitutes the only transparent tissue in the human body. Low-density lipoprotein receptor-related protein 1 (LRP1), a large, multitalented endocytic receptor, is expressed throughout the human cornea, yet its role in the cornea remains unknown. More than 30 years ago, LRP1 was purified by exploiting its affinity for the activated form of the protease inhibitor alpha-2-macroblulin (A2M), and the original purification protocol is generally referred to in studies involving full-length LRP1. Here, we provide a novel and simplified LRP1 purification protocol based on LRP1's affinity for receptor-related protein (RAP) that produces significantly higher yields of authentic LRP1. Purified LRP1 was used to map its unknown interactome in the human cornea. Corneal proteins extracted under physiologically relevant conditions were subjected to LRP1 affinity pull-down, and LRP1 ligand candidates were identified by LC-MS/MS. A total of 28 LRP1 ligand candidates were found, including 22 novel ligands. The LRP1 corneal interactome suggests a novel role for LRP1 as a regulator of the corneal immune response, structure, and ultimately corneal transparency.
人眼角膜大约负责眼睛 70%的光学功能，它与晶状体一起构成了人体唯一的透明组织。低密度脂蛋白受体相关蛋白 1（LRP1）是一种大型多功能的内吞受体，在整个角膜中均有表达，但它在角膜中的作用尚不清楚。三十多年前，人们通过利用其对蛋白酶抑制剂 α-2-巨球蛋白（A2M）激活形式的亲和力来纯化 LRP1，而最初的纯化方案通常用于涉及全长 LRP1 的研究。在这里，我们提供了一种基于 LRP1 与受体相关蛋白（RAP）亲和力的新型简化 LRP1 纯化方案，该方案可显著提高真正的 LRP1 的产量。纯化的 LRP1 用于绘制其在人眼角膜中未知的相互作用组。在生理相关条件下提取角膜蛋白，进行 LRP1 亲和下拉，然后通过 LC-MS/MS 鉴定 LRP1 配体候选物。共发现 28 种 LRP1 配体候选物，包括 22 种新的配体。LRP1 角膜相互作用组表明 LRP1 作为角膜免疫反应、结构，最终是角膜透明度的调节剂具有新的作用。
