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

Abstract

The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC-cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. IntroductionCellular uptake of cobalamin (Cbl, vitamin B 12 ) in mammalian cells is mediated by receptors expressed on the cell surface. 1 Transcobalamin (TC, TCII), a plasma protein secreted by the endothelial cells, binds the Cbl absorbed in the distal ileum and carries between 10% and 30% of the total circulating Cbl. 2,3 TC saturated with Cbl specifically binds to the receptor (TCblR) and is internalized by endocytosis. The TC is degraded in the lysosome, and the free Cbl released is converted into Cbl cofactors. 4,5 Methylcobalamin is a cofactor for the cytosolic enzyme methionine synthase in the conversion of homocysteine to methionine using N 5 -methyltetrahydrofolate 6 ; therefore, homocysteine is elevated in both Cbl and folate deficiency. 7 The mitochondrial enzyme methylmalonyl CoA mutase converts methylmalonyl CoA to succinyl CoA and requires 5Јdeoxyadenosylcobalamin as a cofactor. The elevated methylmalonic acid in Cbl deficiency is a direct consequence of a block in this pathway. 8 The definitive purification of TC 9 followed by the identification of vascular endothelium as the source of TC in blood 10 ultimately led to the cloning of the cDNA and the gene encoding this protein. 11,12 Attempts to purify the receptor have yielded ambiguous results 13,14 ; however, the functional properties of TCblR have been well characterized in cell culture models. 15,16 We have previously described the functional and structural properties of TCblR based on binding of TC-Cbl to TCblR from human placenta and by crosslinking studies. 17,18 Our data on the properties and structure of this receptor differ from 2 other reports describing the purification of this protein. 13,14 The report by Bose et al 14 described a receptor with different structural constituents. 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. This unique receptor has the specificity and affinity required for the cellular uptake of holo TC and differs from the 72/144 kDa monomer/dimer protein reported to be the receptor for TC-Cbl. 14 MethodsActigel ALD agarose (Sterogene Bioseparations, Carlsbad, CA), Ultralink matri...

Proteomics of vitamin B12 processing

“…Vitamin B 12 (cobalamin, Cbl), an ancient vitamin and ' Nature ' s most beautiful cofactor ' [ 1 , 2 ] is required by all cells in the body. Humans rely on dietary supplies of the vitamin since a biosynthesis pathway is lacking in higher organisms.…”

“…The exact mechanisms underlying these non-canonical actions remain largely unexplored. Dietary vitamin B 12 enters the gastrointestinal tract first by complexation with the B 12 -binder haptocorrin (HC), which is present in saliva. Once it reaches the stomach, the vitamin is relayed to a second B 12 -binder, intrinsic factor (IF).…”

“…Dietary vitamin B 12 enters the gastrointestinal tract first by complexation with the B 12 -binder haptocorrin (HC), which is present in saliva. Once it reaches the stomach, the vitamin is relayed to a second B 12 -binder, intrinsic factor (IF). Absorption of B 12 occurs in ileal enterocytes of the lower intestine, where the vitamin dissociates from IF and binds to apo-transcobalamin (TC), the cellular transporter of vitamin B 12 [ 12 ].…”

“…Once it reaches the stomach, the vitamin is relayed to a second B 12 -binder, intrinsic factor (IF). Absorption of B 12 occurs in ileal enterocytes of the lower intestine, where the vitamin dissociates from IF and binds to apo-transcobalamin (TC), the cellular transporter of vitamin B 12 [ 12 ]. The transcobalamin receptor (TCblR) captures holo-TC (TC • XCbl) from circulation and internalizes the complex by absorptive endocytosis [ 12 ].…”

“…Absorption of B 12 occurs in ileal enterocytes of the lower intestine, where the vitamin dissociates from IF and binds to apo-transcobalamin (TC), the cellular transporter of vitamin B 12 [ 12 ]. The transcobalamin receptor (TCblR) captures holo-TC (TC • XCbl) from circulation and internalizes the complex by absorptive endocytosis [ 12 ]. In the acidic milieu of the endosomal compartment, holo-TC dissociates from its receptor and TCblR recycles back to the cell surface [ 13 ].…”