Rapid Purification of Human P-glycoprotein Mutants Expressed Transiently in HEK 293 Cells by Nickel-Chelate Chromatography and Characterization of their Drug-stimulated ATPase Activities (∗)

A full-length MDR1 cDNA was modified to encode for 10 histidine residues the COOH end of the protein. The sequence at the COOH terminus of P-glycoprotein that would normally end as TKRQ now became TKRA(His)₁₀LDPR Q.

Procedures for stable transfection of mouse NIH 3T3 cells, followed by selection in the presence of vinblastine (5 nM) or colchicine (45 nM) have been described previously (Loo and Clarke, 1993a). For purification of P-glycoprotein, twenty (10-cm diameter) culture dishes of subconfluent HEK 293 cells were transfected with MDR1 cDNA, and membranes were prepared 40 h later as described previously (Loo and Clarke, 1994c). The membranes were suspended in 0.3 ml of buffer A (50 mM NaPO₄, pH 8.0, 500 mM NaCl, 50 mM imidazole, and 20% (v/v) glycerol) and solubilized at 4°C by addition of 1 ml of buffer A containing 1% (w/v) n-dodecyl-β-D-maltoside (Sigma). Insoluble material was removed by centrifugation at 16,000 × g for 15 min, and the supernatant was applied onto a nickel spin column (Ni-NTA, Qiagen), which had been pre-equilibrated with buffer B (buffer A containing 0.1% (w/v) n-dodecyl-β-D-maltoside). The column was washed twice with 0.6 ml of buffer B and twice with 0.6 ml of buffer C (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 80 mM imidazole, pH 7, 0.1% (w/v) n-dodecyl-β-D-maltoside, and 20% (v/v) glycerol). P-glycoprotein-(His)₁₀ was then eluted with 0.25 ml of buffer D (buffer C but containing 300 mM imidazole). The eluted material was diluted 6-fold with buffer A lacking imidazole and reapplied onto the same column that had been regenerated by washing with 1 M imidazole, 0.1% (w/v) n-dodecyl-β-D-maltoside followed by equilibration in buffer B. The column was washed as described above, and P-glycoprotein was eluted with 0.25 ml of buffer D. The yield of P-glycoprotein was determined by measuring the amount of protein as described by Bradford(1976) using bovine serum albumin as a standard and subtracting the amount of protein obtained when the same number of vector-transfected cells was carried through the purification procedure. Yields of purified P-glycoprotein ranged from 6 to 12 μg.

Purified P-glycoprotein was diluted with an equal volume of 50 mg/ml crude sheep brain phosphatidylethanolamine (Sigma), which had been previously washed with Tris-buffered saline to remove traces of phosphate and then sonicated. 100 ng of purified P-glycoprotein was incubated with the desired drug and ATPase activity initiated by addition of an equal volume of buffer containing 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl₂, and 10 mM ATP. The samples were incubated at 37°C for 40 min, and the amount of inorganic phosphate liberated was determined by the method of Chifflet et al.(1988).

The cDNA coding for P-glycoprotein was modified to contain 10 histidine residues at the COOH end of the molecule. The presence of the tag did not affect function since transfection of mouse NIH 3T3 cells yielded drug-resistant colonies that had drug-resistant profiles that were similar to those of wild-type enzyme (data not shown).

To purify P-glycoprotein, its cDNA was transiently expressed in HEK 293 cells as they yield a relatively high level of P-glycoprotein. More than 80% of the P-glycoprotein could be extracted from the membranes with the detergent n-dodecyl-β-D-maltoside. The detergent also did not interfere with the binding of P-glycoprotein to the nickel column (>90% of P-glycoprotein was bound to the resin). Ionic detergents or inclusion of phospholipids during the purification procedure prevented binding of P-glycoprotein to the column. Other non-ionic detergents (Triton X-100 or C₁₂E₈ (octaethylene glycol dodecyl ether)) inactivated P-glycoprotein, while octyl glucoside was less effective (<20%) in solubilizing P-glycoprotein. SDS-PAGE of the fractions eluted with 300 mM imidazole (Fig. 1) shows the presence of a single major band of an apparent mass of 170 kDa (lane 6B), which is not present from cells transfected with vector alone (lane 6A). When the purified fractions were subjected to immunoblot analysis with antibody against human P-glycoprotein, the 170-kDa protein was the only immunoreactive band (data not shown). After one round of purification, greater than 50% of the eluted proteins was P-glycoprotein-(His)₁₀. A second round of purification resulted in up to 80% of the protein being P-glycoprotein, with yields of 6-12 μg of P-glycoprotein. Occasionally a minor contaminating band of apparent mass 56 kDa was also present when a regenerated rather than a new nickel column was used during the second round of purification.

Fig. 2 shows that wild-type P-glycoprotein-(His)₁₀ in the presence of phosphatidylethanolamine had an ATPase activity of 0.11 μmol/min/mg of P-glycoprotein. In the absence of lipid, no ATPase activity was detected (<0.015 μmol/min/mg of P-glycoprotein). In the presence of verapamil and lipid, however, the ATPase activity increased about 11-fold (1.20 μmol/min/mg of P-glycoprotein). All ATPase activities were determined in the absence of EGTA and ouabain, as no Ca²⁺-ATPase or Na⁺-K⁺-ATPase activity was detected (assayed ± 2 mM EGTA or ± 2 mM ouabain, respectively). Lower levels of stimulation were observed in the presence of 800 μM colchicine (3.1-fold) and vinblastine (5.8-fold). Maximal stimulation of ATPase activity in the presence of vinblastine occurred at 20-50 μM, and the activity decreased at higher concentrations of vinblastine.

We have previously identified residues in P-glycoprotein, which alter its ability to confer resistance to various cytotoxic drugs in transfected cells (Loo and Clarke, 1993a, 1993b, 1994a, 1994b). For example, mutants G141V or G830V conferred increased resistance to colchicine (about 3-fold) relative to that of wild-type enzyme while mutant F335A conferred decreased resistance to vinblastine. By contrast, mutant F978A conferred decreased resistance to all drugs. In this study, we also included for comparison mutant G185V, which was recently shown by Rao(1995) to have increased verapamil- and colchicine-stimulated ATPase activities (2- and 3.3-fold, respectively). Therefore, to determine the effects of these drugs on the ATPase activities of these mutants, we purified and reconstituted each mutant P-glycoprotein into phospholipid and measured ATPase activity in the presence of various concentrations of vinblastine, colchicine, or verapamil. When analyzed by SDS-PAGE, the major protein in each purified preparation had an apparent mass of 170 kDa (corresponding to the fully glycosylated form of P-glycoprotein) and was present in similar amounts (Fig. 3). The maximal verapamil-stimulated ATPase activities of mutants G141V, G185V, and G830V were all slightly increased (1.4-1.7-fold) relative to that of wild-type enzyme (Fig. 2). The half-maximal stimulation of the ATPase activities of the glycine mutants was 9-16 μM, compared with 40 μM for the wild-type enzyme, suggesting that the mutants had increased affinity for verapamil. Vinblastine-stimulated ATPase activities of all three mutants, however, were similar to that of wild-type enzyme, whereas colchicine-stimulated ATPase activities were markedly increased (2.8-3.7-fold).

Mutant F978A, which confers little resistance to vinblastine, colchicine, doxorubicin, or actinomycin D in transfected cells, also showed little drug-stimulated ATPase activity, except at very high concentrations of verapamil (1.04 μmol/min/mg of P-glycoprotein at 800 μM verapamil). Similarly, no colchicine-stimulated ATPase activity could be detected. Vinblastine-stimulated ATPase activity, however, was detected at low concentrations of vinblastine (10-50 μM) but was 3-4-fold lower than that observed with wild-type enzyme. By contrast, mutation of Phe-335, which is found in an equivalent position to Phe-978, when homologous halves of P-glycoprotein are aligned resulted in a large increase in drug-stimulated ATPase activity. Maximal ATPase activities in the presence of verapamil, vinblastine, or colchicine were 4-9-fold greater than that observed with wild-type enzyme. The basal ATPase activity of mutant F335A was also about 3-fold higher (0.32 μmol/min/mg of P-glycoprotein) than that of wild-type enzyme (0.11 μmol/min/mg of P-glycoprotein). Therefore, the major effect of mutation of Phe-335 was to increase the overall ATPase activity of the enzyme.

P-glycoprotein is predicted to contain two nucleotide binding folds. Each fold contains a homology A consensus sequence (Walker et al., 1982) for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively). We initially tested the ATPase activity of a mutant in which both cysteines in the homology A regions (Cys-431 and Cys-1074) as well as all other cysteines were mutated to alanines. The purified Cys-less P-glycoprotein had a higher amount of the core-glycosylated form (150 kDa) of P-glycoprotein (Fig. 3), which is probably due to the slow maturation of this mutant protein (Loo and Clarke, 1995). Fig. 4 shows that the Cys-less mutant retained approximately 70% of the verapamil-stimulated ATPase activity of wild-type enzyme. To determine the contribution of either nucleotide-binding domain to drug-stimulatable ATPase activity, mutations were made to the core amino acids (G432S, K433M, G1075S, and K1076M, respectively) of the homology A consensus sequences. No drug-resistant colonies were obtained when the cDNAs coding for these mutants were transfected into NIH 3T3 cells and selected in the presence of colchicine (45 nM) or vinblastine (5 nM). These mutants were then transiently expressed in HEK 293 cells and purified using Ni-NTA columns. All four mutants yielded a 170-kDa protein as the major product and in similar amounts to that obtained with wild-type P-glycoprotein-(His)₁₀, suggesting that the mutations did not affect processing of the enzyme (Fig. 3). Mutations to the core amino acids (GK) in either nucleotide-binding domain abolished verapamil-stimulated ATPase activity (Fig. 4), suggesting that both nucleotide-binding domains are essential for coupling drug binding to ATPase activity.