TY - JOUR

T1 - Kinetic analysis of transcellular passage of the cobalamin-transcobalamin complex in Caco-2 monolayers

AU - Juul, Christian Bredgaard

AU - Fedosov, Sergey

AU - Nexø, Ebba

AU - Heegaard, Christian Würtz

PY - 2019/2/15

Y1 - 2019/2/15

N2 - We suggest a novel kinetic approach to quantifying receptor–ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [ 57 Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC–Cbl (K d = 13 nM) and RAP (K d = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.

AB - We suggest a novel kinetic approach to quantifying receptor–ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [ 57 Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC–Cbl (K d = 13 nM) and RAP (K d = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.

KW - Animals

KW - Biological Transport

KW - Caco-2 Cells

KW - Cattle

KW - Cobalt Radioisotopes

KW - Humans

KW - Kinetics

KW - Ligands

KW - Receptors, Cell Surface/metabolism

KW - Transcobalamins/metabolism

KW - Vitamin B 12/metabolism

U2 - 10.1091/mbc.E18-09-0571

DO - 10.1091/mbc.E18-09-0571

M3 - Journal article

C2 - 30565973

VL - 30

SP - 467

EP - 477

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 4

ER -