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GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

ABSTRACT: 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.

SUBMITTER: Ahat E

PROVIDER: S-EPMC9164142 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

Ahat Erpan E Song Yuefan Y Xia Ke K Reid Whitney W Li Jie J Bui Sarah S Zhang Fuming F Linhardt Robert J RJ Wang Yanzhuang Y

Cellular and molecular life sciences : CMLS 20220321 4

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 a ...[more]

PMID: 35312866

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Project description:The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.

Dataset Information

GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

Publications

GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

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