Emmanuel Nsengiyumva, He Qian, Kang Yixin, Zhang Dianbao, Gao Min, Wang Minglin, Fan Kexin, Xiong Jingwen, Wu Shaobo, Fa Botao, Xiao Zhengtao, Niu Yingfang, Yao Jun, Zhang Yilei
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, China.
Department of Biomedical Laboratory Sciences, School of Health Sciences, College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda.
FEBS Open Bio. 2025 Jun;15(6):994-1008. doi: 10.1002/2211-5463.70019. Epub 2025 Mar 24.
The cystine/glutamate antiporter, solute carrier family 7 member 11 (SLC7A11), plays a crucial role in regulating redox homeostasis and cell death processes such as apoptosis and ferroptosis. These processes are implicated in various diseases, including cancer, organ injuries and neurodegenerative disorders. However, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) expression pattern of SLC7A11 varies across studies and remains unclear. In many studies, including ours, SLC7A11 migrates at an atypical molecular weight (MW) of approximately 37 kDa, which is lower than its theoretical molecular mass of 55.4 kDa. This discrepancy raises concerns about the precise molecular mass and expression pattern of SLC7A11 in SDS-PAGE. We confirmed that this fast-migrating band corresponds to SLC7A11 through knockdown of endogenous SLC7A11 or overexpression of exogenous SLC7A11. Furthermore, we ruled out the possibility of proteolytic cleavage after protein translation. We found that the high hydrophobicity of SLC7A11 is a key factor responsible for its anomalous migration. Substituting the non-polar residue isoleucine (Ile) with the polar residue asparagine (Asn) reduced its hydrophobicity and restored normal migration, aligning with its predicted MW of 55 kDa. Additionally, we observed that SLC7A11 migrated faster in SDS-PAGE at lower acrylamide concentrations, whereas higher concentrations (e.g. 12% or 15%) eliminated the gel shift. This study clarifies the expression pattern of SLC7A11 in SDS-PAGE and emphasizes the importance of considering physicochemical properties such as hydrophobicity and gel concentration when characterizing membrane proteins like SLC7A11.
胱氨酸/谷氨酸反向转运体，溶质载体家族7成员11（SLC7A11），在调节氧化还原稳态以及细胞死亡过程（如凋亡和铁死亡）中发挥着关键作用。这些过程与包括癌症、器官损伤和神经退行性疾病在内的多种疾病有关。然而，SLC7A11在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中的表达模式在不同研究中有所不同，仍不清楚。在许多研究中，包括我们的研究，SLC7A11以约37 kDa的非典型分子量（MW）迁移，低于其理论分子量55.4 kDa。这种差异引发了对SLC7A11在SDS-PAGE中精确分子量和表达模式的担忧。我们通过敲低内源性SLC7A11或过表达外源性SLC7A11证实，这条快速迁移带对应于SLC7A11。此外，我们排除了蛋白质翻译后蛋白水解切割的可能性。我们发现SLC7A11的高疏水性是其异常迁移的关键因素。用极性残基天冬酰胺（Asn）取代非极性残基异亮氨酸（Ile）降低了其疏水性并恢复了正常迁移，与预测的55 kDa分子量一致。此外，我们观察到在较低丙烯酰胺浓度下，SLC7A11在SDS-PAGE中迁移得更快，而较高浓度（如12%或15%）消除了凝胶迁移。本研究阐明了SLC7A11在SDS-PAGE中的表达模式，并强调了在表征像SLC7A11这样的膜蛋白时考虑疏水性和凝胶浓度等物理化学性质的重要性。
