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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

Internalization of FITC labeled S. nigra proteins in HeLa cells.(A) Confocal microscopic images of the uptake of SNA-I (25 nM) in HeLa cells after 6 hours incubation. The arrow indicates a spot where protein is accumulating. Nuclei have been delineated be in white. Scale bars represent 10 μm. (B) Uptake of 50 nM FITC labeled SNA-I, SNA-II, SNA-IV, SNA-V and SNLRP by HeLa cells after 0 min, 5 min, 30 min, 1 h, 3 h, 6 h, 9 h and 12 h incubation, based on fluorescence intensity, which was normalized by the FITC labeling efficiency. The pictures for quantification were acquired using identical confocal settings. Data are given as mean ± SE, based on at least 80 individual cell measurements per sample and each treatment was carried out with three independent replicates.
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pone.0132389.g003: Internalization of FITC labeled S. nigra proteins in HeLa cells.(A) Confocal microscopic images of the uptake of SNA-I (25 nM) in HeLa cells after 6 hours incubation. The arrow indicates a spot where protein is accumulating. Nuclei have been delineated be in white. Scale bars represent 10 μm. (B) Uptake of 50 nM FITC labeled SNA-I, SNA-II, SNA-IV, SNA-V and SNLRP by HeLa cells after 0 min, 5 min, 30 min, 1 h, 3 h, 6 h, 9 h and 12 h incubation, based on fluorescence intensity, which was normalized by the FITC labeling efficiency. The pictures for quantification were acquired using identical confocal settings. Data are given as mean ± SE, based on at least 80 individual cell measurements per sample and each treatment was carried out with three independent replicates.

Mentions: To find out whether differences in cytotoxicity between S. nigra proteins were due to differences in cellular protein uptake, HeLa cells were incubated with FITC labeled RIPs and lectins, and monitored using live cell confocal imaging (Fig 3A and S2 Fig). The binding and internalization kinetics of the proteins was quantified at different time points after incubation by measuring the intracellular fluorescence intensities (Fig 3B). Within 0–5 min, we measured fluorescent signals that co-aligned with the plasma membrane, presumably reflecting lectin binding. At later time points, signals were also observed at intracellular locations. All proteins showed time-dependent internalization kinetics with a maximum fluorescent signal after 6 to 9 h of incubation. When fluorescently labelled SNA-I was added to the medium of HeLa cells, the protein attached to the cell surface within minutes. Within the first 40 min, fluorescent SNA-I became internalized and increasingly accumulated in spots close to the nucleus up till 9h (Fig 3A). After 12h a decrease in fluorescent signal was observed, which probably reflects degradation and externalization of internalized lectin. A similar pattern was observed for SNA-V, albeit with a quicker turnover rate (max. at 6h) (S2B Fig). The perinuclear accumulation was maintained even after mitosis. SNA-II (S2A Fig) did not show a distinct tethering to the cell surface but also gradually accumulated inside the cell. Compared to SNA-V, intracellular accumulation of SNA-II was less pronounced. The same holds true for SNA-IV and SNLRP (S2C and S2D Fig, captured with much higher laser power setting to be visible): the amount of bound protein to the HeLa cell surface was almost undetectable and the amount of internalized protein was low compared to SNA-I (Fig 3B). After normalization for labeling efficiency, especially SNLRP internalization was negligible. During the time period of microscopic acquisition, HeLa cells incubated with the S. nigra proteins showed normal growth and cell division (S2B Fig). Only a small subset of cells incubated with SNA-I, SNA-II or SNA-V showed morphological changes characteristic for apoptosis (S2A Fig).


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)

Internalization of FITC labeled S. nigra proteins in HeLa cells.(A) Confocal microscopic images of the uptake of SNA-I (25 nM) in HeLa cells after 6 hours incubation. The arrow indicates a spot where protein is accumulating. Nuclei have been delineated be in white. Scale bars represent 10 μm. (B) Uptake of 50 nM FITC labeled SNA-I, SNA-II, SNA-IV, SNA-V and SNLRP by HeLa cells after 0 min, 5 min, 30 min, 1 h, 3 h, 6 h, 9 h and 12 h incubation, based on fluorescence intensity, which was normalized by the FITC labeling efficiency. The pictures for quantification were acquired using identical confocal settings. Data are given as mean ± SE, based on at least 80 individual cell measurements per sample and each treatment was carried out with three independent replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132389.g003: Internalization of FITC labeled S. nigra proteins in HeLa cells.(A) Confocal microscopic images of the uptake of SNA-I (25 nM) in HeLa cells after 6 hours incubation. The arrow indicates a spot where protein is accumulating. Nuclei have been delineated be in white. Scale bars represent 10 μm. (B) Uptake of 50 nM FITC labeled SNA-I, SNA-II, SNA-IV, SNA-V and SNLRP by HeLa cells after 0 min, 5 min, 30 min, 1 h, 3 h, 6 h, 9 h and 12 h incubation, based on fluorescence intensity, which was normalized by the FITC labeling efficiency. The pictures for quantification were acquired using identical confocal settings. Data are given as mean ± SE, based on at least 80 individual cell measurements per sample and each treatment was carried out with three independent replicates.
Mentions: To find out whether differences in cytotoxicity between S. nigra proteins were due to differences in cellular protein uptake, HeLa cells were incubated with FITC labeled RIPs and lectins, and monitored using live cell confocal imaging (Fig 3A and S2 Fig). The binding and internalization kinetics of the proteins was quantified at different time points after incubation by measuring the intracellular fluorescence intensities (Fig 3B). Within 0–5 min, we measured fluorescent signals that co-aligned with the plasma membrane, presumably reflecting lectin binding. At later time points, signals were also observed at intracellular locations. All proteins showed time-dependent internalization kinetics with a maximum fluorescent signal after 6 to 9 h of incubation. When fluorescently labelled SNA-I was added to the medium of HeLa cells, the protein attached to the cell surface within minutes. Within the first 40 min, fluorescent SNA-I became internalized and increasingly accumulated in spots close to the nucleus up till 9h (Fig 3A). After 12h a decrease in fluorescent signal was observed, which probably reflects degradation and externalization of internalized lectin. A similar pattern was observed for SNA-V, albeit with a quicker turnover rate (max. at 6h) (S2B Fig). The perinuclear accumulation was maintained even after mitosis. SNA-II (S2A Fig) did not show a distinct tethering to the cell surface but also gradually accumulated inside the cell. Compared to SNA-V, intracellular accumulation of SNA-II was less pronounced. The same holds true for SNA-IV and SNLRP (S2C and S2D Fig, captured with much higher laser power setting to be visible): the amount of bound protein to the HeLa cell surface was almost undetectable and the amount of internalized protein was low compared to SNA-I (Fig 3B). After normalization for labeling efficiency, especially SNLRP internalization was negligible. During the time period of microscopic acquisition, HeLa cells incubated with the S. nigra proteins showed normal growth and cell division (S2B Fig). Only a small subset of cells incubated with SNA-I, SNA-II or SNA-V showed morphological changes characteristic for apoptosis (S2A Fig).

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