Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

Abstract

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

Figure 1.

Selenocysteine incorporation and DMNB-Sec structure. (a) Endogenous eukaryotic Sec-incorporation mechanism. (b) Structures of DMNB-Sec and Sec.

Figure 2.

Generation of eGFP-DMNB-Sec39. (a) Fluorescence microscopy and quantification of HEK293T cells transiently transfected with a pAcBac2 plasmid encoding LeuRS BH5 T252A /Leu tRNA/eGFP-39-TAG, in the presence and absence of DMNB-Sec. (b) SDS-PAGE of eGFP-WT and LeuRS BH5 T252A /Leu tRNA/eGFP-39-TAG expressed in the presence and absence of DMNB-Sec (c) ESI-MS analysis of eGFP-DMNB-Sec39 (expected mass: 29865.33) and irradiated eGFP-Sec39 (expected mass: 29669.24)

Figure 3.

Generation of MsrB1-DMNB-Sec95. (a) Immunoblot of MsrB1 expression with a Cys or TAG amber codon at residue 95 in the presence and absence of DMNB-Sec. (b) ESI-MS analysis of MsrB1-Cys95 (expected mass: 13309.97), MsrB1-DMNB-Sec95 (expected mass: 13552.04), and uncaged MsrB1-Sec95 (expected mass: 13356.87). (c) Low pH isoTOP-ABPP analysis showing extracted ion chromatograms for Cys- or Sec-containing peptides from irradiated (red, IA-light) or non-irradiated (blue, IA-heavy) cells expressing MsrB1-DMNB-Sec95. (d) Immunofluorescence of cells transfected with MsrB1-TAG95 in the presence or absence of DMNB-Sec, before and after irradiation. Scale bar represents 10 μm. (e) Methionine sulfoxide reductase activity of buffer alone (negative control), purified uncaged MsrB1-Sec95, and MsrB2 (positive control), measured as the percent reduced dabsyl-Met (dabsyl-MetR) present within the total amount of dabsyl-Met (oxidized and reduced). Immunoblot shows that levels of purified MsrB1-Sec95 is comparable to the amount of MsrB2 used in the activity assay.