Effect of disulfide bonds of transcobalamin II receptor on its activity and basolateral targeting in human intestinal epithelial Caco-2 cells.
Transcobalamin II-receptor (TC II-R) contains 10 half-cysteines, of which 8 are involved in intramolecular disulfide bonding. Reduction followed by alkylation with N-ethylmaleimide (NEM) of the 62-kDa TC II-R monomer in vitro or treatment of human intestinal epithelial Caco-2 cells with low concentrations (10(-6) M) of NEM resulted in TC II-R exhibiting a loss of ligand binding and an increase in its apparent molecular mass by 10 kDa to 72 kDa. Domain-specific biotinylation studies using NEM-treated filter-grown cells revealed loss of TC II-R but not cation-independent mannose 6-phosphate receptor protein at the basolateral cell surface. Pulse-chase labeling of NEM-treated cells with [35S]methionine revealed that the modified 72-kDa TC II-R, like the native 62-kDa TC II-R in untreated cells, turned over rapidly with a t1/2 of 7.5 h and was sensitive to treatment with peptide N-glycosidase F, sialidase alone, or sialidase and O-glycanase but not to treatment with endoglycosidase H. Labeled 72-kDa TC II-R, which was retained intracellularly following treatment of Caco-2 cells with methyl methanethiosulfonate, returned to the basolateral cell surface following withdrawal of cells from methyl methanethiosulfonate treatment and exposure to dithiothreitol. Based on these results, we suggest that formation and maintenance of intramolecular disulfide bonds of TC II-R is important for its acquisition of ligand binding and post-trans-Golgi trafficking to basolateral surface membranes but not for its turnover and exit from the endoplasmic reticulum or trafficking through the Golgi.
Bose
S
S
Department of Biochemistry, Medical College of Wisconsin and Veterans Administration Medical Center, Milwaukee, Wisconsin 53226, USA.
Seetharam
B
B
D018938:Caco-2 Cells; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D014155:Transcobalamins / Q000378:metabolism
D004220:Disulfides; D011956:Receptors, Cell Surface; D014155:Transcobalamins; C028176:transcobalamin receptor; D005033:Ethylmaleimide