Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg482, Gly482, and Thr482 variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg482]ABCG2 transports [3H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly482 and Thr482). Transport of methotrexate by [Arg482]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S-octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg482]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg482 is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

Surface-enhanced Raman scattering (SERS) is an attractive technique in molecular detection with high sensitivity and label-free characteristics. However, its use in protein detection is limited by the large volume of proteins, hindering its approach to the narrow spaces of hotspots. In this study, we fabricated a Au nanoTriangle plate Array on Gel (AuTAG) as an SERS substrate by attaching a Au nanoTriangle plate (AuNT) arrangement on a thermoresponsive hydrogel surface. The AuTAG acts as an actively tunable plasmonic device, on which the interparticle distance is altered by controlling temperature via changes in hydrogel volume. Further, we designed a Gel Filter Trapping (GFT) method as an active protein delivery strategy based on the characteristics of hydrogels, which can absorb water and separate biopolymers through their three-dimensional (3D) polymer networks. On the AuTAGs, fabricated with AuNTs modified with charged surface ligands to prevent the nonspecific adsorption of analytes to particles, the GFT method helped the delivery of proteins to hotspot areas on the AuNT arrangement. This combination of a AuTAG substrate and the GFT method enables ultrahigh sensitivity for protein detection by SERS up to a single-molecule level as well as a wide quantification concentration range of 6 orders due to their geometric advantages.