Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.
P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.
Ichikawa
M
M
Cancer Research Institute, Faculty of Medicine, Kagoshima University, Japan.
Yoshimura
A
A
Furukawa
T
T
Sumizawa
T
T
Nakazima
Y
Y
Akiyama
S
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000311:Adrenal Glands / Q000737:chemistry; D000345:Affinity Labels; D000581:Amidohydrolases / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism; D004351:Drug Resistance; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010777:Photochemistry; D011499:Protein Processing, Post-Translational; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D004095:Dihydropyridines; D008562:Membrane Glycoproteins; D014415:Tunicamycin; C041130:azidopine; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D005944:Glucosamine