N-glycosylation of erythropoietin is critical for apical secretion by Madin-Darby canine kidney cells.
Erythropoietin (Epo) has three N-linked carbohydrate chains at positions 24, 38, and 83 in its 166-amino acid residues. When the human wild-type Epo was expressed in the polarized Madin-Darby canine kidney (MDCK) epithelial cells, Epo was preferentially secreted from the apical domain. The polarized secretion was perturbed by the treatment of the cells with tunicamycin, suggesting the involvement of N-linked carbohydrate chains in the apical sorting mechanism in MDCK cells. Replacement of asparagine residues at all N-glycosylation sites of Epo with glutamine by site-directed mutagenesis resulted in roughly equal secretion from apical and basolateral domains. Comparative studies on MDCK clones expressing the mutant Epos lacking one or two of the three N-glycosylation sites in every possible combination showed that the N-linked carbohydrate chain at position 38 is critical for the polarized secretion. Nocodazole, a microtubule-disrupting drug, reversed the polarized secretion of the wild-type Epo from the apical to basolateral preference with little change in the total secretion. Hepatocyte growth factor, a scatter factor known to induce the tubule-like structure of MDCK cells, caused almost equal secretion of the wild-type Epo into the apical and basolateral sides, although the tight junctions of MDCK cells remained intact.
Kitagawa
Y
Y
Laboratory of Organogenesis, Nagoya University Biosciences Center, Japan.
Sano
Y
Y
Ueda
M
M
Higashio
K
K
Narita
H
H
Okano
M
M
Matsumoto
S
S
Sasaki
R
R
D000818:Animals; D002460:Cell Line; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D004921:Erythropoietin; D017228:Hepatocyte Growth Factor