Development of Isoform-specific Sensors of Polypeptide GalNAc-transferase Activity

Song, Lina; Bachert, Collin; Schjoldager, Katrine T.; Clausen, Henrik; Linstedt, Adam D.

doi:10.1074/jbc.m114.599563

Cited by 16 publications

(35 citation statements)

References 35 publications

(49 reference statements)

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“…This type of glycosylation is initiated by the UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferase (ppGalNAcT) family and formed by GalNAc attachment to the serine/threonine (Ser/Thr) side chains of protein substrates (1)(2)(3). The generation of complex O-linked glycans is involved in a variety of biological molecular processes, such as cell motility, cell growth and death, cell-to-cell communication, the immune response, and host-pathogen interactions (4)(5)(6).…”

mentioning

confidence: 99%

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

Nakamura

Horie

Daidoji

et al. 2016

J Virol

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Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. IMPORTANCEViral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.

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confidence: 99%

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

Nakamura

Horie

Daidoji

et al. 2016

J Virol

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“…Positive controls were cell lines expressing matched sensors with the glycosylation site mutated (∆glycan), specifically T 225 G/T 226 G and T 178 G for the T2 and T3 sensors, respectively (Song et al, 2014). Cells (50,000/well) were seeded in flat bottom 96-well-plates (Corning, NY, Cat#3997) and grown for 24 hr.…”

Section: Methodsmentioning

confidence: 99%

Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels

Song

Linstedt

2017

eLife

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Small molecule inhibitors of site-specific O-glycosylation by the polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) family are currently unavailable but hold promise as therapeutics, especially if selective against individual ppGalNAc-T isozymes. To identify a compound targeting the ppGalNAc-T3 isozyme, we screened libraries to find compounds that act on a cell-based fluorescence sensor of ppGalNAc-T3 but not on a sensor of ppGalNAc-T2. This identified a hit that subsequent in vitro analysis showed directly binds and inhibits purified ppGalNAc-T3 with no detectable activity against either ppGalNAc-T2 or ppGalNAc-T6. Remarkably, the inhibitor was active in two medically relevant contexts. In cell culture, it opposed increased cancer cell invasiveness driven by upregulated ppGalNAc-T3 suggesting the inhibitor might be anti-metastatic. In cells and mice, it blocked ppGalNAc-T3-mediated glycan-masking of FGF23 thereby increasing its cleavage, a possible treatment of chronic kidney disease. These findings establish a pharmacological approach for the ppGalNAc-transferase family and suggest that targeting specific ppGalNAc-transferases will yield new therapeutics.DOI: http://dx.doi.org/10.7554/eLife.24051.001

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“…After processing in the Golgi the sensor accumulates on the cell surface. It takes a minimum of 3 h to develop a strong surface signal, so, after a test condition, a period of at least this duration should be allowed before the cells are analyzed [10]. …”

Section: Methodsmentioning

confidence: 99%

“…Therefore, we developed cell-based fluorescence sensors that are particularly sensitive to inhibition of GalNAc-transferase activity [9, 10]. They are transfectable constructs encoding proteins that traffic to the cell surface and become fluorescent if their glycosylation is inhibited.…”

Section: Introductionmentioning

confidence: 99%

“…At present only T2- and T3-specific versions are available. Their acceptor sequences were derived from known T2 or T3 substrates and then modified to improve selectivity [6, 10, 13, 15, 16]. The second critical aspect is that the starting or ground state signal can be significantly greater than true background (where true background is determined by mutating the furin site).…”

Section: Introductionmentioning

confidence: 99%

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Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo

2016

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Mucin-type O-glycosylation occurring in the Golgi apparatus is an important protein posttranslational modification initiated by up to 20 GalNAc-transferase isozymes with largely distinct substrate specificities. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and misregulation causes human diseases. Here we describe the use of protein-based fluorescence sensors that traffic in the secretory pathway to monitor GalNAc-transferase activity in living cells. The sensors can either be “pan” or isozyme specific.

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Development of Isoform-specific Sensors of Polypeptide GalNAc-transferase Activity

Cited by 16 publications

References 35 publications

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels

Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo

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