Effect of processing inhibitors on cobalamin (vitamin B12) transcytosis in polarized opossum kidney cells

Abstract

The main objectives of the current study were to investigate the effect of tunicamycin and other posttranslational processing inhibitors on the apical brush border expression of intrinsic factor-cobalamin receptor (IFCR) and the apical to basolateral transcytosis of cobalamin (Cbl). Because of the high and selective expression of IFCR in the apical brush border membrane of opossum kidney (OK) cells (K. S. Ramanujam, S. Seetharam, N. Dahms, and B. Seetharam, (1991) J. Biol. Chem. 266, 13135-13140), we have used cultured OK cells to address these issues. When polarized OK cells grown on culture inserts were incubated with tunicamycin, deoxynojirimycin, swainsonine, or cerulenin, the surface binding of the ligand, intrinsic factor-[56Co]Cbl was inhibited by tunicamycin but not by the other inhibitors. However, Cbl transcytosis was inhibited by both tunicamycin and cerulenin but not with deoxynojirimycin or swainsonine. Incubation of cells with tunicamycin decreased the half-life of IFCR from 48 to 24 h, thus causing faster degradation and depletion of the surface receptor. Incubation of cells with cerulenin resulted in the intralysosomal retention of internalized Cbl. Mature receptor labeled with either [35S]methionine or [3H]mannose was sensitive to digestion with both endoglycosidase H and peptide N-glycosidase F and revealed the presence of two or three N-linked oligosaccharides of the high mannose or hybrid type. Metabolic labeling of OK cells with [3H]palmitic acid revealed that IFCR was palmitoylated and the label was sensitive to treatment with hydroxylamine. Based on these results we suggest that IFCR expression in the apical membrane and Cbl transcytosis in polarized OK cells are regulated by core N-glycosylation but not by further processing of the terminal sugars. In addition, we also suggest that the inhibition of Cbl transcytosis by cerulenin is due to inhibition of postinternalization events.