ABCMdb

PMID: 20700710

Wakabayashi-Nakao K, Tamura A, Koshiba S, Toyoda Y, Nakagawa H, Ishikawa T
Production of cells with targeted integration of gene variants of human ABC transporter for stable and regulated expression using the Flp recombinase system.
Methods Mol Biol. 2010;648:139-59., [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCG2 p.Gln141Lys ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Furthermore, certain non-synonymous single nucleotide polymorphisms (SNPs), such as Q141K, F208S, and S441N, were also found to greatly affect the stability of ABCG2 in the ER and to enhance the protein degradation rate via ubiquitination and proteasomal proteolysis in Flp-In-293 cells (22-27) (Fig. 1). Login to comment 226 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn It was of great interest to know how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization of the F208S and S441N variant proteins (see Notes 5 and 6). Login to comment 227 ABCG2 p.Ser441Asn Figure 6a depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 S441N that were incubated with or without 2 mM MG132 for 24 h. Login to comment 228 ABCG2 p.Ser441Asn In the case of the S441N 3.3.4. Login to comment 245 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Figure 3a depicts a schematic illustration of the human ABCG2 protein to indicate the sites of amino acid alteration in the SNP variants of F208S and S441N. Login to comment 247 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Notes F208S, and S441N were individually expressed in Flp-In-293 cells by using the Flp recombinase system. Login to comment 248 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn As shown in Fig. 3b, mRNA levels of ABCG2 WT as well as F208S and S441N were evenly represented in Flp-In-293 cells, where the mRNA levels of ABCG2 and GAPDH were measured by RT-PCR. Login to comment 249 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn On the other hand, ABCG2 WT, F208S, and S441N as well as GAPDH proteins were detected by immunoblotting, and their expression levels were quantified. Login to comment 253 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Schematic illustration of human ABCG2 as well as the expression of ABCG2 WT, F208S, and S441N in Flp-In-293 cells at the mRNA and protein levels. Login to comment 254 ABCG2 p.Phe208Ser ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Ser441Asn (a) Arrows indicate the positions of amino acid substitutions (Phe208Ser and Ser441Asn) in the non-synonymous SNP variants of F208S and S441N. Login to comment 259 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn (b) The mRNA level was analyzed by RT-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, F208S, or S441N. Login to comment 265 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Although mRNA levels were almost the same in the WT and SNP variants (F208S and S441N), protein levels of those variants were markedly decreased (Fig. 3d). Login to comment 268 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Figure 4a shows the effect of ME treatments on the SDS-PAGE migration of ABCG2 WT, F208S, and S441N proteins. Login to comment 270 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Immunoblot detection of ABCG2 WT, F208S, and S441N proteins expressed in Flp-In-293 cells. Login to comment 271 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn (a) Effect of mercaptoethanol (ME) on the status (homodimer or monomer) of ABCG2 WT and the SNP variants. Cell lysate samples (20 mg of protein) were subjected to SDS-PAGE after treatments with or without ME; and, thereafter, ABCG2 WT, F208S, and S441N proteins were detected by immunoblotting with the BXP-21 antibody, as described in Subheading 3.3.3. Login to comment 272 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn By ME treatments, ABCG2 WT, F208S, and S441N proteins were changed from homodimers to monomers. Login to comment 273 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn (b) Effect of Endo H or PNGase F treatments on the glycosylated status of ABCG2 WT and the SNP variants. Cell lysate samples (20 mg of protein) were treated with Endo H or PNGase F, as described in Subheading 3.3.3.ABCG2 WT, F208S, and S441N proteins in the resulting samples were analyzed by immunoblotting with the BXP-21 antibody.The apparent molecular weights of mature and non-glycosylated ABCG2 WT were 81,000 and 72,000, respectively. Login to comment 280 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn These results provide evidence that F208S and S441N proteins form homodimers through a cysteinyl disulfide bond as does the ABCG2 WT protein. Login to comment 284 ABCG2 p.Ser441Asn On the other hand, for the S441N variant, two protein bands of ABCG2 were detected around the molecular weight of 81,000. Login to comment 285 ABCG2 p.Phe208Ser In the case of the F208S variant, one faint band was detected at the apparent molecular weight of 74,000 by the same sample processing and immunoblotting experiment. Login to comment 289 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Although the major band (M.W. = 81,000) of ABCG2 WT was not at all affected by the Endo H treatment, the apparent molecular weights of the smaller protein bands of F208S (M.W. = 74,000) and S441N (M.W. = 78,000) decreased after the Endo H treatment. Login to comment 292 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn The matured N-linked oligosaccharide of ABCG2 WT may have a structure resistant to Endo H, whereas the immature N-linked oligosaccharides of the F208S and S441N variant proteins are susceptible to this endoglycosidase. Login to comment 295 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Flp-In-293 cells expressing F208S or S441N were incubated in the presence of MG132 at different concentrations (0, 0.4, 2.0 mM) for 24 h, and then cell lysate samples were immediately prepared. Login to comment 296 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Protein expression levels of the F208S and S441N variants were determined by immunoblotting after PNGase F treatments in the same way as described above. Login to comment 299 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn After the 24-h treatment with 2 mM MG132, F208S and S441N protein levels were increased 12-and 6-fold, respectively. Login to comment 301 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Effects of MG132 on the glycosylation status and protein levels of ABCG2 F208S and S441N variants expressed in Flp-In-293 cells. Login to comment 302 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn (a) Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4, 2 mM for 24 h. Login to comment 304 ABCG2 p.Phe208Ser ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Ser441Asn ABCG2 F208S and S441N variant proteins were analyzed by immunoblotting with the ABCG2-specific monoclonal antibody (BXP-21) either directly or after PNGase F treatment.The signal intensity of the non-glycosylated form of the ABCG2 F208S or S441N variant was measured after PNGase F treatment.The intensities are represented as ratios relative to the ABCG2 level in MG132-untreated cells. Login to comment 308 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn (b) Detection of aggregated forms (M.W. > 200,000) of ABCG2 F208S and S441N variants. Login to comment 309 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Flp-In-293/ABCG2 (F208S) and Flp-In-293/ ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4, 2 mM for 24 h. Login to comment 310 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Cell lysate samples (20 mg of protein) were prepared in the presence of ME.ABCG2 F208S and S441N variant proteins were prepared from MG132-treated cells as described above and analyzed by immunoblotting with the BXP-21 antibody without PNGase F treatment. Login to comment 311 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn The aggregated forms of ABCG2 F208S and S441N are indicated by arrowheads. Login to comment 315 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn non-glycosylated form (M.W. = 72,000) for both F208S and S441N variants, where those cell lysate samples were not treated with PNGase F (Fig. 5a). Login to comment 316 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn More importantly, upon the immunoblot analysis without PNGase F treatments, aggregated forms (indicated by arrowheads in Fig. 5b) of both F208S and S441N variants were detected in the high molecular weight range of over 200,000. Login to comment 318 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn It was of great interest to know how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization of the F208S and S441N variant proteins. Login to comment 319 ABCG2 p.Ser441Asn Figure 6a depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 S441N that were incubated with or without 2 mM MG132 for 24 h. Login to comment 320 ABCG2 p.Ser441Asn In the case of the S441N variant, MG132 treatments enhanced the localization of the ABCG2 variant protein at the plasma membrane and in intracellular compartments. By immunoelectron microscopy, we could detect ABCG2 aggresome formation adjacent to the nuclei when Flp-In-293 cells expressing the F208 variant were treated with 2 mM MG132 for 24 h (Fig. 6b). Login to comment 323 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Immunocytochemical staining of Flp-In-293 cells expressing ABCG2 S441N protein (a) and immunoelectron microscopic image of Flp-In-293 cells expressing ABCG2 F208S protein (b). Login to comment