Development of a Sel-tag for Multimodality Imaging and Studies of Mammalian Thioredoxin Reductase 1

Abstract: Selenocysteine (Sec; U in one-letter code), the 21st amino acid existing in all selenoproteins, has unique biochemical properties due to its selenium atom, including a low pKa and a high reactivity with many electrophilic agents. When a selenoprotein is translated, the insertion of Sec occurs at a UGA codon, normally yielding translational termination. Here UGA can be recoded as Sec through the presence of a species-specific downstream mRNA sequence called Sec insertion sequence (SECIS) element. As one of about 25 mammalian selenoproteins, thioredoxin reductase 1 (TrxR1) contains one Sec residue within its C-terminal tetrapeptide motif, -Gly-Cys- Sec-Gly-COOH. By utilizing an engineered bacterial SECIS element in the rat TrxR1 cDNA, we are able to produce recombinant rat TrxR1 with both high yields and specific activities, which gives us an international leading edge, as selenoprotein production is a common bottleneck for researchers in the field of selenoprotein biology. Based on the studies on TrxR1, we have developed a Sel-tag for recombinant proteins using the C-terminal tetrapeptide motif of TrxR1. We have subsequently demonstrated that the selenolate of a reduced Sel-tag provides a handle which can be utilized for a wide range of selenolate-targeted applications, including a one-step protein purification procedure, residue-specific fluorescent labeling and radiolabeling with either gamma emitters (75Se) or positron emitting radionuclides (11C). We first utilized Sel-tag to investigate the mechanism of antigen presentation through carbohydrate based particles (CBP) by tracking the selenium-75 labeled Sel-tagged Fel d 1, the major cat allergen, in a mouse model. We further applied this technique for studies of apoptosis with a particular interest in utilizing Sel-tagged annexin A5 to accomplish multimodality imaging. We demonstrate that Seltagged annexin A5 labeled either with fluorescent 5-iodoacetamidofluorescein (5-IAF) or the positron emitter carbon11 remains functional and specifically binds apoptotic cells. This proved to be useful for (i) utilizing fluorescently labeled annexin A5 in either the conventional annexin A5 affinity assay with flow cytometry or in fluorescent microscopy of cells undergoing cell death, (ii) PET imaging of apoptotic liver of health BALB/c mice treated with anti-FAS antibody, and (iii) PET imaging of xenograft tumors developed from FaDu cells in SCID mice. We have solved the crystal structure of wild type rat TrxR1 and probed its catalytic mechanism. We have shown that the oxidized protein presents a stabilized selenenylsulfide motif in cis-configuration, with the Sec residue coordinated to the planar surface of a tyrosine side chain (Tyr116), thus located far from the redox active moieties of the enzyme proposed to be involved in electron transport to the C-terminal motif during catalysis. We believe that Tyr116 plays an important role for catalysis of TrxR1, possibly by involvement in electron transfer during the reductive half reaction, or by stabilizing the selenenylsulfide configuration of oxidized TrxR1. It has been shown that TrxR1 has pro-oxidant roles especially upon redox cycling with certain low molecular weight substrates. Thus we have investigated the potential of ROS generation by TrxR1 using wild type enzyme as well as several TrxR1 mutants. Utilizing Electron Spin Resonance (ESR) spin trapping with 5-Diethoxyphos-phoryl-5-methyl-1pyrroline-N-oxide (DEPMPO), we found that TrxR1 could generate free radicals including both hydroxyl radicals (HO.) and superoxide (O2.-) upon NADPH reduction in the absence of other substrates, and the HO. generation was largely dependent upon prior enzyme-catalyzed O2.-generation, indicating an inherent peroxidase activity of TrxR1. Closer analyses revealed that the the DEPMPO/HOO. adduct was a direct substrate of TrxR that could be reduced to DEPMPO/HO. in a Sec-dependent manner.