Bipolar Functional Expression of Transcobalamin II Receptor in Human Intestinal Epithelial Caco-2 Cells

Abstract

The plasma transport of cobalamin (Cbl 1 ; vitamin B 12 ) to all tissues/cells occurs bound to a plasma transporter, transcobalamin II (TC II), by receptor-mediated endocytosis (1) via transcobalamin II receptor (TC II-R). Recent studies (2) have shown that TC II-R is expressed as a non-covalent homodimer of molecular mass of 124 kDa in all human (2), rat (3), and rabbit (4) tissue plasma membranes. The plasma membrane expression of TC II-R is important for the tissue/cellular uptake of Cbl, since its functional inactivation in vivo by its circulatory antiserum causes intracellular deficiency of Cbl, which in turn results in the development of Cbl deficiency of the animal as a whole (4). Although TC II-R is expressed in the plasma membrane of all cells, its polarity of expression in epithelial cells is not known. Recent immunoblot studies (3) have revealed that in the rat kidney, TC II-R protein is expressed in both the isolated apical and basolateral membranes with an enrichment in the basolateral membranes by about 8-fold. However, how TC II-Cbl internalized via the TC II-R from either the apical or basolateral surface is processed is not known. Recent TC II-[ 57 Co]Cbl uptake studies (4) using filter-grown polarized Caco-2 cells have shown that [ 57 Co]Cbl taken up from the basolateral side in these cells was utilized as Cbl coenzymes, suggesting that these cells derive Cbl essential for their use from the basolateral side.Despite these studies, the details of intracellular sorting of Cbl and TC II by a polarized epithelial cell are poorly understood. The present studies were undertaken to address the issues related to polarized expression and function of TC II-R in human intestinally derived Caco-2 cells, a well established cell model used extensively to study nutrient transport and general intestinal epithelial cell biology (5, 6).The results of this study show that TC II-R is asymmetrically expressed in the ratio of 1:7 and 1:6.8 in the apical and the basolateral membranes of human intestinal mucosa and intestinally derived Caco-2 cells, respectively. Furthermore, when TC II-Cbl is presented on the apical side of Caco-2 cells or to the intestinal lumen of rats, TC II is transcytosed across the epithelial cell by a non-lysosomal pathway. In addition, these studies also demonstrate that when presented on the basolateral side, TC II is processed via the lysosomal pathway, resulting in the degradation of TC II and the utilization of intracellularly released Cbl as coenzymes.

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.…”

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

“…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).…”

Efficient Peroral Delivery of Insulin via Vitamin B12 Modified Trimethyl Chitosan Nanoparticles

“…Most studys use Caco-2 cell model to compare modified and unmodified NPs. Human intestinal Caco-2 cell line, originally obtained from a human colon adenocarcinoma, is the most widely used cell model of intestinal barrier (28)(29)(30)(31). It is reported that caco-2 cells express several morphological and functional characteristics of the mature enterocytes.…”