Galectin-3 bound cell membrane proteins identified by LC-MS/MS

The HMEC-1 membrane proteins were divided into two fractions: one for His-Gal-3 with a His-tag antibody, and the other for the His-tag antibody alone. Subsequently, SDS-PAGE and silver staining were performed. Mass spectrometry was conducted to analyze potential Gal-3 binding candidates, resulting in the identification of a total of 64 distinct proteins. Given that Gal-3 is believed to bind to cell surface glycoproteins, thereby mediating various functions, the selected proteins were those with the cell membrane. Among these, 15 proteins were primarily localized in the cell membrane, each with unique peptides exceeding two. In our study, we found that the top five proteins bound with Gal-3 identified in HMEC-1 were CD166, CD146, ECE1, integrin β1, and catenin α-1. Taken together, this study identifies the candidate proteins bound to Gal-3 and suggest that the proteins binding to Gal-3 vary on the cell types from different tissues.

将HMEC-1细胞的膜蛋白分为两组：一组使用结合组氨酸标签（His-tag）的抗体，用于捕获与His-Gal-3结合的蛋白；另一组仅使用该组氨酸标签抗体作为对照。随后分别进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及银染实验。通过质谱分析筛选潜在的半乳糖凝集素3（Gal-3）结合候选蛋白，最终共鉴定出64种不同的蛋白质。鉴于半乳糖凝集素3（Gal-3）可结合细胞表面糖蛋白并介导多种生理功能，本研究选取的候选蛋白均定位于细胞膜，其中15种蛋白主要定位于细胞膜且其特异性肽段数量均超过2条。本研究发现，在HMEC-1细胞中鉴定出的与Gal-3结合的前五大蛋白依次为CD166、CD146、ECE1、整合素β1（integrin β1）和连环蛋白α-1（catenin α-1）。综上，本研究明确了与Gal-3结合的候选蛋白，并提示不同组织来源的细胞类型中，与Gal-3结合的蛋白存在差异。