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The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

Shang C, Chen Q, Dell A, Haslam SM, De Vos WH, Van Damme EJ - PLoS ONE (2015)

Bottom Line: In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins.Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula.Our data suggest that one of these pathways involves the induction of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ABSTRACT
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

No MeSH data available.


Related in: MedlinePlus

Comparative analysis of the relative intensities of LacNAc antenna and sialylated LacNAc antenna in all complex glycans in HeLa and NHDF cells (A), and in Sialidase S treated (B) and Sialidase A treated (C) samples.Black colour, relative intensity of α2–3 and α2–6 sialylated LacNAc antenna; blue colour, relative intensity of LacNAc antenna; red colour, relative intensity of α2–6 sialylated LacNAc antenna.
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pone.0132389.g007: Comparative analysis of the relative intensities of LacNAc antenna and sialylated LacNAc antenna in all complex glycans in HeLa and NHDF cells (A), and in Sialidase S treated (B) and Sialidase A treated (C) samples.Black colour, relative intensity of α2–3 and α2–6 sialylated LacNAc antenna; blue colour, relative intensity of LacNAc antenna; red colour, relative intensity of α2–6 sialylated LacNAc antenna.

Mentions: To assess whether differences in cytotoxicity between cell types and proteins were due to differences in carbohydrate binding to the cell surface, we analysed the glycome patterns on both glycoproteins and glycolipids of HeLa and NHDF cell samples using mass spectrometric methodologies [40]. These experiments confirmed the presence of high mannose and complex glycans in both cell types. The major complex N-glycans in both HeLa and NHDF cells are sialylated with N-acetylneuraminic acid (NeuAc) or are terminated with uncapped galactose (Gal) (S3 Fig). To determine the sialic acid linkages, the glycans were digested with sialidase S, which specifically removes α2–3 linked sialic acid, or sialidase A, which cleaves all non-reducing terminal sialic acid residues. Quantification of terminal Gal and terminal NeuAc on glycans from each cell line was performed by comparing the relative abundance of LacNAc (Gal-GlcNAc) antenna and sialylated LacNAc antenna. The result (Fig 7) showed that before Sialidase S digestion, the percentage of glycans terminated with Gal was approximately 54% in NHDF cells, which is significantly higher than in HeLa cells (approximately 30%). After Sialidase S digestion, the percentage of glycans terminated with α2–6 linked NeuAc in HeLa cells is around 17%, which is considerably higher than the 3% observed in NHDF cells, Sialidase A digestion removed all NeuAc supporting the glycomic assignments.


The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

Shang C, Chen Q, Dell A, Haslam SM, De Vos WH, Van Damme EJ - PLoS ONE (2015)

Comparative analysis of the relative intensities of LacNAc antenna and sialylated LacNAc antenna in all complex glycans in HeLa and NHDF cells (A), and in Sialidase S treated (B) and Sialidase A treated (C) samples.Black colour, relative intensity of α2–3 and α2–6 sialylated LacNAc antenna; blue colour, relative intensity of LacNAc antenna; red colour, relative intensity of α2–6 sialylated LacNAc antenna.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493096&req=5

pone.0132389.g007: Comparative analysis of the relative intensities of LacNAc antenna and sialylated LacNAc antenna in all complex glycans in HeLa and NHDF cells (A), and in Sialidase S treated (B) and Sialidase A treated (C) samples.Black colour, relative intensity of α2–3 and α2–6 sialylated LacNAc antenna; blue colour, relative intensity of LacNAc antenna; red colour, relative intensity of α2–6 sialylated LacNAc antenna.
Mentions: To assess whether differences in cytotoxicity between cell types and proteins were due to differences in carbohydrate binding to the cell surface, we analysed the glycome patterns on both glycoproteins and glycolipids of HeLa and NHDF cell samples using mass spectrometric methodologies [40]. These experiments confirmed the presence of high mannose and complex glycans in both cell types. The major complex N-glycans in both HeLa and NHDF cells are sialylated with N-acetylneuraminic acid (NeuAc) or are terminated with uncapped galactose (Gal) (S3 Fig). To determine the sialic acid linkages, the glycans were digested with sialidase S, which specifically removes α2–3 linked sialic acid, or sialidase A, which cleaves all non-reducing terminal sialic acid residues. Quantification of terminal Gal and terminal NeuAc on glycans from each cell line was performed by comparing the relative abundance of LacNAc (Gal-GlcNAc) antenna and sialylated LacNAc antenna. The result (Fig 7) showed that before Sialidase S digestion, the percentage of glycans terminated with Gal was approximately 54% in NHDF cells, which is significantly higher than in HeLa cells (approximately 30%). After Sialidase S digestion, the percentage of glycans terminated with α2–6 linked NeuAc in HeLa cells is around 17%, which is considerably higher than the 3% observed in NHDF cells, Sialidase A digestion removed all NeuAc supporting the glycomic assignments.

Bottom Line: In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins.Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula.Our data suggest that one of these pathways involves the induction of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ABSTRACT
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

No MeSH data available.


Related in: MedlinePlus