PDF(Pubmed)
Abstract：
Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes. VCF analysis revealed high-affinity binding of D-glucose and D-galactose, which are known transported substrates. D-fructose, which is not a transported substrate, also bound to SGLT1, suggesting potential recognition despite the lack of transport activity. VCF analysis using the T287N mutant of the substrate-binding pocket, which has reduced D-glucose transport capacity, showed that its D-galactose-binding affinity exceeded its D-glucose-binding affinity. This suggests that the change in the VCF signal was due to substrate binding to the binding pocket. Both D-fructose and L-sorbose showed similar binding affinities, indicating that SGLT1 preferentially binds to pyranose-form sugars, including D-fructopyranose. Electrophysiological analysis confirmed that D-fructose binding did not affect the SGLT1 transport function. The significance of the VCF assay lies in its ability to measure sugar-protein interactions in living cells, thereby bridging the gap between structural analyses and functional characterizations of sugar transporters. Our findings also provide insights into SGLT substrate selectivity and the potential for developing medicines with reduced side effects by targeting non-glucose sugars with low bioreactivity.
摘要：
糖的吸收对生命至关重要，依赖于葡萄糖转运蛋白，包括钠-葡萄糖共转运蛋白(SGLTs)。虽然SGLT的结构已经解决，由于复杂的底物识别过程和有限的分析方法，尚未确定SGLTs在各种同种型中的底物选择性。因此,这项研究使用电压钳荧光法（VCF）来探索人SGLT1在非洲爪鱼卵母细胞中的底物结合亲和力。VCF分析显示D-葡萄糖和D-半乳糖的高亲和力结合，它们是已知的运输基底。D-果糖,它不是运输的基底，也与SGLT1有关，这表明尽管缺乏运输活动，但仍有潜在的认可。使用底物结合袋的T287N突变体进行VCF分析，降低了D-葡萄糖的转运能力，显示其D-半乳糖结合亲和力超过其D-葡萄糖结合亲和力。这表明VCF信号的变化是由于底物与结合袋结合。D-果糖和L-山梨糖都显示出相似的结合亲和力，表明SGLT1优先结合吡喃糖形式的糖，包括D-吡喃果糖。电生理分析证实D-果糖结合不影响SGLT1转运功能。VCF测定的意义在于它能够测量活细胞中的糖-蛋白质相互作用，从而弥合了糖转运蛋白的结构分析和功能表征之间的差距。我们的发现还提供了对SGLT底物选择性的见解，以及通过靶向具有低生物活性的非葡萄糖糖来开发具有减少副作用的药物的潜力。
