N-glycosylation and deletion mutants of the human MDR1 P-glycoprotein.
P-glycoproteins are heavily glycosylated plasma membrane proteins, which confer multidrug resistance by pumping a range of different drugs from the cell. To investigate the significance of the conserved N-glycosylation sites present in the putative first extracellular loop of P-glycoproteins, we mutated one, two, or all three of these sites present in the human MDR1 P-glycoprotein. We also deleted a stretch of 20 amino acids, containing two of the three N-glycosylation sites. The effects of these mutations were studied by transfection into drug-sensitive cells. In vincristine-resistant transfected clones selected for similar steady state levels of membrane-bound P-glycoprotein, the absence of N-glycosylation did not alter the level or pattern of (cross-)resistance. However, the absence of N-glycosylation sites drastically reduced the efficiency with which drug-resistant clones could be generated. These findings suggest that N-glycosylation contributes to proper routing or stability of P-glycoprotein but not to drug transport per se. The deletion mutants demonstrated a clearly decreased and altered drug resistance pattern, even with a high level of P-glycoprotein in the plasma membrane. This, and possibly the observed lack of glycosylation of the remaining intact glycosylation sequence, suggests a constrained P-glycoprotein structure. Our findings support the current model for P-glycoprotein structure.
Schinkel
A H
AH
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
Kemp
S
S
Dollé
M
M
Rudenko
G
G
Wagenaar
E
E
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D004351:Drug Resistance / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D008545:Melanoma; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D004247:DNA