G protein-coupled receptor mediated trimethylamine sensing
Introduction
Trimethylamine (TMA) is a volatile low molecular weight tertiary aliphatic amine that has been identified widely in many animal and plant tissues and, together with other amines, is a degradation product of nitrogenous organic material. Noticeably, TMA measurements in seafood can be used as an indicator of freshness (Mitsubayashi et al., 2004). TMA is produced by metabolism of the precursor trimethylamine N-oxide (TMAO) by microorganisms and its concentration rapidly increases in marine products during putrefaction. Industrial use of TMA includes e.g. application both as a catalyst and an intermediate in chemical industry, especially for the manufacture of quaternary ammonium compounds. There is a considerable risk for human exposure from industrial sources.
In humans, TMA toxicity includes headache and nausea, severe skin burns, as well as irritation to the eyes and the respiratory tract (ACGIH, 1991). Moreover, animal studies have demonstrated effects on nervous function (Anthoni et al., 1991). Such observations have led to the establishment of exposure limits and a maximum allowable concentration of the gas in the air of industrial plants. The National Institute for Occupational Safety and Health (NIOSH) has established a recommended exposure limit (REL) for trimethylamine of 10 ppm (24 mg/m3) as a time weighted average concentration (TWA) for up to a 10-h workday and a 40-h workweek and 15 ppm (36 mg/m3) as a 15-min TWA short-term exposure limit (STEL, NIOSH, 1992).
Methods to control atmospheric TMA levels traditionally require TMA sampling/collection, subsequent microextraction, and gas or liquid chromatography for analysis (Cháfer-Pericás et al., 2006, Chien et al., 2000). These methods are rarely field-applicable or simple for routine analysis. In recent literature, efforts creating simpler TMA vapor sensors were presented. These TMA sensors are based on piezoelectric crystals (Li et al., 2007), semiconducting metal-oxide (Kwon et al., 1998), chemiluminescence (Zhang et al., 2005), and flavin containing monooxygenase (Mitsubayashi and Hashimoto, 2002). Two microbial sensors, based on the mould Penicillium decumbens and the bacteria Pseudomonas aminovorans were also presented (Gamati et al., 1991, Li et al., 1994). In the effort of developing a simple, low cost, and more sensitive sensor with high selectivity, we were inspired by our own TMA detection system, the sense of smell. TMA has a pungent ammonia-like odor, and since the sense of smell depends on available sensory receptors that respond to airborne chemicals, odor receptors sensitive to TMA have to exist.
Recently, it was demonstrated that trace amine-associated receptors (TAARs) function as chemosensory receptors in the olfactory epithelium in mice and are activated by several volatile amines (Liberles and Buck, 2006). TMA was shown to specifically activate TAAR5 from mice (mTAAR5). Variants of the TAAR proteins are present in vertebrates and belong to the superfamily of G protein-coupled receptors (GPCRs), which are seven transmembrane proteins (Berghard and Dryer, 1998, Hashiguchi and Nishida, 2007). GPCRs are challenging to target for biosensing applications, because they are often very difficult to purify from cells in their native conformation and are usually unstable in solution. Using whole cell-based systems is up to date the best option, but sufficient levels of cell membrane localized expression required for functional assays is far from trivial to obtain, which can be exemplified by difficulties in achieving functional odorant receptor expression (Zhuang and Matsunami, 2007 and references therein).
Xenopus laevis melanophores constitute a very convenient expression system for GPCRs and their potential for cell-based sensing has been demonstrated (Karlsson et al., 2002, Suska et al., 2005). In these specialized cells, GPCR activation leads to the dispersion or aggregation of intracellular pigment granules, i.e. melanin filled vesicles termed melanosomes. Both states (dispersion or aggregation) of intracellular melanosome distribution are easily detectable by either measuring the change in light transmittance through the cells or by imaging the cells as they are responding (Potenza and Lerner, 1992, McClintock et al., 1993).
In the present investigation, we report on the functional expression of mTAAR5 in Xenopus melanophores, and its application for TMA detection. The performance of this sensor in detection of TMA in solution as well as in air was evaluated and a focused chemical screen specified the sensors selectivity.
Section snippets
Cell culture and reagents
Fibroblasts and melanophores from X. laevis were propagated in cell culture as previously described (McClintock and Lerner, 1997). The melanophore and fibroblast cell lines were kind gifts from Dr. Michael R. Lerner, Arena Pharmaceuticals, Inc. (San Diego, CA, USA). Cell culture reagents were from Life Technologies (Carlsbad, CA, USA), and cell culture plastics were from Sarstedt (Nümbrecht, Germany). All chemical compounds, including trimethylamine (25% in water) were from Sigma (St. Louis,
Evaluation of the mTAAR5 based sensor
We created a X. laevis melanophore-based sensor for TMA, by genetically engineering melanophores to express mTAAR5, a GPCR previously shown to mediate sensitivity to TMA. In order to achieve this, cells were transfected with an expression vector with the mTAAR5 coding sequence and the hph gene, allowing the selection of a stable mTAAR5 expressing cell line using hygromycin B. In Xenopus melanophores, GPCR activation results in the collective translocation of melanosomes along the microtubules
Conclusions
We have demonstrated a novel cell-based sensor for the determination of TMA in liquid and air. The sensor is based on mTAAR5 expressing Xenopus melanophores, and shows high sensitivity and specificity to a very limited number of tertiary amines, including TMA. As the system does not show sensitivity to the TMA precursor TMAO, it offers the possible application for fast and convenient determination of TMA in fish containing products. Moreover, a platform for the detection of gaseous substances
Acknowledgements
This research has been financially supported by GOSPEL, the European Network of Excellence in Artificial Olfaction, the Swedish Research Council FORMAS (to A.B.) and Carl Trygger Foundation (to A.B.), whom the authors gratefully acknowledge. Dr. V. Vedin is gratefully acknowledged for sharing expertise in expression vector construction.
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