Effects of tunicamycin on insect cells

Butters, Terry D.; Hughes, R. Colin

doi:10.1042/bst0080170

Cited by 10 publications

(3 citation statements)

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“…To confirm that NS1 in the supernatants of infected insect cells ( Fig. 1A ) was not caused by cell lysis, DENV-infected C6/36 cells (at 24 h post-infection) were washed extensively to remove NS1-containing culture medium and treated with fresh medium containing tunicamycin, an inhibitor of N-linked glycosylation (35, 36) ( Fig. 2 ).…”

Section: Resultsmentioning

confidence: 99%

Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin–Mediated Neutralization

Thiemmeca

Tamdet

Punyadee

et al. 2016

The Journal of Immunology

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Flavivirus nonstructural protein 1 (NS1) is a unique secreted non-structural glycoprotein. Although it is absent from the flavivirus virion, intracellular and extracellular forms of NS1 have essential roles in viral replication and the pathogenesis of infection. The fate of NS1 in insect cells has been more controversial, with some reports suggesting it is exclusively cell-associated. Here, we confirm NS1 secretion from cells of insect origin and characterize its physical, biochemical, and functional properties in the context of dengue virus (DENV) infection. Unlike mammalian cell-derived NS1, which displays both high mannose and complex type N-linked glycans, soluble NS1 secreted from DENV-infected insect cells contains only high mannose glycans. Insect cell-derived secreted NS1 also has different physical properties including smaller and more heterogeneous sizes, and the formation of less stable NS1 hexamers. Both mammalian and insect cell-derived NS1 bind to complement proteins C1s, C4 and C4 binding protein as well as to a novel partner, mannose-binding lectin (MBL). Binding of NS1 to MBL protects DENV virus against MBL-mediated neutralization by the lectin pathway of complement activation. As we detected secreted NS1 and DENV virus together in the saliva of infected Aedes aegypti mosquitoes, these findings suggest a mechanism of viral immune evasion at the very earliest phase of infection.

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Section: Resultsmentioning

confidence: 99%

Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin–Mediated Neutralization

Thiemmeca

Tamdet

Punyadee

et al. 2016

The Journal of Immunology

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“…All purified fractions were assayed for 5′-nucleotidase, a plasma membrane marker (Butters and Hughes, 1980;Norris and Cary, 1981), by incubating 50 μl aliquots of crude fraction (and homogenate) in a solution of 200 mmol l −1 glycine (pH 8.5), 200 mmol l −1 MgSO 4 and 66 mmol l −1 5′-AMP for 60 min at 37°C. An aliquot of the incubation mixture was removed, added to 10% trichloroacetic acid (w/v), and centrifuged at 1000 g for 15 min.…”

Section: Subcellular Localization Of Actagsmentioning

confidence: 99%

Seasonal accumulation of acetylated triacylglycerols by a freeze-tolerant insect

Marshall

Thomas

Roxin

et al. 2014

Journal of Experimental Biology

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Most animals store energy as long-chain triacylglycerols (lcTAGs). Trace amounts of acetylated triacylglycerols (acTAGs) have been reported in animals, but are not accumulated, likely because they have lower energy density than lcTAGs. Here we report that acTAGs comprise 36% of the neutral lipid pool of overwintering prepupae of the goldenrod gall fly, Eurosta solidaginis, while only 17% of the neutral lipid pool is made up of typical lcTAGs. These high concentrations of acTAGs, present only during winter, appear to be synthesized by E. solidaginis and are not found in other freezetolerant insects, nor in the plant host. The mixture of acTAGs found in E. solidaginis has a significantly lower melting point than equivalent lcTAGs, and thus remains liquid at temperatures at which E. solidaginis is frozen in the field, and depresses the melting point of aqueous solutions in a manner unusual for neutral lipids. We note that accumulation of acTAGs coincides with preparation for overwintering and the seasonal acquisition of freeze tolerance. This is the first observation of accumulation of acTAGs by an animal, and the first evidence of dynamic interconversion between acTAGs and lcTAGs during development and in response to stress. INTRODUCTIONLipids fulfill diverse biological functions including cell membrane structure, signalling and energy storage (Canavoso et al., 2001;Reue, 2011;Yen et al., 2008). Long-chain triacylglycerols (lcTAGs) are generally used as fuel stores by animals because of their high energy density, and are consequently the most abundant lipid class in animals (Williams et al., 2011;Yen et al., 2008). No other neutral lipid has been reported as a storage molecule in terrestrial animals.Instead of having three long-chain fatty esters (as in lcTAGs), acetylated triacylglycerols (acTAGs) have an acetyl group esterified at the 3-position of the glycerol backbone. This distinct structure results in the reduced viscosity and the significantly lower energy density of acTAGs compared with lcTAGs (Durrett et al., 2010). Trace amounts of acTAGs have been described in a broad range of organisms, including the antlers of Japanese deer (Cervus nippon) (Yang et al., 2004), the udders of domestic cows (Bos primigenius) (Limb et al., 1999) and the seeds of the burning bush plant (Euonymus alatus) (Durrett et al., 2010). acTAGs constitute a very small proportion of the total lipid pool in the two mammal species investigated [<0.1% by mass Limb et al., 1999;Yang et al., 2004)], but predominate (92% by moles) in E. alatus seed oils (Durrett et al., 2010). Euonymus alatus synthesises acTAGs via an unusual diacylglycerol acyltransferase (DGAT) by esterifying an acetyl group, rather than a third long-chain fatty acid, to diacylglycerol (DAG) (Durrett et al., 2010). Although trace acTAGs are present in a few animals, we are not aware of any clear hypotheses for a physiological role of acTAGs in biological systems. Here we report that the goldenrod gall fly, Eurosta solidaginis (Fitch 1855), accumulates significan...

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“…Tunicamycin is an antibiotic isolated from Streptomyces lysosuperificus that inhibits the formation of lipid-linked sugar intermediates involved in the synthesis of complex carbohydrates. It has proved useful for studying the role of such intermediates in glycosylation reactions in many systems, including bacteria (Takatsuki et al, 1972;Ward, 1977), fungi , insects (Butters & Hughes, 1980) and mammalian tissues (Struck et al, 1978;Speake & White, 1979).…”

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confidence: 99%

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

King¹,

Tabiowo²

1981

Biochemical Journal

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1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[(3)H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45-55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-(14)C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[(3)H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[(3)H]glucosamine but not of (U-(14)C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[(3)H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[(3)H]glucosamine and (35)SO(4) (2-) into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl beta-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.

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Effects of tunicamycin on insect cells

Cited by 10 publications

References 1 publication

Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin–Mediated Neutralization

Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin–Mediated Neutralization

Seasonal accumulation of acetylated triacylglycerols by a freeze-tolerant insect

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

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