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The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Heinz LX, Baumann CL, Köberlin MS, Snijder B, Gawish R, Shui G, Sharif O, Aspalter IM, Müller AC, Kandasamy RK, Breitwieser FP, Pichlmair A, Bruckner M, Rebsamen M, Blüml S, Karonitsch T, Fauster A, Colinge J, Bennett KL, Knapp S, Wenk MR, Superti-Furga G - Cell Rep (2015)

Bottom Line: Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis.Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling.Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus

Enzymatic Activity and Inducibility of SMPDL3B(A) Substrate velocity curve and Lineweaver-Burk diagram for murine SMPDL3B produced in Sf9 insect cells.(B) Influence of pH on the enzymatic activity of murine SMPDL3B produced in Sf9 insect cells.(C) Coomassie staining of SDS-PAGE gel containing equal amounts of human or mouse SMPDL3B and H135A point mutants. Bar graphs show phosphodiesterase activity of the indicated proteins.(D) Measurement of phosphodiesterase activity on HEK293T cells stably expressing an empty vector (mock) or murine SMPDL3B.(E) Phosphodiesterase activity on control (shCTRL) and Smpdl3b-depleted (shSMPDL3B) RAW264.7 cells.(F) Wild-type (wt) or Smpdl3b-deficient (ko) BMDMs were stimulated or not with 100 ng/ml LPS, 1 μM CpG, 2.5 μM IMQ, 200 ng/ml Pam3Csk4 (P3C4), and 1,000 U/ml IFN-β or 1,000 U/ml IFN-γ for 16 hr, and SMPDL3B or Actin protein levels were analyzed by western blotting. Bar graphs represent phosphodiesterase activity measured after stimulation.(A and C) Data are representative of at least two independent experiments. (B and D–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. See also Figure S3.
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fig3: Enzymatic Activity and Inducibility of SMPDL3B(A) Substrate velocity curve and Lineweaver-Burk diagram for murine SMPDL3B produced in Sf9 insect cells.(B) Influence of pH on the enzymatic activity of murine SMPDL3B produced in Sf9 insect cells.(C) Coomassie staining of SDS-PAGE gel containing equal amounts of human or mouse SMPDL3B and H135A point mutants. Bar graphs show phosphodiesterase activity of the indicated proteins.(D) Measurement of phosphodiesterase activity on HEK293T cells stably expressing an empty vector (mock) or murine SMPDL3B.(E) Phosphodiesterase activity on control (shCTRL) and Smpdl3b-depleted (shSMPDL3B) RAW264.7 cells.(F) Wild-type (wt) or Smpdl3b-deficient (ko) BMDMs were stimulated or not with 100 ng/ml LPS, 1 μM CpG, 2.5 μM IMQ, 200 ng/ml Pam3Csk4 (P3C4), and 1,000 U/ml IFN-β or 1,000 U/ml IFN-γ for 16 hr, and SMPDL3B or Actin protein levels were analyzed by western blotting. Bar graphs represent phosphodiesterase activity measured after stimulation.(A and C) Data are representative of at least two independent experiments. (B and D–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. See also Figure S3.

Mentions: The similarity to other phosphodiesterases prompted us to assess SMPDL3B activity by monitoring phosphodiesterase-dependent hydrolysis of chromogenic bis(4-nitrophenyl)phosphate (bis-pNPP) (Figure S3A). Recombinant SMPDL3B lacking the C-terminal GPI signal, purified from supernatants of Sf9 insect cells or HEK293T cells, efficiently hydrolyzed the substrate, exerting highest activity at neutral pH (Figures 3A, 3B, and S3B). This indicated that the enzyme is fully active at the pH found at the plasma membrane location. Confirming specificity, a mutant (H135A) replacing a conserved histidine residue of SMPDL3B predicted to be involved in substrate hydrolysis (Seto et al., 2004), showed reduced enzymatic activity (Figure 3C). In line with the abundant expression on the plasma membrane, substrate hydrolysis could also be detected on HEK293T cells stably expressing murine or human SMPDL3B, indicating that this assay was well suited for measuring activity on the surface of intact cells (Figures 3D, S3C, and S3D). Indeed, RAW264.7 cells showed robustly detectable enzymatic activity, which was strongly reduced in the Smpdl3b knockdown cells, highlighting SMPDL3B as an important phosphodiesterase enzyme significantly contributing to substrate hydrolysis on macrophages (Figures 3E and S3E).


The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Heinz LX, Baumann CL, Köberlin MS, Snijder B, Gawish R, Shui G, Sharif O, Aspalter IM, Müller AC, Kandasamy RK, Breitwieser FP, Pichlmair A, Bruckner M, Rebsamen M, Blüml S, Karonitsch T, Fauster A, Colinge J, Bennett KL, Knapp S, Wenk MR, Superti-Furga G - Cell Rep (2015)

Enzymatic Activity and Inducibility of SMPDL3B(A) Substrate velocity curve and Lineweaver-Burk diagram for murine SMPDL3B produced in Sf9 insect cells.(B) Influence of pH on the enzymatic activity of murine SMPDL3B produced in Sf9 insect cells.(C) Coomassie staining of SDS-PAGE gel containing equal amounts of human or mouse SMPDL3B and H135A point mutants. Bar graphs show phosphodiesterase activity of the indicated proteins.(D) Measurement of phosphodiesterase activity on HEK293T cells stably expressing an empty vector (mock) or murine SMPDL3B.(E) Phosphodiesterase activity on control (shCTRL) and Smpdl3b-depleted (shSMPDL3B) RAW264.7 cells.(F) Wild-type (wt) or Smpdl3b-deficient (ko) BMDMs were stimulated or not with 100 ng/ml LPS, 1 μM CpG, 2.5 μM IMQ, 200 ng/ml Pam3Csk4 (P3C4), and 1,000 U/ml IFN-β or 1,000 U/ml IFN-γ for 16 hr, and SMPDL3B or Actin protein levels were analyzed by western blotting. Bar graphs represent phosphodiesterase activity measured after stimulation.(A and C) Data are representative of at least two independent experiments. (B and D–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. See also Figure S3.
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fig3: Enzymatic Activity and Inducibility of SMPDL3B(A) Substrate velocity curve and Lineweaver-Burk diagram for murine SMPDL3B produced in Sf9 insect cells.(B) Influence of pH on the enzymatic activity of murine SMPDL3B produced in Sf9 insect cells.(C) Coomassie staining of SDS-PAGE gel containing equal amounts of human or mouse SMPDL3B and H135A point mutants. Bar graphs show phosphodiesterase activity of the indicated proteins.(D) Measurement of phosphodiesterase activity on HEK293T cells stably expressing an empty vector (mock) or murine SMPDL3B.(E) Phosphodiesterase activity on control (shCTRL) and Smpdl3b-depleted (shSMPDL3B) RAW264.7 cells.(F) Wild-type (wt) or Smpdl3b-deficient (ko) BMDMs were stimulated or not with 100 ng/ml LPS, 1 μM CpG, 2.5 μM IMQ, 200 ng/ml Pam3Csk4 (P3C4), and 1,000 U/ml IFN-β or 1,000 U/ml IFN-γ for 16 hr, and SMPDL3B or Actin protein levels were analyzed by western blotting. Bar graphs represent phosphodiesterase activity measured after stimulation.(A and C) Data are representative of at least two independent experiments. (B and D–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. See also Figure S3.
Mentions: The similarity to other phosphodiesterases prompted us to assess SMPDL3B activity by monitoring phosphodiesterase-dependent hydrolysis of chromogenic bis(4-nitrophenyl)phosphate (bis-pNPP) (Figure S3A). Recombinant SMPDL3B lacking the C-terminal GPI signal, purified from supernatants of Sf9 insect cells or HEK293T cells, efficiently hydrolyzed the substrate, exerting highest activity at neutral pH (Figures 3A, 3B, and S3B). This indicated that the enzyme is fully active at the pH found at the plasma membrane location. Confirming specificity, a mutant (H135A) replacing a conserved histidine residue of SMPDL3B predicted to be involved in substrate hydrolysis (Seto et al., 2004), showed reduced enzymatic activity (Figure 3C). In line with the abundant expression on the plasma membrane, substrate hydrolysis could also be detected on HEK293T cells stably expressing murine or human SMPDL3B, indicating that this assay was well suited for measuring activity on the surface of intact cells (Figures 3D, S3C, and S3D). Indeed, RAW264.7 cells showed robustly detectable enzymatic activity, which was strongly reduced in the Smpdl3b knockdown cells, highlighting SMPDL3B as an important phosphodiesterase enzyme significantly contributing to substrate hydrolysis on macrophages (Figures 3E and S3E).

Bottom Line: Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis.Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling.Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus