Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis

Willems, Anke P.; Sun, Lingbo; Schulz, Morten Alder; Tian, Weihua; Ashikov, Angel; Scherpenzeel, Monique van; Hermans, Esther; Clausen, Henrik; Yang, Zifeng; Lefeber, Dirk

doi:10.1016/j.bbagen.2019.05.011

Cited by 22 publications

(22 citation statements)

References 29 publications

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“…Four top candidates identified in the CRISPR screen following T3SA+ infection were Nans, St3gal4, Slc351, and Cmas. Nans is required for the phosphorylation of all types of SAs (27). St3gal4 is required for the sialylation of α2,3-linked glycoconjugates (28).…”

Section: Discussionmentioning

confidence: 99%

Cytidine Monophosphate N -Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection

Urbanek

Sutherland

Orchard

et al. 2020

J Virol

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Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

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Section: Discussionmentioning

confidence: 99%

Cytidine Monophosphate N -Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection

Urbanek

Sutherland

Orchard

et al. 2020

J Virol

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“…However, and rather unexpectedly, neither RNA-Seq analysis nor qPCR approaches detected expression of N-Acetylneuraminic Acid Phosphatase (NANP), an enzyme that dephosphorylates sialic acid 9phosphate to free sialic acid as part of the required intermediate biosynthetic metabolites. As sialylated glycans are undeniably present in SFs, alternative biosynthesis pathways might be operating in these cells, perhaps as in recent observations in CHO cells 29 . To identify possible mechanisms behind changes in sialylation, we used qPCR to evaluate expression of selected genes (as in Fig.…”

Section: Sfs From Arthritic Mice Exhibit Down-regulatedmentioning

confidence: 85%

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in Rheumatoid Arthritis

Wang

Khan

Antonopoulos

et al. 2020

Preprint

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In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis but are recognized to adopt a pathological role in rheumatoid arthritis (RA), promoting the infiltration and activation of immune cells to perpetuate local inflammation, pain and joint destruction.Carbohydrates (glycans) attached to cell surface proteins are fundamental regulators of cellular interactions between stromal and immune cells, but very little is known about the glycome of SFs or how glycosylation regulates their biology. Here we fill these gaps in our understanding of stromal guided pathophysiology by systematically mapping glycosylation pathways in healthy and arthritic SFs. We used a combination of transcriptomic and glycomic analysis to show that transformation of fibroblasts into pro-inflammatory cells in RA is associated with profound glycan remodeling, a process that involves reduction of a2-6 terminal sialylation that is mostly mediated by TNFa-dependent inhibition of the glycosyltransferase ST6Gal1. We also show that sialylation of SFs correlates with distinct disease stages and SFs functional subsets in both human RA and models of mouse arthritis. We propose that pro-inflammatory cytokines in the joint remodel the SF-glycome, transforming a regulatory tissue intended to preserve local homeostasis, into an under-sialylated and highly pro-inflammatory microenvironment that contributes to an amplificatory inflammatory network that perpetuates chronic inflammation. These results highlight the importance of cell glycosylation in stromal immunology.

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“…Batch-to-batch variations in sialylation of EPO have been a challenge ( 301 ), and secreted neuraminidases ( NEU1-3 ) from CHO cells can pose issues and KO of these can improve sialylation ( 302 ). Several reports have suggested that CHO cells may add minor amounts of the immunogenic α1-3Gal epitope as well as NeuGc sialic acids ( 303 , 304 , 305 ), although these findings are disputed and CHO cells with KO of these genes are available ( 306 ).…”

Section: Production Of Therapeutic Glycoproteins In Glycoengineered Mmentioning

confidence: 99%

Genetic glycoengineering in mammalian cells

Narimatsu

Büll

Chen³

et al. 2021

Journal of Biological Chemistry

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Advances in nuclease-based gene-editing technologies have enabled precise, stable, and systematic genetic engineering of glycosylation capacities in mammalian cells, opening up a plethora of opportunities for studying the glycome and exploiting glycans in biomedicine. Glycoengineering using chemical, enzymatic, and genetic approaches has a long history, and precise gene editing provides a nearly unlimited playground for stable engineering of glycosylation in mammalian cells to explore and dissect the glycome and its many biological functions. Genetic engineering of glycosylation in cells also brings studies of the glycome to the single cell level and opens up wider use and integration of data in traditional omics workflows in cell biology. The last few years have seen new applications of glycoengineering in mammalian cells with perspectives for wider use in basic and applied glycosciences, and these have already led to discoveries of functions of glycans and improved designs of glycoprotein therapeutics. Here, we review the current state of the art of genetic glycoengineering in mammalian cells and highlight emerging opportunities.

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Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis

Cited by 22 publications

References 29 publications

Cytidine Monophosphate N -Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection

Cytidine Monophosphate N -Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in Rheumatoid Arthritis

Genetic glycoengineering in mammalian cells

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