Kosuge Hirofumi, Nakakido Makoto, de Vega Susana, Ohnuma Shin-Ichi, Tsumoto Kouhei
Department of Bioengineering, School of Engineering, The University of Tokyo, 7- 3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Sci Rep. 2025 Jul 9;15(1):24634. doi: 10.1038/s41598-025-09018-7.
A small leucine-rich repeat proteoglycan PRELP is responsible for various biological functions. Here, to quantitatively assess the ligand binding of PRELP and its relevance to physiological activities, we validated the premise that PRELP multi-specifically binds to TGFβ1, IGFI-R, and p75NTR with relatively weak, micromolar range of affinities using surface plasmon resonance analysis. Results of a direct binding assay using N-terminal-truncated PRELP and chimeric PRELP and a dual injection assay to evaluate the binding regions and competitiveness suggested that PRELP interacts with the ligands via different but partially overlapping regions in the leucine-rich repeat domain. RNA-seq analysis revealed that PRELP greatly promotes gene expression of various extracellular matrix (ECM) components in A549 lung carcinoma cells, also at micromolar concentration. Since we reasoned that ECM anchoring contributes to an increase of apparent local concentrations of PRELP required for the weak affinity interactions, we validated the direct binding and co-localization of PRELP with ECM proteins using ELISA analysis and immunofluorescence staining. Results of this study suggest that PRELP modulates multiple interactions with intrinsically weak binding affinities through the anchoring to ECM proteins and also promotes the ECM protein expression to maintain the preferred environment to exert the molecular functions.
一种富含亮氨酸的小分子重复蛋白聚糖PRELP具有多种生物学功能。在此，为了定量评估PRELP的配体结合及其与生理活动的相关性，我们验证了以下前提：使用表面等离子体共振分析，PRELP以相对较弱的微摩尔亲和力范围多特异性结合TGFβ1、IGFI-R和p75NTR。使用N端截短的PRELP和嵌合PRELP进行的直接结合测定以及评估结合区域和竞争性的双注射测定结果表明，PRELP通过富含亮氨酸重复结构域中不同但部分重叠的区域与配体相互作用。RNA测序分析显示，PRELP在微摩尔浓度下也能极大地促进A549肺癌细胞中各种细胞外基质（ECM）成分的基因表达。由于我们推断ECM锚定有助于增加弱亲和力相互作用所需的PRELP的表观局部浓度，我们使用酶联免疫吸附测定分析和免疫荧光染色验证了PRELP与ECM蛋白的直接结合和共定位。本研究结果表明，PRELP通过锚定到ECM蛋白上调节多种具有内在弱结合亲和力的相互作用，并且还促进ECM蛋白表达以维持发挥分子功能的理想环境。
