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PubMed1995

Membrane expression and interactions of human transcobalamin II receptor.

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.

Biopolymers↗
PubMed1996

Dimerization of transcobalamin II receptor. Requirement of a structurally ordered lipid bilayer.

Transcobalamin II receptor (TC II-R) exists as a monomer and a dimer of molecular masses of 62 and 124 kDa in the microsomal and plasma membranes, respectively, and in vitro, pure TC II-R monomer dimerizes upon insertion into egg PC/cholesterol (molar ratio, 4:1) liposomes (Bose, S., Seetharam, S., and Seetharam, B. (1995) J. Biol Chem. 270, 8152-8157 and Bose, S., Seetharam, S., Hammond, T., and Seetharam, B. (1995) Biochem. J. 310, 923-929). The current studies were carried out to define the mechanism of TC II-R dimerization. Both the mature TC II-R (62 kDa) and the enzymatically deglycosylated TC II-R (45-47 kDa) demonstrated optimal association and formed dimers of molecular masses of 95 and 124 kDa, respectively, at 22 degrees C when bound to egg PC vesicles containing at least 10 mol % of cholesterol. Mature TC II-R dimerized upon insertion into synthetic phosphatidylcholine vesicles of different fatty acyl chain length (dimyristoyl, dipalmitoyl, and disteroyl phosphatidylcholine) in the absence or the presence of cholesterol at temperatures below or above their transition temperatures, respectively. Dimerization of TC II-R also occurred with vesicles prepared using lipid extract from the plasma but not microsomal membranes. Cholesterol depletion of native intestinal plasma membranes or its enrichment in the microsomal membranes resulted in the in situ conversion of the 124-kDa dimer to the 62-kDa monomer or of the monomer into the dimer form, respectively. Treatment of plasma membranes with phospholipase A2 resulted in the conversion of the dimer form of the receptor to the monomer form and spin label studies using 1-palmitoyl, 12 doxylsteroyl phosphatidylcholine revealed that interactions of TC II-R with PC vesicles increased order around the probe. Based on these results we suggest that dimerization of TC II-R is mediated by its interactions with a rigid more ordered lipid bilayer membrane, is regulated in plasma membranes by cholesterol levels, and is independent of glycosylation-mediated folding.

Animals↗
PubMed1997

Bipolar functional expression of transcobalamin II receptor in human intestinal epithelial Caco-2 cells.

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.

Animals↗
PubMed1998

Brefeldin A (BFA) inhibits basolateral membrane (BLM) delivery and dimerization of transcobalamin II receptor in human intestinal epithelial Caco-2 cells. BFA effects on BLM cholesterol content.

Brefeldin A (BFA) treatment of Caco-2 cells (5 microg/ml for 12 h) reduced by 90% the cholesterol, but not the phospholipid (PL), levels of the basolateral membrane (BLM), thus altering its PL/cholesterol molar ratio from 2.6 to 22.0, and decreasing its steady state fluorescent anisotropy (rs) from 0.27 to 0.15. BFA treatment for 12 h also resulted in complete loss of transcobalamin II receptor (TC II-R) activity/protein levels in the BLM and the disappearance of trans-Golgi network (TGN) morphology as revealed by confocal immunofluorescence microscopy using antibody to TGN 38. However, BFA treatment had no effect on either total cellular cholesterol, TC II-R activity, or PL levels. When cells treated with BFA for 12 h were exposed to BFA-free medium for 0-24 h, all of the effects were reversed, including reappearance of normal TGN morphology. TC II-R delivered to the BLM during this period was progressively sialylated and changed its physical state from a monomer (8 h) to a dimer (12 h), coinciding with increased delivery (11-53 pmol) of cholesterol to the BLM and an increase in the BLM rs from 0.15 to 0.21. These results indicate that cholesterol, but not PL, delivery to the BLM of Caco-2 cells is BFA-sensitive, and cholesterol, by influencing the higher order of the BLM, is essential for TC II-R dimerization.

Anti-Bacterial Agents↗
PubMed2002

Effects of interferon beta on transcobalamin II-receptor expression and antitumor activity of nitrosylcobalamin.

BACKGROUND: The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl. METHODS: Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-beta, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively. RESULTS: Cancer cell lines were more sensitive to NO-Cbl (with ID(50)s [the dose that inhibits growth by 50%] as low as 2 microM) than normal cell lines (with ID(50)s of 85-135 microM). Single-agent NO-Cbl and IFN-beta treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-beta treatment increased TC II-R expression in vitro and uptake of [(57)Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-beta were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl. CONCLUSION: NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-beta resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.

Animals↗
PubMed1972

Transport function of transcobalamin II.

The uptake of free and bound (57)CoB(12), principally to transcobalamin II (TC II), was studied in isolated, perfused liver and kidney of the dog. (1) There was good uptake of canine TC II-B(12) by both organs. (2) In the liver TC II enhanced uptake over that of free B-12. (3) Renal uptake of free B-12 was greater than that of TC II-B(12). Free B-12 was neither lost in the urine nor returned to the circulation. (4) On a per gram tissue basis, renal uptake of TC II-B(12) was greater than hepatic. (5) There was renal release or production of TC II (6) Some TC II but more of a larger molecular size binder came from the liver. (7) Passing free B-12 through the kidney enhanced its uptake by the liver. (8) Passing free B-12 through the liver depressed its uptake by the kidney. (9) It is postulated that the distribution of B-12 can be modified by (a) different responses of tissue to TC II-B(12), (b) synthesis of TC II by an organ, and (c) the effects of B-12 passing through one organ to another.

Animals↗