N-GLYCOSYLATION OF ERYTHROPOIETIN IS CRITICAL FOR APICAL SECRETION BYMADIN-DARBY CANINE KIDNEY-CELLS

Erythropoietin (Epo) has three N-linked carbohydrate chains at positio ns 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 (MDC K) epithelial cells, Epo was preferentially secreted from the apical d omain. The polarized secretion was perturbed by the treatment of the c ells with tunicamycin, suggesting the involvement of N-linked carbohyd rate chains in the apical sorting mechanism in MDCK cells. Replacement of asparagine residues at all N-glycosylation sites of Epo with gluta mine by site-directed mutagenesis resulted in roughly equal secretion from apical and basolateral domains. Comparative studies on MDCK clone s expressing the mutant Epos lacking one or two of the three N-glycosy lation sites in every possible combination showed that the N-linked ca rbohydrate chain at position 38 is critical for the polarized secretio n. Nocodazole, a microtubule-disrupting drug, reversed the polarized s ecretion of the wildtype 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 cel ls, caused almost equal secretion of the wild-type Epo into the apical and basolateral sides, although the tight junctions of MDCK cells rem ained intact. (C) 1994 Academic Press, Inc.