Effect of Disulfide Bonds of Transcobalamin II Receptor on Its Activity and Basolateral Targeting in Human Intestinal Epithelial Caco-2 Cells

Abstract

Transcobalamin II-receptor (TC II-R)1 is a single chain glycoprotein with a molecular mass of 62 kDa (1) containing about 27% carbohydrate. It promotes plasma transport of cobalamin (Cbl; vitamin B 12 ) bound to transcobalamin II (TC II) (2). TC II-R functions as a noncovalent homodimer with a molecular mass of 124 kDa in tissue membranes (1), and its dimerization occurs not in the ER, where it is a monomer, but rather in the plasma membrane (3). In addition, the dimerization of TC II-R is a membrane fluidity-driven event requiring a highly ordered rigid bilayer (4). In polarized epithelial Caco-2 cells, TC II-R is predominantly present in basolateral membranes (5), where it functions in the delivery of Cbl to be utilized as coenzymes (6). Treatment of the basolateral surface of Caco-2 cells with TC II-R antiserum in vitro or circulating TC II-R antibody in vivo results in a failure of TC II-Cbl uptake, causing intracellular Cbl deficiency (6). These studies have suggested that plasma membrane delivery of TC II-R is extremely important for cells to obtain their Cbl from the circulation to maintain their normal metabolic, differentiation, and proliferation status. Despite the importance of plasma membrane expression of TC II-R in the tissue transport of Cbl, no information is available regarding its intracellular trafficking and plasma membrane expression.Recent studies have shown that formation of intracellular inter-or intrachain disulfide bonds and their final rearrangement by thiol-disulfide exchange reaction is one of the co/posttranslational modifications of some proteins (7,8). The correct formation of intrachain and, in the case of oligomeric proteins, interchain disulfide bonds is crucial for the proper assembly of secretory and plasma membrane proteins, which in turn determines their stability, intracellular transport, maturation, and function (9 -11). Inhibition of disulfide bond formation by DTT treatment has been shown to affect secretion of albumin and H1 subunit of asialoglycoprotein receptor from fibroblasts (12) and of gp80 (clusterin, apolipoprotein J) in polarized MadinDarby canine kidney cells (13). However, the secretion of nondisulfide bond-containing proteins such as ␣ 1 -antitrypsin (14) in HepG2 cells or of a 20-kDa peptide derived from osteoponin in Madin-Darby canine kidney cells (13) is not affected. These studies have proposed that the retention of proteins in the ER following treatment with DTT is due to a failure of these proteins to form disulfide bonds, which then fold improperly. As a consequence, these misfolded proteins fail to achieve a conformation essential for their function as well as their exit from the ER for further processing and targeting (15).Human TC II-R contains 10 half-cysteines (16), and recent immunoblot studies of placental plasma membrane proteins under nonreducing and reducing conditions (1) have shown that TC II-R dimers contain intramolecular disulfide bonds that, upon reduction, increase the apparent molecular mass of the dimer by 20 kDa. Taken together, ...

The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin

“…13 They identified a 72 kDa/144 kDa monomer/dimer as the receptor protein 14 and followed with a number of publications describing the complex physiochemical properties of this receptor. [19][20][21][22][23][24][25] The lack of progress in identifying the gene encoding the receptor for nearly 10 years after their first report claiming purification of substantial quantities of the receptor protein motivated us to reevaluate our original data and attempt to purify the protein. Extensive modifications to the solubilization, affinity chromatography, and elution procedures finally yielded a pure functional protein for amino acid sequence analysis and the gene encoding this sequence was identified.…”

“…Since their first report, numerous publications by this group have described the structural and functional characterization of a putative receptor from human placenta. [19][20][21][22][23][24][25] However, they did not establish the functional specificity of their receptor for TC-Cbl and have not identified the primary structure and the gene encoding the receptor. This report describes the purification and definitive identification of the primary structure and the gene encoding a receptor for the cellular uptake of TC-Cbl.…”

Brefeldin A (BFA) Inhibits Basolateral Membrane (BLM) Delivery and Dimerization of Transcobalamin II Receptor in Human Intestinal Epithelial Caco-2 Cells

“…Cell Surface Biotinylation-Biotinylation of BLM of Caco-2 cells recovering from the effects of BFA was carried out by adding disuccinimidyl suberate sulfosuccinimido biotin (0.5 mg/ml) to the basolateral compartments of filter-grown monolayers (12-day growth) and was performed a total of three times for 30 min each essentially as described recently (6,18).…”

“…Pulse-Chase Labeling of Caco-2 Cells-Postconfluent cells untreated and treated with BFA (5 g/ml) for 12 h were first incubated with methionine-free DMEM for 30 min and then pulsed for 1 h with S-labeled TC II-R isolated at each time interval by immunoprecipitation was further processed for nonreducing SDS-PAGE as described before (18).…”

“…The radioactive counts were liberated by boiling with SDS (1%), and the liberated radioactivity was subjected to acetone precipitation to remove SDS. The precipitated radioactivity was further processed for treatment with various glycosidases essentially as described earlier (18).…”