Institute of Biochemistry II, Medical Faculty, University of Cologne, 50931 Koeln, Germany.
Department of Chemistry, Laboratory of Bio-organic Chemistry, University of Namur (FUNDP), B-5000 Namur, Belgium.
Cells. 2020 Mar 3;9(3):607. doi: 10.3390/cells9030607.
Genetic defects of human galactose-1-phosphate uridyltransferase (GALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by GALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant GALT with a K of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.
人类半乳糖-1-磷酸尿苷酰转移酶（GALT）的遗传缺陷和酶功能的部分丧失导致半乳糖代谢改变，经典半乳糖血症患者的器官长期发育受损。在寻找半乳糖这一应激源诱导的细胞病理机制时，我们试图通过抑制 GALT 在 HEK293 细胞中诱导类似半乳糖血症的表型。在动力学研究中，我们提供了证据表明 2-氟代半乳糖-1-磷酸（F-Gal-1-P）能够以 0.9mM 的 K 值竞争性抑制重组 GALT。与肝细胞不同，MALDI-MS 显示诱导分泌 netrin-1 探针时，MIG（半乳糖血症的代谢诱导）-HEK293 细胞中的 N-糖蛋白图谱没有改变。荧光激活细胞分选显示，表面表达的 N-糖基化 CD109、EGFR、DPP4 和 rMUC1 减少。膜筏蛋白质组显示出明显的改变，表明 unfolded protein response 和靶向蛋白运输受到影响。最显著的是，在还原 Gal/F-Gal 时 NADPH 池耗竭期间，发现氧化应激受到负调控，可能作为一种反应。正常上皮细胞中氟代半乳糖诱导的细胞扰动类似于半乳糖血症成纤维细胞中揭示的蛋白质组变化。总之，在健康上皮/神经元细胞中诱导类似半乳糖血症的表型可以支持经典半乳糖血症中分子病理机制的研究，特别是在低半乳糖应激和残留 GALT 活性的条件下。
