Related Experiment Videos
Bipolar functional expression of transcobalamin II receptor in human intestinal epithelial Caco-2 cells
S Bose1, S Seetharam, N M Dahms
1Department of Biochemistry, Medical College of Wisconsin and Veterans Administration Medical Center, Milwaukee, Wisconsin 53226, USA.
Insights
Transcobalamin II (TC II)-cobalamin (Cbl) is transported differently across intestinal cells. Apical uptake allows TC II-Cbl transcytosis, while basolateral uptake degrades TC II, releasing Cbl.
Area of Science:
- Cell Biology
- Gastroenterology
- Nutritional Science
Background:
- The Transcobalamin II (TC II) receptor is present in human intestinal membranes.
- Caco-2 cells exhibit a higher concentration of TC II receptors on basolateral membranes.
Purpose of the Study:
- To investigate the distinct processing and transport pathways of TC II-cobalamin (Cbl) at the apical and basolateral surfaces of intestinal epithelial cells.
- To elucidate the role of lysosomal and non-lysosomal pathways in TC II-Cbl intestinal absorption.
Main Methods:
- Utilized human intestinal epithelial Caco-2 cells grown on culture inserts.
- Quantified binding and internalization of TC II-[57Co]cobalamin (Cbl) and 125I-TC II at apical and basolateral surfaces.
- Assessed the effects of chloroquine and leupeptin on TC II-Cbl processing and transcytosis.
- Administered 125I-TC II-Cbl orally to rats and detected labeled TC II in portal blood.
Main Results:
- Apical uptake of TC II-Cbl led to internalization and subsequent transcytosis of the intact complex.
- Basolateral uptake resulted in internalization, TC II degradation, and Cbl transfer to other cellular proteins.
- Lysosomal inhibitors (chloroquine, leupeptin) blocked TC II degradation after basolateral internalization, promoting TC II-Cbl secretion.
- Intact TC II was detected in rat portal blood after oral administration, suggesting luminal to systemic transport.
Conclusions:
- TC II-Cbl utilizes a non-lysosomal pathway for apical/luminal transcytosis, transporting both TC II and Cbl intact.
- Basolateral processing involves lysosomal degradation of TC II, with subsequent utilization of released Cbl.
- These distinct pathways highlight differential handling of vitamin B12 uptake in the intestine.
Abstract:
Transcobalamin II (TC II) receptor is expressed in the apical and basolateral membranes of human intestinal mucosa and in post-confluent human intestinal epithelial Caco-2 cells with a 6-7-fold enrichment in basolateral membranes. Caco-2 cells grown on culture inserts bound (at 5 degrees C) 30 and 180 fmol of the ligand, TC II-[57Co]cobalamin (Cbl), to the apical and the basolateral surfaces, respectively. Within 5 h at 37 degrees C, all apically bound Cbl was internalized and subsequently transcytosed as TC II-Cbl. In contrast, all basolateral surface-bound Cbl was internalized and retained by the cells, but transferred from TC II to other cellular proteins. Chloroquine or leupeptin had no effect on the apical to basolateral transcytosis of either [57Co]Cbl or 125I-TC II. In contrast, following basolateral internalization of the ligand, both chloroquine and leupeptin inhibited the intracellular degradation of 125I-TC II, which resulted in secretion of 60-65% of TC II-Cbl complex into the basolateral medium. When 125I-TC II-Cbl was orally administered to rats, intact labeled TC II was detected in the portal blood 4 and 8 h later. These studies suggest that TC II-Cbl is processed when presented to the (a) apical/luminal side by a hitherto unrecognized non-lysosomal pathway in which both TC II and Cbl are transcytosed and (b) basolateral side by the lysosomal pathway in which TC II is degraded and the released Cbl is utilized.