Limits...
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

Confocal microscopic images and quantitative analysis of the colocalization.(A) Double immunofluorescence analysis of FITC-labeled SNA-I (a, e, i and m), and marker for ER (b, c and d), Golgi (f, g and h), endosomes (j, k and l) and lysosomes (n, o and p) in HeLa cells. The merged reconstructed images are shown in (d, h, l and p) with the green dots from FITC-labeled SNA-I and the red dots from the marker. The arrow indicates SNA-I dots overlapping with the marker. Scale bars represent 10 μm. (B) Manders’ coefficients and object-based colocalization graphs of the colocalization image analysis study. Asterisks denote values significantly different from the lysosome (*: p < 0,05; **: p< 0,01; ***: p < 0,001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493096&req=5

pone.0132389.g004: Confocal microscopic images and quantitative analysis of the colocalization.(A) Double immunofluorescence analysis of FITC-labeled SNA-I (a, e, i and m), and marker for ER (b, c and d), Golgi (f, g and h), endosomes (j, k and l) and lysosomes (n, o and p) in HeLa cells. The merged reconstructed images are shown in (d, h, l and p) with the green dots from FITC-labeled SNA-I and the red dots from the marker. The arrow indicates SNA-I dots overlapping with the marker. Scale bars represent 10 μm. (B) Manders’ coefficients and object-based colocalization graphs of the colocalization image analysis study. Asterisks denote values significantly different from the lysosome (*: p < 0,05; **: p< 0,01; ***: p < 0,001).

Mentions: To find out where the FITC-labeled S. nigra proteins are targeted to in the cell, a quantitative colocalization analysis was performed on confocal microscopy images after co-labeling various endovesicles (Fig 4). Irrespective of the measurement method (Manders’ coefficients or object-based colocalized coefficients), the analysis showed that a few discrete dots overlap with markers for ER or Golgi but the majority of the lectin/RIP-positive dots colocalized with the endosomes and lysosomes. In the case of SNA-I and SNA-IV higher Manders’ coefficients were obtained for the ER, indicating relatively more colocalisation. It is also worth noting that the presence of SNA-IV and SNLRP in the Golgi compartment was clearly lower than for the other proteins.


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)

Confocal microscopic images and quantitative analysis of the colocalization.(A) Double immunofluorescence analysis of FITC-labeled SNA-I (a, e, i and m), and marker for ER (b, c and d), Golgi (f, g and h), endosomes (j, k and l) and lysosomes (n, o and p) in HeLa cells. The merged reconstructed images are shown in (d, h, l and p) with the green dots from FITC-labeled SNA-I and the red dots from the marker. The arrow indicates SNA-I dots overlapping with the marker. Scale bars represent 10 μm. (B) Manders’ coefficients and object-based colocalization graphs of the colocalization image analysis study. Asterisks denote values significantly different from the lysosome (*: p < 0,05; **: p< 0,01; ***: p < 0,001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132389.g004: Confocal microscopic images and quantitative analysis of the colocalization.(A) Double immunofluorescence analysis of FITC-labeled SNA-I (a, e, i and m), and marker for ER (b, c and d), Golgi (f, g and h), endosomes (j, k and l) and lysosomes (n, o and p) in HeLa cells. The merged reconstructed images are shown in (d, h, l and p) with the green dots from FITC-labeled SNA-I and the red dots from the marker. The arrow indicates SNA-I dots overlapping with the marker. Scale bars represent 10 μm. (B) Manders’ coefficients and object-based colocalization graphs of the colocalization image analysis study. Asterisks denote values significantly different from the lysosome (*: p < 0,05; **: p< 0,01; ***: p < 0,001).
Mentions: To find out where the FITC-labeled S. nigra proteins are targeted to in the cell, a quantitative colocalization analysis was performed on confocal microscopy images after co-labeling various endovesicles (Fig 4). Irrespective of the measurement method (Manders’ coefficients or object-based colocalized coefficients), the analysis showed that a few discrete dots overlap with markers for ER or Golgi but the majority of the lectin/RIP-positive dots colocalized with the endosomes and lysosomes. In the case of SNA-I and SNA-IV higher Manders’ coefficients were obtained for the ER, indicating relatively more colocalisation. It is also worth noting that the presence of SNA-IV and SNLRP in the Golgi compartment was clearly lower than for the other proteins.

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