Membrane Expression and Interactions of Human Transcobalamin II Receptor

Abstract

Antiserum raised to purified 62-kDa human placental transcobalamin II receptor (TC II-R) has been used to study its synthesis and membrane expression. The antiserum immunoprecipitated a 45-kDa protein from the cell-free translation using human kidney mRNA and recognized a single 124-kDa band on immunoblotting of placental and other human tissue membranes, and quantitation of the blots revealed high levels of TC II-R expression in the human kidney followed by placenta, intestine, and liver. Triton X-100 extraction of placental membranes resulted in the complete (100%) solubilization of the receptor, and immunoblotting of the Triton X-100-soluble fraction revealed a single band of 62 kDa. Lipid extraction of placental membranes with a mixture of chloroformmethanol (2:1) followed by immunoblotting revealed a single band of molecular mass 62 kDa. The molecular mass of the pure Triton X-100-bound receptor increased on SDS-polyacrylamide gel electrophoresis from 62 to 124 kDa upon its insertion in liposomes prepared using egg phosphatidylcholine and cholesterol. Chemical cross-linking of native membrane-or lipid vesicle-bound TC II-R or detergent-soluble extracts of the membrane with 125I-TC II-cobalamin revealed that both the 124- and 62-kDa forms of the receptor were active in ligand binding. Based on these results we suggest that TC II-R is synthesized as a single polypeptide of 45 kDa, and following its maturation (involving N- and O-glycosylation) the 62-kDa mature receptor is expressed in plasma membranes as a noncovalent dimer of 124 kDa. The dimerization of TC II-R in the plasma membranes is due to its interactions with annular lipids.

In Vitro and in Vivo Inactivation of Transcobalamin II Receptor by Its Antiserum

“…Cbl Receptor Activity and Protein Determination-TC II-R activity in the Triton X-100 extracts of normal and affected rabbit tissue total membranes or isolated intestinal apical and basolateral membranes was determined as described earlier (3). In some experiments, prior to detergent extraction and ligand binding assay, the total membranes were treated as follows.…”

“…57 Co]Cbl to pure human TC II-R was carried out as described earlier (3). The extracts were neutralized to pH 7.4 and dialyzed against 4 liters of 10 mM Tris-HCl buffer, pH 7.4, for 24 h, with one 2-liter exchange of the dialysis buffer at the end of 12 h, prior to use.…”

“…The plasma transport of absorbed dietary and biliary cobalamin (Cbl: vitamin B 12 ) 1 bound to plasma transporter, transcobalamin II (TC II) occurs by receptor mediated endocytosis (1) via TC II-receptor (2) (TC II-R), which is expressed as a noncovalent dimer of molecular mass of 124 kDa in all tissue plasma membranes (3). Disruption in the cellular uptake of TC II-Cbl will ultimately result in intracellular Cbl deficiency and decreased synthesis of coenzyme forms of Cbl, methyl-Cbl, and adenosyl-Cbl (4).…”

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

“…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.…”

“…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.…”