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. 2017 Jun 29;7(1):4391.
doi: 10.1038/s41598-017-04586-9.

Aminoglycoside-driven Biosynthesis of Selenium-Deficient Selenoprotein P

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Free PMC article

Aminoglycoside-driven Biosynthesis of Selenium-Deficient Selenoprotein P

Kostja Renko et al. Sci Rep. .
Free PMC article

Abstract

Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
In vitro effects of G418 and gentamicin on selenoprotein biosynthesis. (A) HepG2 cells were incubated for 48 h in FCS-free medium with supplemental selenite (100 nM), G418 or gentamicin. Western blot analysis indicates increased secreted SELENOP protein levels in response to selenite and G418 treatment. Intracellular protein levels of selenoenzymes GPX1 and GPX2 were marginally affected. (B) The effect of gentamicin was verified in a dose-dependent analysis. (C) The effects of G418 on SELENOP secretion were replicated in human Hep3B, and (D) murine Hepa1-6 hepatocytes.
Figure 2
Figure 2
Interplay between AG and supplemental selenite on SELENOP concentrations. HepG2 cells were incubated for 48 h in the presence of G418 or gentamicin with or without supplemental selenite (100 nM). Secreted SELENOP (SEPP1) was quantified by ELISA analyses. G418 strongly induced SELENOP concentrations in the conditioned medium, whereas gentamicin showed no effect. The effect of G418 was augmented by supplemental selenite. (Mean ± SEM, n = 6, ANOVA followed by Dunnett’s).
Figure 3
Figure 3
Effects of AG treatment on selenoprotein transcript levels. HepG2 cells were treated with G418, gentamicin or Se for 48 h and mRNA expression was determined by qRT-PCR using 18 S rRNA as reference. (A) DDIT3 served as control gene for AG effects, and transcript levels increased in response to AG treatment in a dose-dependent manner. (B–C) Supplemental selenite and G418 increased SELENOP (SEPP1) and GPX1 transcript levels. (D) Supplemental selenite or gentamicin had no effect on GPX2 transcript levels, whereas G418 showed a moderate effect. (Mean ± SEM, n = 4, ANOVA followed by Dunnett’s).
Figure 4
Figure 4
Se-content of SELENOP in relation to G418 and supplemental selenite. (A) The protein fraction of conditioned media from HepG2 cells was immobilized on a nitrocellulose membrane before and after immuno-precipitation of SELENOP (SEPP -IP). The SELENOP-IP procedure efficiently removed SELENOP (SEPP1), and with it all the detectable Se from the culture medium. (B) Supplemental selenite and G418 increased immuno-detectable SELENOP (SEPP1). (C) Se content of SELENOP preparations varied strongly between the different incubation conditions. (D) A comparison of the Se/SELENOP (Se/SEPP1) ratio indicates that G418 induces Sec-free SELENOP variants, whereas selenite increased the Se content of SELENOP in the presence of G418. (Mean ± SEM, n = 3).
Figure 5
Figure 5
Amino acid insertion at UGA codons SEC3, SEC4 and SEC5 in SELENOP. HepG2 cells were treated with 100 nM Se, 200 µg/mL G418 or their combination. SELENOP was purified by immuno-affinity and subjected to LC-MS/MS analysis. (A) Sec in SELENOP was detected almost exclusively at the positions SEC3, SEC4 and SEC5 when cells were supplemented with selenite. (B) The pattern of amino acids inserted at the three Sec codons varied strongly when cells were grown in the presence of 100 nM selenite and 200 µg/mL G418. (C) SELENOP synthesized in the absence of supplemental selenite but in presence of 200 µg/mL G418 was devoid of Sec residues at the three positions available for analysis (SEC3-5). The amino acids replacing Sec were mainly tryptophan (W), cysteine (C) and arginine (R), but their relative proportions were Sec-codon specific.
Figure 6
Figure 6
Effects of AG treatment on selenoprotein expression in mice. Mice fed Se-deficient or Se-sufficient diets were treated with G418 or gentamicin. (A) Ddit3 was analyzed as an AG-responsive gene in kidney and liver, and significantly increased Ddit3 mRNA concentrations were observed in kidney upon G418 injection (ANOVA followed by Dunnett’s). (B) Western blot analyses of Gpx3 and Selenop showed no differences in expression levels in response to G418 or gentamicin in Se-treated or Se-deficient mice. (C) Se concentrations were higher in the selenite-supplemented mice than in mice raised on a Se-deficient diet. No effect of AG treatment on serum Se was detectable. (D) Se concentrations of precipitated protein from serum were higher in Se supplemented than in Se-deficient mice. Differences between the AG treatment groups were not detectable. (Mean ± SEM, n = 7).

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