Department of Molecular, Cellular and Developmental Biology, University of Michigan, 1105 North University Avenue, Ann Arbor, MI, 48109-1085, USA.
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Cell Mol Life Sci. 2022 Mar 21;79(4):199. doi: 10.1007/s00018-022-04223-3.
Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNAcT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans.
糖胺聚糖（如硫酸乙酰肝素（HS）和硫酸软骨素（CS））的合成发生在高尔基体腔中，但高尔基体结构完整性与糖胺聚糖合成之间的关系尚不清楚。在这项研究中，我们通过敲除 GRASP55 和 GRASP65 破坏了高尔基体结构，并确定了其对 HS 和 CS 合成、硫酸化和分泌的影响。我们发现，GRASP 耗竭增加了细胞中 HS 的合成，同时减少了 CS 的合成，改变了 HS 和 CS 的硫酸化，并减少了 HS 和 CS 的分泌。通过蛋白质组学、RNA-seq 和生化方法，我们鉴定出 EXTL3，这是 HS 合成途径中的关键酶，其水平在 GRASP 敲除细胞中上调；而 GalNAcT1，一种必需的 CS 合成酶，被强烈下调。此外，我们发现 GRASP 耗竭通过降低 HS 硫酸化中的双功能酶 PAPSS2 来减少 HS 硫酸化。我们的研究首次提供了证据，表明高尔基体结构缺陷可能显著改变糖胺聚糖的合成和分泌。
