Uptake of transcobalamin II-bound cobalamin by isolated rat kidney tubule cells

Abstract

The uptake and intracellular processing of transcobalamin II-bound cobalamin by isolated rat kidney tubule cells were studied. The cells absorbed the complex in a temperature-and calcium-dependent process, which could be inhibited by monensin, an inhibitor of endocytosis. Cells, loaded with a mixture of 125I- and 57Co-labelled transcobalamin II-vitamin B12, released 125I-labelled protein-degradation products, while keeping the 57Co-labelled vitamin. Protein degradation was inhibited by chloroquine and monensin, which is further evidence for a process of endocytosis, followed by intralysosomal hydrolysis of the transport protein. Transcobalamin II-vitamin B12 uptake was not fully saturable and other proteins, for example, haemoglobin, inhibited the uptake in a concentration-dependent way. Apparently the uptake proceeds through relatively unspecific protein-binding sites, probably involved in the reabsorption of filtrated proteins, although the affinity for transcobalamin II seems relatively high. Consequently, elevated urinary excretion of cobalamin is expected in patients with overflow proteinuria, and was indeed found in a patient with paroxysmal nocturnal haemoglobinuria.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

“…The Ca2+-sensitive binding sites previously described (10,11) on renal proximal tubule cells may be identical to megalin, whereas TC-B12 binding sites on several nonepithelial cell types not expressing megalin must be of different origin. A protein with unknown primary structure has been purified by ligand-affinity chromatography (35,37,38).…”

“…It is suggested to be a homodimer with an estimated size of 120-130 kDa (35) as judged by SDS/gel electrophoresis. The protein has also been detected by immunoblotting of isolated rat Mg2+-precipitated kidney basolateral membranes (40), and this (5,6,8,10,12) showing that the proportional uptake in the kidney of dietary B12 or intravenously administrated TC-B12 is higher at a high B12 load in the organism (5,6,8,12 were reported to inhibit the binding to this receptor, which might be identical to megalin. The binding of both the apo-and holo TC seems biological meaningful, because binding of only the holoform would result in loss of TC in the urine.…”

“…Binding sites for TC-B12 have previously been described (10,11,(34)(35)(36)(37)(38)(39) on several different cell types including fibroblasts, hepatocytes, renal tubule cells, and syncytiotrophoblasts. The Ca2+-sensitive binding sites previously described (10,11) on renal proximal tubule cells may be identical to megalin, whereas TC-B12 binding sites on several nonepithelial cell types not expressing megalin must be of different origin.…”

“…In agreement with this fact, high-affinity binding sites for complexes, but not for free B12, have been shown on the luminal surface of isolated rat (10) and hog tubule cells (11). Interestingly, the proportional uptake in the kidney of dietary B12 or intravenously administrated TC-B12 is higher at a high B12 load in the organism, and a regulatory mechanism for uptake and storage of B12 in the kidney has been suggested (5,6,8,10,12).…”

“…In agreement with this fact, high-affinity binding sites for complexes, but not for free B12, have been shown on the luminal surface of isolated rat (10) and hog tubule cells (11). Interestingly, the proportional uptake in the kidney of dietary B12 or intravenously administrated TC-B12 is higher at a high B12 load in the organism, and a regulatory mechanism for uptake and storage of B12 in the kidney has been suggested (5,6,8,10,12).Megalin [previously designated glycoprotein 330 (gp330) or Heymann Nephritis autoantigen] is a 600-kDa endocytosismediating membrane protein expressed in the tubule epithelium (13-15) and some other absorptive epithelia, e.g., in yolk sac, lung, retina, and inner ear (16,17). Megalin belongs to the low density lipoprotein receptor gene family (18) and has been shown to bind different substances including clusterin (19), polybasic aminoglycoside drugs (20), and several of the proteins binding to the homologous 600-kDa a2-macroglobulin receptor/low density lipoprotein receptor-related protein (a2MR/LRP)-e.g., the 39-44 kDa receptor-associated protein (RAP) and plasminogen activators (free or in complex with type-1 inhibitor) (for reviews, see refs.…”