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Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Yang J, Qin N, Zhang H, Yang R, Xiang B, Wei Q - Sci Rep (2016)

Bottom Line: These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS.Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB.These properties of CnB support its potential for development as an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.

ABSTRACT
Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.

No MeSH data available.


Related in: MedlinePlus

MST measurements of the interaction between CnB and the TLR4 receptor complexes.(a) Measurement of rhCnB binding to the purified TLR4 ectodomain. (b) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. (c) Measurement of rhCnB binding to purified soluble CD14. (d) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. (e) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. (f) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. (g) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.
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f7: MST measurements of the interaction between CnB and the TLR4 receptor complexes.(a) Measurement of rhCnB binding to the purified TLR4 ectodomain. (b) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. (c) Measurement of rhCnB binding to purified soluble CD14. (d) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. (e) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. (f) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. (g) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.

Mentions: The affinity of the interaction between CnB and TLR4 receptor complexes was determined by MST (microscale thermophoresis). MST is a technique based on the motion of a molecule (fluorescently labelled or with a fluorescent protein tag) in an infrared-laser-induced microscopic temperature gradient, an effect termed thermophoresis. The thermophoretic motion yields a fluorescence time trace from which a normalized fluorescence value (FNorm) is recorded. Upon ligand binding, the thermophoretic mobility of the molecule changes, leading to shifts of the Fnorm values. The shifts are used to quantify the affinity of the interactions. MST can also be utilized to analyse the interactions between proteins and small molecules in complex biological liquids such as serum or cell lysates3637. The present interaction was evaluated in the buffered salt solution of purified proteins or cell lysates from transfected cells. The KD between CnB and the labelled ectodomain of TLR4 was approximately 370 nM (Fig. 7a and Table 1). In the cell lysates, the KD was approximately 6.5 μM (Fig. 7b and Table 1). We also assessed the KD between LPS and TLR4. Using the purified proteins, the KD was approximately 134 nM (Supplementary Fig. S8a and S8f), whereas in cell lysates, the KD was approximately 3.2 μM (Supplementary Fig. S8b and S8f). The affinity between TLR4 and LPS was slightly higher than between CnB and TLR4. The MST results for LPS were consistent with those determined using other methods38. In the cell lysates, competitive interactions decreased the affinity between the molecules, but they closely mimicked the binding situation in vivo. The KD for the interaction between CnB and labelled, soluble CD14 was approximately 1 μM (Fig. 7c and Table 1), and the KD in the cell lysates was approximately 34.8 μM (Fig. 7d and Table 1). The KD for the interaction between LPS and purified sCD14 was approximately 270 nM (Supplementary Fig. S8c and S8f), and the KD between LPS and membrane-anchored CD14 was approximately 1 μM in the cell lysates (Supplementary Fig. S8d and 8f). The affinity between LPS and sCD14 was about 3.5-fold greater than that between sCD14 and CnB. The interaction between secreted MD2 and CnB was also evaluated in the supernatants of MD2-GFP-transfected 293 cells grown in suspension. A clear association was detected, and the KD was approximately 74 μM (Fig. 7e and Table 1); the KD between CnB and TLR4-bound MD2 in the cell lysates was approximately 7.4 μM (Fig. 7f and Table 1), and an approximately 10-fold increase in affinity was observed between sMD2 and CnB. These results demonstrated that sMD2 also participated in the recognition of CnB and probably played an important role in avoiding severe inflammatory responses. The GFP tag did not interact with CnB in cell lysates (Supplementary Fig. S8e). In the MST experiment, some binding curves were shifted up or down as the amount of ligand increased, and the results depended on the different thermophoretic effects of the unbound molecules and the complex. If the MST signal increased as the concentration ratio of the binding partners increased, the binding curve exhibited a characteristic s-shape in which the unbound and saturated state formed lower and upper plateaus. In the other case, the MST signal decreased as the concentration ratio of the binding partners increased, and the binding curve showed a mirrored s-shape.


Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Yang J, Qin N, Zhang H, Yang R, Xiang B, Wei Q - Sci Rep (2016)

MST measurements of the interaction between CnB and the TLR4 receptor complexes.(a) Measurement of rhCnB binding to the purified TLR4 ectodomain. (b) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. (c) Measurement of rhCnB binding to purified soluble CD14. (d) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. (e) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. (f) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. (g) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.
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f7: MST measurements of the interaction between CnB and the TLR4 receptor complexes.(a) Measurement of rhCnB binding to the purified TLR4 ectodomain. (b) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. (c) Measurement of rhCnB binding to purified soluble CD14. (d) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. (e) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. (f) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. (g) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.
Mentions: The affinity of the interaction between CnB and TLR4 receptor complexes was determined by MST (microscale thermophoresis). MST is a technique based on the motion of a molecule (fluorescently labelled or with a fluorescent protein tag) in an infrared-laser-induced microscopic temperature gradient, an effect termed thermophoresis. The thermophoretic motion yields a fluorescence time trace from which a normalized fluorescence value (FNorm) is recorded. Upon ligand binding, the thermophoretic mobility of the molecule changes, leading to shifts of the Fnorm values. The shifts are used to quantify the affinity of the interactions. MST can also be utilized to analyse the interactions between proteins and small molecules in complex biological liquids such as serum or cell lysates3637. The present interaction was evaluated in the buffered salt solution of purified proteins or cell lysates from transfected cells. The KD between CnB and the labelled ectodomain of TLR4 was approximately 370 nM (Fig. 7a and Table 1). In the cell lysates, the KD was approximately 6.5 μM (Fig. 7b and Table 1). We also assessed the KD between LPS and TLR4. Using the purified proteins, the KD was approximately 134 nM (Supplementary Fig. S8a and S8f), whereas in cell lysates, the KD was approximately 3.2 μM (Supplementary Fig. S8b and S8f). The affinity between TLR4 and LPS was slightly higher than between CnB and TLR4. The MST results for LPS were consistent with those determined using other methods38. In the cell lysates, competitive interactions decreased the affinity between the molecules, but they closely mimicked the binding situation in vivo. The KD for the interaction between CnB and labelled, soluble CD14 was approximately 1 μM (Fig. 7c and Table 1), and the KD in the cell lysates was approximately 34.8 μM (Fig. 7d and Table 1). The KD for the interaction between LPS and purified sCD14 was approximately 270 nM (Supplementary Fig. S8c and S8f), and the KD between LPS and membrane-anchored CD14 was approximately 1 μM in the cell lysates (Supplementary Fig. S8d and 8f). The affinity between LPS and sCD14 was about 3.5-fold greater than that between sCD14 and CnB. The interaction between secreted MD2 and CnB was also evaluated in the supernatants of MD2-GFP-transfected 293 cells grown in suspension. A clear association was detected, and the KD was approximately 74 μM (Fig. 7e and Table 1); the KD between CnB and TLR4-bound MD2 in the cell lysates was approximately 7.4 μM (Fig. 7f and Table 1), and an approximately 10-fold increase in affinity was observed between sMD2 and CnB. These results demonstrated that sMD2 also participated in the recognition of CnB and probably played an important role in avoiding severe inflammatory responses. The GFP tag did not interact with CnB in cell lysates (Supplementary Fig. S8e). In the MST experiment, some binding curves were shifted up or down as the amount of ligand increased, and the results depended on the different thermophoretic effects of the unbound molecules and the complex. If the MST signal increased as the concentration ratio of the binding partners increased, the binding curve exhibited a characteristic s-shape in which the unbound and saturated state formed lower and upper plateaus. In the other case, the MST signal decreased as the concentration ratio of the binding partners increased, and the binding curve showed a mirrored s-shape.

Bottom Line: These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS.Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB.These properties of CnB support its potential for development as an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.

ABSTRACT
Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.

No MeSH data available.


Related in: MedlinePlus