Abstract
The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.
Highlights
The predicted cytoplasmic orientation and two-do- tive sites and its global structure is not destroyed by main structure of the multidrug effluxpump P-glyco- tryptic cleavage. protein were demonstrated with sequence-specific antibodies
Zdentification and Cytoplasmic Topologyof P-glycoprotein with Anti-synthetic Peptide Antibodies-To detect P-glycoprotein in multidrug-resistant cells, we produced antibodies against synthetic peptides corresponding to the amino acid sequences of human P-glycoprotein, G l ~ ~ ' ~ - L(aynsti~-P~)
Each duplicate segment includes a consensus nucleotide-binding site (NB-I andNB-2 in Fig. 1).From the primary structure of P-glycoprotein, anti-C antibody might recognize another corresponding position, L e ~ ~ ~ ' - T(hanrt~i-C~ 'lin Fig. 1) in the first half, since 18 out of 21 amino acids of this region were identical to thecorresponding region of the second half
Summary
Zdentification and Cytoplasmic Topologyof P-glycoprotein with Anti-synthetic Peptide Antibodies-To detect P-glycoprotein in multidrug-resistant cells, we produced antibodies against synthetic peptides corresponding to the amino acid sequences of human P-glycoprotein, G l ~ ~ ' ~ - L(aynsti~-P~). Leu'2ffi-Thr'226(anti-C), deduced from humanmdrl cDNA sequence [8].These regions are near the two putative nucleotide binding sites and may be in the intracellular domain of P-glycoprotein (Fig. l). Each duplicate segment includes a consensus nucleotide-binding site (NB-I andNB-2 in Fig. 1).From the primary structure of P-glycoprotein, anti-C antibody might recognize another corresponding position, L e ~ ~ ~ ' - T(hanrt~i-C~ 'lin Fig. 1) in the first half, since 18 out of 21 amino acids of this region were identical to thecorresponding region of the second half. Anti-P will probably recognize only one site, OUT xsugars. 1226 (anti-C)are the sites for synthetic peptides, and 561-581 (antic’) was the correspondingsequence of the first half to anti-C recognition site (1206-1226) of the second half
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